Name: large-animal model of donation after circulatory death and normothermic regional perfusion for cardiac assessment
Uploaded: 2022-05-10T16:00:00
Description: Watch this Scientific Journal Video about Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment at JoVE.com

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 in...

<ol>
	<li>Yusen, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Registry+of+the+International+Society+for+Heart+and+Lung+Transplantation:+thirty-third+adult+heart+transplantation+report-2016;+focus+theme:+primary+diagnostic+indications+for+transplant.">The Registry of the International Society for Heart and Lung Transplantation: thirty-third adult heart transplantation report-2016; focus theme: primary diagnostic indications for transplant.</a> The Journal of Heart and Lung Transplantation: the Official Publication of the International Society for Heart Transplantation. 35 (10), 1170-1184 (2016).</li><li>Hornby, K., Ross, H., Keshavjee, S., Rao, V., Shemie, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-utilization+of+hearts+and+lungs+after+consent+for+donation:+a+Canadian+multicentre+study.">Non-utilization of hearts and lungs after consent for donation: a Canadian multicentre study.</a> Canadian Journal of Anaesthesia. 53 (8), 831-837 (2006).</li><li>Messer, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+and+transplantation+of+the+donor+heart+after+circulatory+death.">Functional assessment and transplantation of the donor heart after circulatory death.</a> The Journal of Heart and Lung Transplantation: the Official Publication of the International Society for Heart Transplantation. 35 (12), 1443-1452 (2016).</li><li>Messer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcome+after+heart+transplantation+from+donation+after+circulatory-determined+death+donors.">Outcome after heart transplantation from donation after circulatory-determined death donors.</a> The Journal of Heart and Lung Transplantation: the Official Publication of the International Society for Heart Transplantation. 36 (12), 1311-1318 (2017).</li><li>Dhital, K. K., Chew, H. C., Macdonald, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donation+after+circulatory+death+heart+transplantation.">Donation after circulatory death heart transplantation.</a> Current Opinion in Organ Transplantation. 22 (3), 189-197 (2017).</li><li>Ardehali, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex-vivo+perfusion+of+donor+hearts+for+human+heart+transplantation+(PROCEED+II):+a+prospective,+open-label,+multicentre,+randomised+non-inferiority+trial.">Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomised non-inferiority trial.</a> The Lancet. 385 (9987), 2577-2584 (2015).</li><li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+donor+heart+viability+during+ex+vivo+heart+perfusion.">Assessment of donor heart viability during ex vivo heart perfusion.</a> Canadian Journal of Physiology and Pharmacology. 93 (10), 893-901 (2015).</li><li>Xin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+multi-mode+perfusion+system+for+ex+vivo+heart+perfusion+study.">A new multi-mode perfusion system for ex vivo heart perfusion study.</a> Journal of Medical Systems. 42 (2), 25 (2017).</li><li>Robinson, N., et al., Iaizzo, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Cardiac Anatomy, Physiology, and Devices. , 469-491 (2015).</li><li>Swindle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques. , (2007).</li><li>Nasir, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+inhibitor+improves+lung+protection+in+porcine+model+of+donation+after+circulatory+arrest.">HSP90 inhibitor improves lung protection in porcine model of donation after circulatory arrest.</a> The Annals of Thoracic Surgery. 110 (6), 1861-1868 (2020).</li><li>Aceros, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+heat+shock+protein+90+inhibitor+improves+cardiac+recovery+in+a+rodent+model+of+donation+after+circulatory+death.">Novel heat shock protein 90 inhibitor improves cardiac recovery in a rodent model of donation after circulatory death.</a> The Journal of Thoracic and Cardiovascular Surgery. 163 (2), 187-197 (2022).</li><li>Der Sarkissian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+90+inhibition+and+multi-target+approach+to+maximize+cardioprotection+in+ischaemic+injury.">Heat shock protein 90 inhibition and multi-target approach to maximize cardioprotection in ischaemic injury.</a> British Journal of Pharmacology. 177 (15), 3378-3388 (2020).</li><li>Aceros, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celastrol-type+HSP90+modulators+allow+for+potent+cardioprotective+effects.">Celastrol-type HSP90 modulators allow for potent cardioprotective effects.</a> Life Sciences. 227, 8-19 (2019).</li><li>Aceros, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+model+of+cardiac+donation+after+circulatory+death.">Pre-clinical model of cardiac donation after circulatory death.</a> Journal of Visualized Experiments. (150), e59789 (2019).</li><li>Der Sarkissian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celastrol+protects+ischaemic+myocardium+through+a+heat+shock+response+with+up-regulation+of+haeme+oxygenase-1.">Celastrol protects ischaemic myocardium through a heat shock response with up-regulation of haeme oxygenase-1.</a> British Journal of Pharmacology. 171 (23), 5265-5279 (2014).</li></ol>

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-i...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#39;&#39; x 120&#39;&#39;</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 perfusio...

