Considerations for the use of porcine organ donation models in preclinical organ donor intervention research

Abstract Use of animal models in preclinical transplant research is essential to the optimization of human allografts for clinical transplantation. Animal models of organ donation and preservation help to advance and improve technical elements of solid organ recovery and facilitate research of ischemia–reperfusion injury, organ preservation strategies, and future donor‐based interventions. Important considerations include cost, public opinion regarding the conduct of animal research, translational value, and relevance of the animal model for clinical practice. We present an overview of two porcine models of organ donation: donation following brain death (DBD) and donation following circulatory death (DCD). The cardiovascular anatomy and physiology of pigs closely resembles those of humans, making this species the most appropriate for pre‐clinical research. Pigs are also considered a potential source of organs for human heart and kidney xenotransplantation. It is imperative to minimize animal loss during procedures that are surgically complex. We present our experience with these models and describe in detail the use cases, procedural approach, challenges, alternatives, and limitations of each model.


| INTRODUC TI ON
Transplantation is the most efficient therapy for end-stage organ failure.However, the unavailability of adequate organs for transplantation to meet existing demand has resulted in major organ shortage crises. 1 Despite the growing gap between the supply of donor organs and increasing number of recipients on the transplant waiting lists, data show that only a fraction of offered organs are ultimately accepted for transplantation. 2,3Expanding the donor pool by increasing utilization of marginal organs together with optimizing the preservation methods for out-of-body organ transport are key components of the strategy for ameliorating the organ shortage.5][6] Despite clinical success of DCD from Maastricht category III donors, there remain concerns about the effect of warm ischemia on post-transplantation graft performance. 710][11] Animal models play an indispensable role in enabling biomedical studies to address these aims. 124][15][16] The reproducibility of pig models has also permitted investigation into optimal donor organ preservation. 17,18udies using large animal models for donor organ research can be grouped into three distinct categories.In the first category (Table 2A) are studies evaluating hemodynamic, metabolic, and hormonal changes associated with brain death and the effects of brain death on donor organ function [19][20][21][22][23][24][25] or pharmacologic interventions to mitigate the adverse effects of brain death. 26,279][30] In the second category (Table 2B) are studies that evaluate experimental methods of DBD graft preservation and technical and pharmacological modifications of DCD graft recovery. 17,31,32,33,34,35,36,37,38,39,40,41[44][45][46][47][48][49][50][51]

| Donation after brain death
Most large animal models of DBD involve the use of a subdural balloon to elevate intracranial pressure (ICP), thereby compromising brain perfusion and causing global brain and brainstem ischemia and herniation.Cushing reflex, hyperdynamic state, catecholamine storm, vasopressin and adrenocorticotropic hormone cessation, total cerebral necrosis, and diabetes insipidus are consistent findings after intracranial pressure rises above brain perfusion pressure for at least 30 min. 19,27,44

| Donation after circulatory death
The most clinically relevant animal DCD model is based on asphyxiation and circulatory arrest subsequent to the withdrawal of mechanical ventilation in a paralyzed animal. 39To a certain degree, the model allows for control over the duration of hypoxic and hypotensive warm ischemia and thus facilitates studies focused on the impact of these intervals on graft injury and postreperfusion performance.This model can also be used to produce allografts for in-situ (inside of the body) reanimation of multiple organs with either abdominal or thoracoabdominal normothermic perfusion and ex-situ (outside of the body) single organ machine perfusion after direct procurement. 34,40,41,47,52These platforms enable organs to be evaluated after a period of ischemia, and facilitate further research aimed at reanimation and preservation of DCD allografts.

| Veterinary care, anesthesia induction, and common instrumentation
All animal work needs approval from the Institutional Animal Care and Use Committee before commencing the experiments.For both DBD and DCD procedures, veterinary care, anesthesia induction, and certain instrumentation and vascular access lines are similar.
The common initial steps are described here, followed by DBD-and DCD-specific protocols.Essential and optional measurements are summarized in Table 3. Vascular access is summarized in Table 4.A complete pharmacopeia for DBD and DCD procedures is provided in Table 5.

