Name: normothermic ex situ heart perfusion in working mode: assessment of cardiac function and metabolism
Uploaded: 2019-01-12T12:30:00
Description: Watch this Scientific Journal Video about Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism at JoVE.com

This work was supported by grants from the Canadian National Transplant Research Program. SH is the recipient of a Faculty of Medicine and Dentistry Motyl Graduate Studentship in Cardiac Sciences. DHF is a recipient of a Collaborative Research Projects (CHRP) grant in aid from the National Sciences and Engineering Research Council and Canadian Institutes of Health Research.

All the procedures in this manuscript were performed in compliance with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The protocols were approved by the institutional animal care committee of the University of Alberta. This protocol has been applied in female juvenile Yorkshire pigs between 35&#8211;50 kg. All individuals involved in ESHP procedures had received proper biosafety training.
1. Pre-surgical Preparations
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K., 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=Adult+heart+transplantation+with+distant+procurement+and+ex+vivo+preservation+of+donor+hearts+after+circulatory+death:+A+case+series.">Adult heart transplantation with distant procurement and ex vivo preservation of donor hearts after circulatory death: A case series.</a> Lancet. 385 (9987), 2585-2591 (2015).</li><li>Messer, S., Ardehali, A., Tsui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+donor+heart+perfusion:+Current+clinical+experience+and+the+future.">Normothermic donor heart perfusion: Current clinical experience and the future.</a> Transplant International. 28 (6), 634-642 (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>Messer, S. 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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=Ex+vivo+perfusion+in+a+loaded+state+improves+the+preservation+of+donor+heart+function.">Ex vivo perfusion in a loaded state improves the preservation of donor heart function.</a> Canadian Journal of cardiology. 31 (10), s202 (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=A+wholeblood-based+perfusate+provides+superior+preservation+of+myocardial+function+during+ex+vivo+heart+perfusion.">A wholeblood-based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion.</a> Journal of Heart and Lung Transplantation. (14), (2014).</li><li>Lips, D. 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=Left+ventricular+pressure-volume+measurements+in+mice:+comparison+of+closed-chest+versus+open-chest+approach.">Left ventricular pressure-volume measurements in mice: comparison of closed-chest versus open-chest approach.</a> Basic Research in Cardiology. 99 (5), 351-359 (2004).</li><li>Morita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+there+a+crystal+ball+for+predicting+the+outcome+of+cardiomyopathy+surgery?+Preload+recruitable+stroke+work,+may+be+a+possible+candidate.">Is there a crystal ball for predicting the outcome of cardiomyopathy surgery? Preload recruitable stroke work, may be a possible candidate.</a> Journal of Cardiology. 71 (4), 325-326 (2018).</li><li>Hatami, 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=.">.</a> Canadian Society for Transplantation. , (2017).</li><li>Anthony, C., 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+coronary+angiographic+evaluation+of+a+beating+donor+heart.">Ex vivo coronary angiographic evaluation of a beating donor heart.</a> Circulation. 130 (25), e341-e343 (2014).</li><li>Sandha, J. K., 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=Steroids+Limit+Myocardial+Edema+During+Ex+vivo+Perfusion+Of+Hearts+Donated+After+Circulatory+Death.">Steroids Limit Myocardial Edema During Ex vivo Perfusion Of Hearts Donated After Circulatory Death.</a> Annals of Thoracic Surgery. , (2018).</li></ol>

Successful perfusion is defined according to the aims of the study; however, this should include uninterrupted ESHP for the desired amount of time and complete collection of the data on cardiac function during the perfusion. For this purpose, a few critical steps in the protocol must be followed.
The heart is an organ with high oxygen and energy demands, and minimizing the ischemic time before cannulation and perfusion is an important principle that must be followed. The process of procurement...