Watch this Scientific Journal Video about Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment at JoVE.com

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 ...

This protocol describes a large animal model of donation after circulatory death followed by thoracoabdominal normothermic regional perfusion that precisely mimics the clinical heart transplantation. This could be used to evaluate potential strategies in translational research. The main advantage of this protocol is that it provides a suitable preclinical model for cardiac transplantation given the anatomical and surgical techniques similarities between the boar swine and the human hearts.Begin by using a cautery pen to perform a midline incision on an anesthetized animal from the mid cervical region to the xiphoid. Then, divide the subcutaneous fat, layer by layer, along the midline using electrocautery to reach the peristernum. After cutting around the sternal notch, use a finger to retract and sweep soft tissues from under the sternum.Open the sternum with a bone saw, ensuring that the sternum is completely divided, while being careful with the vein beneath the upper part of the sternal bone. Then, cauterize the sternum or apply bone wax to ensure adequate hemostasis. Next, using electrocautery, dissect and remove the thymus by lifting it from the pericardium and cauterize the vessels that supply the thymus from the aorta and the superior vena cava to prevent bleeding.Carefully cut the pericardium open with cautery. While cutting, insert fingers under the pericardium to avoid injury to the heart. Then, administer 300 units per kilogram of heparin intravenously to achieve systemic anticoagulation.If the test is available, ensure the activated clotting time is more than 300 seconds. Administer a bolus of three milligrams per kilogram propofol intravenously. Then, stop mechanical ventilation and disconnect the endotracheal tube.Monitor arterial pressure and peripheral oxygen saturation. When systolic pressure is less than 50 millimeters of mercury, start functional warm ischemia time. Establish circulatory arrest when there is asystole and an absence of arterial pulsatility.After the withdrawal of life sustaining therapies and confirmation of death, retract the right ventricle outflow tract inferiorly, the pulmonary artery to the left and the aorta to the right. Then, carefully dissect the aorta pulmonary space using cautery. Apply a large cross clamp over the supraaortic vessels to avoid and exclude cerebral perfusion.Next, using the Metzenbaum and right angle forceps, dissect delicately between the superior vena cava and the innominate artery and between the inferior vena cava and pericardium. And circle the superior and inferior vena cava using an umbilical tape or a simple zero silk suture and secure them with a tourniquet. Using a 3-0 suture, place two concentric purse-string sutures on the distal ascending aorta adventitia, avoiding full thickness sutures.Then, place a purse-string suture on the right atrium before securing all sutures with a tourniquet. Next, set up and prime the normothermic regional perfusion system. Then, cannulate the aorta using a 17 to 21 French arterial cannula and secure it in place by tightening the tourniquet, holding the purse-string suture.Create a five millimeter incision at the center of the purse-string on the right atrium and dilate it using a small angled instrument, like a right angle or snap. Cover the incision with a finger to avoid excessive bleeding. Next, use a double stage venous cannula to cannulate the right atrium.Then, secure the cannula by tightening the purse-string sutures using a tourniquet. Connect the cannulas to the venous line of the normothermic regional perfusion circuit using a half to three over eight inch connector, ensuring complete de-airing to avoid an airlock in the system. Ensuring that a minimum of 15 minutes has passed between the declaration of death and the initiation of normothermic regional perfusion to encompass the time needed in clinical practice preparation, draping and having access to the heart.15 minutes after starting functional warm ischemia time, initiate the normothermic regional perfusion and adjust flow rates progressively to reach a perfusion index of 2.5 liters per minute per square meter. Restart mechanical ventilation with a 50%fraction of inspired oxygen and six milliliters per kilogram tidal volume. When weaning off criteria are met, stop normothermic regional perfusion.Remove the cannula from the right atrium and quickly tighten the purse-string suture to minimize blood loss. Secure the suture with knots. Then follow the same procedure to remove the aortic cannula.Analyze cardiac function through echocardiography using a standard transesophageal probe and a transthoracic probe placed directly on the heart. Representative results of cardiac function demonstrated a significant decline in heart function following donation after circulatory death. However, these organs demonstrated similar functional recovery post transplantation when compared to conventionally transplanted hearts.Cerebral oximetry measurements during normothermic regional perfusion with the supraaortic vessels clamped confirmed the absence of adequate cerebral perfusion, while lung compliance measurements during normothermic regional perfusion and after weaning from support shown no significant change from baseline. The development of this technique introduced the importance of assessing the transplantability of other organs like the lungs, the kidneys, and the liver in the context of donation after circulatory death and normothermic regional perfusion.

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Medicine

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 ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

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 ...

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 Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...