Donation after brain death (DBD)
1. Study the impact of brain death on the quality of donor organs 2. Evaluate different preservation strategies for DBD organs Donation after circulatory death (DCD)
TA B L E 2 C Studies using large animal models in pre-clinical donor organ research evaluating post transplantation outcomes.7. Normal saline is infused following the 4/2/1 rule (4 mL/kg/h for the first 10 kg, 2 mL/kg/h for the next 10 kg, and 1 mL/kg/h for each kg after the first 20 kg).

| Donation after brain death
1.After induction of general anesthesia, intubation, insertion of percutaneous arterial and venous lines, and placement of noninvasive monitoring, the animals are positioned prone.Care is taken to avoid displacing vascular access and non-invasive monitors as the animal is repositioned.
Coronal and sagittal sutures are identified (Figure 1B), and two burr holes are drilled through the skull in the lateral frontotemporal regions using a rotary drill (Figure 1B,C).One burr hole is enlarged to approximately 6 mm to accommodate the balloon of a Foley catheter.4. A 20G angiocath is inserted through the smaller burr hole for ICP measurement.The catheter must be flushed to ensure the appropriate tracing for accurate ICP measurement.A 14-Fr 2-way balloon-tipped Foley catheter is introduced through the larger burr hole and positioned inside the subdural/subarachnoid space.
5. The Foley balloon is gradually inflated with water to elevate ICP above mean arterial pressure (MAP), typically in 5 cc increments with a 5-min pause to assess physiological response.

Brainstem death (BSD) is established after a 30-min interval when
ICP pressure is maintained above MAP.This is typically preceded by transient hypertension and tachycardia (Cushing response).
Absence of a Cushing response in the presence of continuously elevated ICP confirms brainstem death.7. Animals often require vasoactive management following BSD due to marked hypotension.Our initial intervention is vasopressin infusion at 0.02 U/min and saline boluses of 10 mL/kg (Figure 3).Additionally, vasopressin infusion may be increased to 0.04 U/min and norepinephrine infusion ranging from 0.02 to 0.06 mcg/kg/ min may be initiated as necessary.
8. After brain death is confirmed, the animals are turned supine for organ recovery.5) is administered immediately prior to planned discontinuation from ventilatory support.Anesthesia with inhalation isoflurane is switched to an IV continuous infusion of Propofol (14-20 mg/kg/h).A bolus of vecuronium (1 mg/kg IV) is delivered, followed by discontinuation of mechanical ventilation.

Heparin (Table
The breathing circuit from the anesthesia machine is then disconnected to prevent apneic oxygenation.
6. Hypoxic cardiac arrest is declared when central venous pressure equals mean arterial pressure and mechanical asystole is noted.These events are commonly preceded by a brief elevation of heart rate and blood pressure which is presumed to be the result of perimortem release of endogenous catecholamines.
7. The time interval from the withdrawal of ventilatory support to cardiac arrest varies from animal to animal.After death declaration, there may be a variable period of warm ischemia added before organs are removed to standardize the total length of warm ischemia for all study animals.The method of organ procurement is dependent on whether ex-situ or in-situ organ resuscitation is planned.There are several challenges with using the DBD model.While venous access is required for the induction and management of the animal outside the specifics of this model, additional vascular access can be challenging as it is our experience that the porcine vasculature is more prone to intimal dissection than human vessels.The use of ultrasound can be very helpful in obtaining access with less trauma to the vessels.The alternative to percutaneous approach is surgical cutdown with cannulation of the vessels under direct vision.
The identification of the cranial sutures is enhanced by using an electrosurgical pencil to cauterize the exposed skull's surface, resulting in the sutures being much more pronounced.Our experience with the intracranial balloon does not support the description in prior publications that the balloon could be advanced in a subdural position.It is technically challenging to enter the cranium without breaching the dura and it may be necessary to position the balloon in the subarachnoid or intraparenchymal space. 24,44me authors suggest using the absence of oculo-pupillary and corneal reflexes as confirmatory criteria for brain death. 53The interpretation of these tests in a paralyzed animal is very unreliable and the time required to fully allow the return of neuromuscular function can be lengthy.Loss of the Cushing reflex response in the presence of elevated ICP (above MAP) and the corresponding decrease of heart rate and blood pressure can be used to determine the start of brain ischemia (Figure 3).Keeping the intracranial pressure greater than mean arterial pressure for at least 30 min ensures the brain remains without blood supply and inevitably leads to irreversible brain structural damage.In our opinion, the confirmatory tests are not necessary since non-perfused brain and brainstem cannot survive.This is supported by observation The hemodynamic stability of the animals following brain death could be very challenging to manage.Initial attempts to maintain mean arterial pressure with volume and phenylephrine (ease of acquisition and low cost) may not always be successful.The additional infusion of vasopressors and inotropic agents is invariably necessary to maintain adequate hemodynamics (Figure 3 and Table 5).
Organ recovery within 1 hour from the diagnosis of brain death does not mirror clinical practice in which usually many hours pass in between declaration of brain death and organ procurement.We believe that this difference between clinical and research scenarios is not significant since the evidence suggests that hemodynamic, metabolic, and hormonal changes that are being attributed to the donor myocardial injury occur as early as 15 min after brain death. 54other limitation is the potential impact of brain death kinetics (velocity) on post-transplantation outcomes.Using an allotransplantation porcine model, Kerforne et al. 43 demonstrated that renal primary graft function was less satisfactory in pigs grafted with kidneys procured after slow-induced brain death as opposed to rapidinduced brain death.This is in keeping with the observation that heart transplant recipients who receive organs from donors after traumatic brain injury (rapid-induced) experience reduced mortality, rejections and cardiac allograft vasculopathy in comparisons to recipients who received organs from donors after non-traumatic brain death (slow-induced). 55