Clinical relevance
While most aspects of cardiac transplantation have evolved significantly since the first heart transplant in 1967, cold storage (CS) remains the standard for donor heart preservation1. CS exposes the organ to a period of cold ischemia that limits the safe preservation interval (4&#8211;6 hours) and increases the risk of primary graft dysfunction2,3,4. Due to the static nature of CS, assessments of function or therapeutic interventions are not possible in the time between the organ procurement and transplantation. This is a particular limitation in extended criteria donors including hearts donated after circulatory death (DCD), creating an obstacle for overcoming the considerable gap between demand and the current donor pool5,6. To address this limitation, ex situ heart perfusion has been proposed as a novel, semi-physiologic method of preserving donated hearts, minimizing exposure to cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart during preservation time1,7,8.
Ex situ heart perfusion
One of the most frequently used methods for ex situ examination of the isolated heart is Langendorff perfusion. In this method, introduced by Oskar Langendorff in 1895, the blood flows into the coronary arteries and out the coronary sinus of the isolated heart, with the heart in an empty and beating state9,10. Clinical ESHP in a Langendorff mode with the Transmedics Organ Care System apparatus (OCS) has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts1, and has facilitated the clinical transplantation of DCD hearts11. However, there are concerns about the ability of the device to evaluate organ viability, as a number of donor hearts initially thought to be transplantable were discarded after perfusion on the OCS3. The OCS supports the heart in the Langendorff (non-working) mode, and thus possesses a limited capacity for evaluation of the pumping function of the heart3,12. A growing body of evidence suggests that functional parameters offer a better way to assess organ viability, suggesting that assessments of cardiac function may become a reliable tool for the evaluation and selection of hearts for transplantation during ESHP3,12,13,14, Furthermore, our studies on ex situ perfused porcine hearts suggest that ESHP in working mode provides enhanced functional preservation of the heart during the perfusion interval15,16.
An ESHP apparatus capable of preserving the heart in a working mode must possess a level of automation to safely and precisely maintain preload, afterload and flow rates. Also, such a system should possess the flexibility to facilitate comprehensive assessments of cardiac function to be undertaken. The ESHP apparatus used here is equipped with custom software that 1) provides and maintains desired aortic (Ao) and left atrial (LA) pressure/flow and 2) provides real-time analysis of functional parameters and visual evaluation of pressure waveforms with minimal need for supervision. Pressure data is acquired with standard fluid-filled pressure transducers, and flow data is acquired with transit-time doppler flow probes. These signals are digitized with a bridge and analog input, respectively. The heart is positioned horizontally with a slight elevation to the great vessels on a soft silicone membrane. The cannulation attachments pass through the membrane, incorporating a compliance chamber for dampening ventricular ejection. The goal of this work is to provide researchers in the field of cardiac transplantation with a protocol for ex situ perfusion and evaluation of the heart, under normothermic, semi-physiologic conditions in working mode, in a large mammal (Yorkshire pig) model.

<table>
	<tbody>
		<tr>
			<td>Debakey-Metzenbaum dissecting scissors</td>
			<td>Pilling</td>
			<td>342202</td>
			<td></td>
		</tr>
		<tr>
			<td>MAYO dissecting scissors</td>
			<td>Pilling</td>
			<td>460420</td>
			<td></td>
		</tr>
		<tr>
			<td>THUMB forceps</td>
			<td>Pilling</td>
			<td>465165</td>
			<td></td>
		</tr>
		<tr>
			<td>Debakey straight vascular tissue forceps&#160;</td>
			<td>Pilling</td>
			<td>351808</td>
			<td></td>
		</tr>
		<tr>
			<td>CUSHING Gutschdressing forceps</td>
			<td>Pilling</td>
			<td>466200</td>
			<td></td>
		</tr>
		<tr>
			<td>JOHNSON needle holder</td>
			<td>Pilling</td>
			<td>510312</td>
			<td></td>
		</tr>
		<tr>
			<td>DERF needle holder</td>
			<td>Pilling</td>
			<td>443120</td>
			<td></td>
		</tr>
		<tr>
			<td>Sternal saw</td>
			<td>Stryker</td>
			<td>6207</td>
			<td></td>
		</tr>
		<tr>
			<td>Sternal retractor</td>
			<td>Pilling</td>
			<td>341162</td>
			<td></td>
		</tr>
		<tr>
			<td>Vorse tubing clamp</td>
			<td>Pilling</td>
			<td>351377</td>
			<td></td>
		</tr>
		<tr>
			<td>MORRIS ascending aorta clamp</td>
			<td>Pilling</td>
			<td>353617</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical snare (tourniquet) set</td>
			<td>Medtronic</td>
			<td>CVR79013</td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 SILK black 12 X 18&#34; strands</td>
			<td>ETHICON</td>
			<td>A185H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PROLENE blue 18&#34; PS-2 cutting</td>
			<td>ETHICON</td>
			<td>8687H</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomedicus pump drive (modified)</td>
			<td>Medtronic</td>
			<td>540</td>
			<td>Modified to allow remote electronic control of pump speed</td>
		</tr>
		<tr>
			<td>Biomedicus pump</td>
			<td>Maquet</td>
			<td>BPX-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane oxigenator D 905</td>
			<td>SORIN GROUP</td>
			<td>50513</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing flow module&#160;&#160;</td>
			<td>Transonic</td>
			<td>Ts410</td>
			<td></td>
		</tr>
		<tr>
			<td>PXL clamp-on flow sensor</td>
			<td>Transonic</td>
			<td>ME9PXL-BL37SF</td>
			<td></td>
		</tr>
		<tr>
			<td>TruWave pressure transducer</td>
			<td>Edwards</td>
			<td>VSYPX272</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercept tubing 3/8&#34; X 3/32&#34; X 6&#39;</td>
			<td>Medtronic</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercept tubing 1/4&#34; X 1/16&#34; X 8&#39;</td>
			<td>Medtronic</td>
			<td>3108</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated/Refrigerated Bath Circulator&#160;</td>
			<td>Grant</td>
			<td>TX-150</td>
			<td></td>
		</tr>
		<tr>
			<td>ABL 800 FLEX Blood Gas Analyzer</td>
			<td>Radiometer</td>
			<td>989-963</td>
			<td></td>
		</tr>
		<tr>
			<td>5F Ventriculr straight pigtail cathter</td>
			<td>CORDIS</td>
			<td>534550S</td>
			<td></td>
		</tr>
		<tr>
			<td>5F AVANTI+ Sheath Introducer</td>
			<td>CORDIS</td>
			<td>504605A</td>
			<td></td>
		</tr>
		<tr>
			<td>Emerald Amplatz Guidewire</td>
			<td>CORDIS</td>
			<td>502571A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual chamber pace maker</td>
			<td>Medtronic</td>
			<td>5388</td>
			<td></td>
		</tr>
		<tr>
			<td>Defibrilltor</td>
			<td>CodeMaster</td>
			<td>M1722B</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion pump</td>
			<td>Baxter</td>
			<td>AS50</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical electrocautery device</td>
			<td>Kls Martin</td>
			<td>ME411</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas mixer</td>
			<td>SECHRIST</td>
			<td>3500 CP-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical oxygen tank</td>
			<td>praxair</td>
			<td>2014408</td>
			<td></td>
		</tr>
		<tr>
			<td>Cabon dioxide tank</td>
			<td>praxair</td>
			<td>5823115</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP biomedicals</td>
			<td>218057791</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

At the start of the perfusion (in non-working mode), the heart will normally resume a sinus rhythm when the temperature of the system and perfusate approaches normothermia. When entering working mode, as the LA pressures are approaching the desired values, ejection on the Ao pressure tracing should be observed and the LA flow (a reflection of cardiac output) should increase gradually. In a Yorkshire pig model (35&#8211;50 kg) and a starting heart weight of 180&#8211;220 grams, the initial...

Watch this Scientific Journal Video about Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism at JoVE.com

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

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 ...