| Donation after circulatory death
This model is technically very demanding and requires advanced surgical and wire skills to minimize animal loss.When in-situ recovery is planned, some technical nuances need to be considered.For example, occlusion of the head/neck vessels before initiation of insitu organ perfusion significantly impacts monitoring devices placed on cranial structures or inserted into neck vessels.The presence of lower extremity monitors and access is important.
Porcine tissues (as opposed to humans) are very friable, and the porcine ascending aorta is short.This poses some technical challenges when handling the tissues and inserting the catheters.
The pressure-volume (PV) loop catheter is extremely difficult to advance across the aortic valve into the LV.We found that positioning the sheath directly into LV allows a more straightforward path for the catheter across the valve.When in-situ reperfusion of organs is planned, the head and neck vessels must be occluded before initiation of perfusion to preserve an uninterrupted process of brain ischemia after the flow to the thoracic and abdominal organs is restored.This requires dissection around the carotid artery and the occlusion of the vessel around the PV loop catheter.
Monitoring vital signs after the occlusion of the head and neck vessels presents difficulties, as many of the common clinical monitors and access points are using these vessels.Monitoring of the pulse oximetry cannot be accomplished from the head, ears, snout, etc. and must be placed caudally.Additionally, the venous access via the ear vein will no longer function and therefore cannot be used.
It is important to establish venous and arterial access that permits use throughout the experiment, generally through the femoral vessels.Some animals may demonstrate refractory ventricular arrhythmia that is difficult to control with antiarrhythmic medications and external defibrillation may be required.Manipulation of the heart during placement of the sheaths and catheters must be done carefully as the cardiac function may be significantly impaired by this process.
A major limitation of the hypoxic circulatory arrest model of DCD is an unpredictable length of agonal period, i.e. the time interval from the withdrawal of respiratory support until circulatory arrest.From our experience, the agonal time in paralyzed animals ranges between 5 and 8 min.There is less variability in the agonal time when using animal models in comparison to clinical practice since paralyzed animals lack residual respiratory drive.To obtain consistent hypoxic intervals for all DCD study animals, it may be necessary to implement a variable period of no-touch time following circulatory arrest prior to delivery of cardioplegia or initiation of in-situ reperfusion.
It is currently not known whether the porcine heart has the same tolerance for warm and cold ischemia as the human heart.
Experience from the pig xenotransplant experiments showed that non-ischemic porcine heart preservation (as opposed to cold static storage) is crucial for the survival of the transplanted hearts. 51,56,57is suggests that pig hearts might be more susceptible to cold ischemic injury than human hearts.Whether this difference in tolerance to cold ischemia between humans and pigs also applies to warm ischemia is currently not known (Figure 4).

| Donation after brain death
An alternative to using a Foley catheter to increase intracranial pressure is the infusion of autologous blood in the brain parenchyma through a catheter placed in the subdural space. 27In this model, animals developed the typical Cushing reflex between 60 and 90 min after the start of the intracranial blood infusion (1.5 mL/min).Cooper et al. described a model in which brain death was induced by clamping or ligation of both carotid and subclavian arteries.When compared to the method of the sudden increase of intracranial pressure with the balloon, both models showed identical hemodynamic changes. 58

| Donation after circulatory death
Martin et al. 45 described a large animal model of circulatory arrest by means of exsanguination.However, this mode of death does not reflect a real clinical DCD situation.Additionally, there is some evidence that death from asphyxiation causes more myocardial damage than death from exsanguination. 48,59 alternative to the hypoxic pulmonary arrest DCD model is to achieve organ hypoperfusion through a combined pharmacological and technical approach to decrease mean arterial pressure. 30,50though such alternative models are very reliable and reproducible, they do not account for the effect of hypoxia, specifically hypoxic pulmonary vasoconstriction, on the physiologic changes in thoracic organs during the agonal period. 29