This method can help answer key questions about the optimal preservation of donor hearts, as well as their functional and metabolic evaluation for subsequent transplantation suitability. The main advantage of this technique is it allows a functional evaluation of the donor heart that is not based solely on indirect metabolic assessments. The implications of this technique extend towards the therapy of dysfunctional donor hearts, as the heart function can be easily and repeatedly assessed over time.Generally, individuals new to this matter will struggle because some technical expertise is required for connecting the heart in a safe and secure manner. Visual demonstration of this method is critical as the technical aspects of the heart retrieval and connection to the device can be challenging to learn by text instruction alone. After confirming a lack of response to noxious stimulus, perform a median sternotomy on the anesthetized pig and once the heart has been exposed, use Metzenbaum scissors to open the pericardium.Use 1-0 silk sutures to fix the pericardial edges to the sternum and gradually collect 750 milliliters of whole blood from the two-stage venous cannula placed in the right atrium in an autoclaved glass container over a period of 15 minutes while simultaneously replacing the volume with one liter of an isotonic crystalloid solution. Add the blood to a perfusion circuit primed with 750 milliliters of Krebs-Henseleit buffer supplemented with 8%albumin and place a 14 gauge cardioplegia needle and cannula into the ascending aorta. Secure the cardioplegia needle on the aorta with a snare and connect the cardioplegia cannula to the cardioplegia bag.Add 100 milliliters of blood to 400 milliliters of cardioplegia. After the end of exsanguination, cross-clamp the ascending aorta with an aortic clamp. Next, deliver the cardioplegic solution into the aortic root.When all the solution has been administered, remove the clamp and excise the heart, including all of the aortic arch vessels and a segment of the descending aorta. To prevent the deleterious effects of prolonged ischemia, the heart should be procured and mounted on the apparatus for perfusion as quickly as possible. After removing any excess tissue around the left atrium, use a 3-0 polypropylene suture to place a purse string suture around the left atrial orifice.Suture and close the inferior vena cava with a 3-0 polypropylene suture. Place the left atrial cannula into the left atrial orifice and secure the cannula with a snare. Place the left atrial cannula over the silicone membrane embedded magnet of the apparatus, taking care that the magnet and the corresponding metal ring in the left atrial cannula are properly engaged.Attach the aorta to the aortic cannula embedded in the silicone membrane and use a silk tie to secure the aorta around the cannula. Turn the aorta to achieve a proper lie without tension or kinking and increase the aortic pump speed to about 1600 RPM and gently squeeze the ventricles to de-air the heart. The remaining air in the aortic root will be ejected through the innominate and subclavian branches.Connect the aortic purge line to the innominate artery and secure the connection with a silk tie. Snare the left subclavian artery orifice with a silk tie and secure the closure with a snare and a snap. Place a 5 French introducer sheath through the orifice of the subclavian artery, adjusting the length and orientation of the catheter so that the catheter does not interfere with the aortic valve function.Adjust the aortic pump speed to reach a mean pressure of 30 millimeters of mercury to start the perfusion in the non-working Langendorff mode. The appearance of a dark, deoxygenated perfusate in the pulmonary arterial line indicates the re-establishment of coronary flow. Turn the heat exchanger to 38 degrees Celsius to warm the perfusate and use a blood gas analyzer to check the dissolved gas status, adjusting the gas mixture to the appropriate parameters.After an hour in non-working mode, enter a suitable left atrial pressure in the desired left atrial pressure section of the software and initiate the feedback loop. The activated working mode will appear as a green button and the left atrial pump speed will automatically increase and decrease to reach and maintain the selected left atrial pressure. For the assessment and recording of the steady-state data, place a fluid-filled pigtail catheter through the introducer sheath into the left ventricle.For assessment of the preload recruitable stroke work, remove the pigtail catheter from the left ventricle and adjust the desired rate of drop in the left atrial pump speed during the analysis and the desired time period during which the analysis will take place. Click record pressure volume loop. The software will automatically exit the working mode and gradually reduce the left atrial pump RPM, while simultaneously recording the left ventricle stroke work and the left atrial pressure.At the conclusion of data collection, the software will perform a linear regression on the newly acquired data set to yield the preload recruitable stroke work. After the ex situ apparatus heart software has completed the analysis, a message will appear on the main page showing the correlation coefficient of the analysis. Click okay if the coefficient is greater than 0.95.The preload recruitable stroke work analysis results will be recorded. To assess the metabolic state of the heart and the perfusate, use the information obtained from the blood gas analysis of the perfusate samples collected from both the arterial and pulmonary artery lines every one to two hours. In a Yorkshire Pig model with a starting heart weight of 180 to 220 grams, the initial left atrial flow will be approximately 2, 000 milliliters minute, typically approaching about 2, 250 milliliters per minute during the first one to two hours of perfusion in the working mode.Blood gas analysis and metabolic assessments performed on the perfusate samples obtained during ex situ heart perfusion provide extensive information on the metabolic status of the heart over time, indicating the perfusate lactate concentration and the information needed to calculate the myocardial oxygen consumption. In the absence of the organs that naturally metabolize their clear cardiac biomarkers, the accumulation of these factors within the perfusate solution over time is typically observed. In addition to load dependent parameters, the left ventricle preload recruitable stroke work can be evaluated during the computer-controlled linear reduction in the left atrial pressure, as demonstrated.Echocardiography during ex situ heart perfusion can also provide additional information about myocardial function and anatomical parameters. While attempting this procedure, it's important to remember that ex situ heart perfusion is still in its infancy and that more studies are warranted to design better metabolic heart support for better preservation of cardiac viability and function. Following this procedure, other methods like angiographic imaging can be performed to answer additional questions about donor heart's coronary vascular status.After it's development, this technique paved the way for researchers in the field of organ transplantation to explore the possibility of the safe transplantation of extended criteria donated hearts in patients awaiting a heart transplant. Don't forget that working with biomaterials can be extremely hazardous and that precautions, such as wearing gloves and protective glasses, should always be taken while performing this procedure.