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
Large animal models of DBD and DCD organ recovery are highly important to translational research groups working on strategies to optimize ischemia-reperfusion injury and organ preservation.They also provide an opportunity to facilitate and improve recovery technique, team technical skills, communication, and collaboration during innovative surgical procedures employed in organ recovery such as in-situ reperfusion after circulatory death.Models can also allow for concurrent and collaborative research programs involving thoracic and abdominal organs.
Porcine models of DBD and DCD organ donation have been described previously. 25,30,43The purpose of this article was to present an overview of both models with the emphasis on key conceptual and practical considerations.We outlined step-by-step description of procedural details, instrumentation, and pharmacopeia of current DBD and DCD models.We described common pitfalls, limitations, and challenges inherent to these procedures, and offered alternatives.We believe that this work will provide a reliable resource for re-

FU N D I N G I N FO R M ATI O N
This study was funded by a Team Seed Grant (#26685) from the University of Nebraska Collaborative Initiative Program; S. Merani, P.I.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that there is no conflict of interest regarding the publication of this article.

1 .
The porcine model of circulatory death is based on the hypoxic circulatory arrest after controlled withdrawal from respiratory support in a paralyzed animal.Essential monitoring and optional measurements during withdrawal in DCD model are described TA B L E 5 Pharmacopeia for porcine organ donation experimental models. .The rationale for essential monitoring is to define the time intervals for hypoxic and hypotensive warm ischemia periods.Optional measurements can provide additional invasive hemodynamic data.2. After induction of general anesthesia and obtaining femoral vascular access (as described in Section 2.1: Veterinary Care, Anesthesia Induction, and Common Instrumentation) an 8 Fr sheath is inserted percutaneously under ultrasound guidance in F I G U R E 1 Catheters and craniotomy landmarks for DBD procedure.(Ai) External craniotomy landmarks on dorsal skull.(Aii) Parasagittal cross-section of burr hole locations relative to the brain parenchyma.(Bi) 5 × 5 cm skin flap is raised to identify the coronal and sagittal sutures.Two contralateral burr holes are drilled.One burr hole is to admit the 14 Fr balloon-tipped Foley catheter, and the other is sized to admit a 20G angiocath for ICP measurement.(Bii) Parasagittal view of the 20G angiocath ICP monitor and Foley placement prior to Foley inflation.(Ci) Dorsal view of inserted angiocath and Foley catheters.(Cii) the Foley balloon tip is inflated with water until an appropriate ICP and hemodynamic response is observed.Sulci are compressed.Gray matter may be observed extruding from the Foley burr hole site.the right common carotid artery.A 7 Fr Pressure-volume impedance catheter (Transonic Systems, Ithaca, NY, USA) is inserted in left ventricle (LV) via the sheath in the right common carotid artery (Figure 2).Transesophageal ultrasound (Phillips iE33 Ultrasound with X7-2t TEE Probe) is used to confirm the correct placement of the cannulae/catheters and to evaluate cardiac function after manipulation.3. Lidocaine (50 mg IV bolus) is administered before sternotomy to prevent ventricular arrhythmias.A median sternotomy is performed.The pericardial sac is opened, and a pericardial well is created with stay sutures using 0-silk.4. A multi-lumen catheter (14 Fr) is inserted under direct visualization in the right atrium through the superior vena cava.The catheter is used for pressure monitoring and drug delivery.An 8.5 Fr Swan-Ganz pulmonary arterial catheter with mixed venous oxygen saturation (Edwards Lifescience, Irvine, CA, USA) is inserted through the distal port of the multilumen catheter and advanced into the PA.

8 .
If ex-situ reperfusion is the intended recovery method, median sternotomy and laparotomy are performed, organs are flushed with the organ specific cold storage solution, removed from the donor, and instrumented on ex-situ apparatus.9. In-situ reperfusion requires placement of the drainage (venous) and return (arterial) cannulae for the extracorporeal mechanical circulatory support to drive the circulation with oxygenated blood in the absence of the native heart and lung function.The venous cannula is placed into the inferior vena cava through right atrial appendage, the arterial cannula is placed in the ascending aorta.The brain is excluded from circulation by clamping the supra-aortic vessels before the perfusion is initiated.As opposed to real clinical practice when instrumentation is only permitted after death, in the animal model it is advisable to cannulate great vessels before ventilation is stopped.This promotes animal stability and avoids animal loss from surgical misadventures that may occur because of the haste to initiate extracorporeal circulation in effort to minimize the time sensitive organ damage.

F I G U R E 2
LV catheter placement for DCD procedure.(A) Fourchamber cross section view of porcine heart.An 8 Fr introducer sheath fully enclosing a 7 Fr pressure conductance catheter has been advanced from the right common carotid artery past the aortic valve.(B) The 7 Fr pressure conductance catheter has been advanced from the introducer sheath into the left ventricle.(C) The introducer sheath is withdrawn to leave the pressure conductance catheter positioned in the left ventricle.

4 | 4 . 1 |
LIMITATI ON S AND CHALLENG E S OF ANIMAL MODEL S Donation after brain deathThe model of brain death based on elevating intracranial pressure using the balloon of a Foley catheter inflated inside the skull is very reproducible and technically not demanding.The required personnel include a surgeon and an intensivist/anesthesiologist coordinating anesthesia care with veterinary staff.Necessary procedures include vascular access, monitoring ICP, managing hemodynamic disturbances during brain death, timekeeping, scribing, recording data, and obtaining samples.

of
Chen et al. who recorded a flat-line electroencephalogram after 8-20 min after inflation of the balloon in every subject in their brain death model.The average time for all brainstem reflexes (including corneal, pupillary, oculomotor, and breathing reflexes) to disappear varied in each animal and generally occurred at an average time of 13.3 ± 1.5 min after balloon inflation.It is noteworthy that our initial experiments involving the establishment of brain death were performed in non-paralyzed animals.These animals had significant seizure and tonic-clonic motion when the intracranial balloon was F I G U R E 3 Event timeline and summary of hemodynamics during typical DBD procedure.Event timeline following vascular access during DBD procedure Pl(Section 2.2).ICP, intracranial pressure; MBP, mean blood pressure; SBP, systolic blood pressure.X-axis: time (minutes) following vascular instrumentation.inflated.This created difficulties in maintaining stable vascular access.
producible pre-clinical donor organ intervention research and enable new investigators to address unmet needs in transplant medicine.AUTH O R CO NTR I B UTI O N S FH, SM, NM, KD, MM, LF, WT, TW, MA, and MU contributed to the conception and design of the work; FH, SM, NM, KD, MM, LF, RS, and MU contributed to data acquisition; FH, SM, NM, and MU contributed to data analysis and interpretation; FH, SM, NM, and MU wrote the draft manuscript; FH, SM, NM, MM, LF, and MU revised the manuscript; all authors approved the final version for publication.

F I G U R E 4
Event timeline and summary of hemodynamics during typical DCD procedure.Event timeline following vascular access during DCD procedure.CPB, cardiopulmonary bypass; PEA, pulseless electrical activity.X-axis: time (minutes) following vascular instrumentation.

journal, date Model Death induction Study group Main findings
1. Study the impact of warm ischemia on the quality of DCD organs 2. Evaluate different recovery techniques, interventions, and preservation strategies to improve DCD organ quality 3. Facilitate and improve recovery team technical skills, communication, and cooperation during clinical DCD organ recoveriesTA B L E 2 A Studies using large animal models in pre-clinical donor organ research evaluating hemodynamic, metabolic, and hormonal changes during brain death and circulatory death.Author,

TA B L E 2 B Studies
using large animal models in pre-clinical donor organ research evaluating procurement techniques and preservation methods.

Author, journal, date Model Death induction Study group Main findings
Vascular access catheters for porcine organ recovery models.
42ttner et al.42Ann Thorac Surg 1995DBD Inflation of a subdurally placed balloon 34 dog heart transplantationsBrain death causes a significant loss of right and left ventricular function.These injuries are greater in the right ventricle and may contribute to early right ventricular failure after transplantation.Brain death and cardiac preservation interact significantly to impair right ventricular function further 3. Animals are placed in the prone position.General anesthesia is induced with mask-inhaled isoflurane to effect followed by oral intubation and mechanical ventilation with inhaled isoflurane 1%-4% (Table5).Respiratory rate is adjusted to target end-tidal CO 2 of 35-40 mmHg and oxygen saturation above 95%.4.Animals are placed in a lateral position to place five ECG electrodes: left shoulder (black); right shoulder (white); left lower artery for invasive blood pressure monitoring and sampling.In the same manner, a multi-lumen catheter is inserted percutaneously into the superficial femoral vein for sampling and drug delivery (Table4).6.Vital signs are monitored, and data recorded using dedicated software (VitalRecorder, Vital DB, Seoul, South Korea).TA B L E 3 Essential and optional monitoring for porcine DBD and DCD models.TA B L E 4