normothermic-ex-situ-heart-perfusion-working-mode-assessment-cardiac

Research

JoVE Journal

Medicine

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

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 ...

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, ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

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 ...

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.

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 ...

Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured between fascial borders at a given time point during an exercise. This selected time point produces a one-dimensional image resulting in real-time, live observation of anatomy. Ultrasound used during functional movement can be referred to as dynamic ultrasound; this is feasible and reliable with the use of a linear transducer, elastic belt, and foam block to secure consistent transducer placement. The lateral abdominal wall is commonly investigated using ultrasound due to the overlapping nature of the muscles. Surface electromyography (sEMG) can complement M-mode ultrasound imaging because it measures the electrical representation of muscle activation. There is minimal evidence using M-mode ultrasound and sEMG simultaneously during core exercise. Exercises that challenge the core musculature involve both isometric holds (e.g., side plank), as well as oscillatory extremity movements (e.g., dead bug). In this study, both instruments will be used simultaneously to measure core muscle function during exercise. Ultrasound measurements will be obtained using a linear transducer and ultrasound unit, and sEMG measurements will be acquired from a wireless sEMG system. To make comparisons between participants and exercises, normalization methods using static, exercise starting positions for both instruments will be used. An activation ratio will be used for ultrasound and calculated by dividing the contracted thickness (thickness during a time point of exercise) by the rested (starting position) thickness. Muscle thickness will be measured in centimeters from the superior inferior fascial border to inferior superior fascial border. These methods aim to offer an innovative and practical measurement of muscle function with M-mode ultrasound and sEMG during core endurance exercises.

This protocol uses motion mode ultrasound and surface electromyography simultaneously to measure muscle function of the core. Muscle thickness and activation of the local stabilizers (e.g., transverse abdominis, internal oblique) and global movers (e.g., external oblique) is achievable during specific time points of the side plank and dead bug exercises.

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured ...

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

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

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

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

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

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Coronary Artery Assessment During Ex-Situ Heart Perfusion

Coronary Angiography During Ex-Situ Heart Perfusion in a Porcine Model

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the current organ shortage. Coronary angiography is recommended in ECD at risk for coronary artery disease but is not systematically performed. These hearts are, therefore, either declined for transplant or procured without screening for coronary artery disease. Coronary angiography during normothermic ex-situ heart perfusion (NESP) could be an interesting approach to enhance the rate of ECD procurement and to reduce the risk of primary graft failure in the absence of coronary angiography in ECD. The present protocol aims to provide material details along with optimal imaging views for coronary angiography during NESP. Reproducible angiographic views were observed, including one dedicated to the right coronary artery, two for the left anterior descending artery, two for the circumflex artery, and a spider view. Continuous lactate extraction was observed in all procedures with a final median concentration of 1.10 mmol/L (0.61-1.75 mmol/L) two hours after coronary angiography, consistent with myocardial viability. The median contrast agent volume used for ex-situ imaging of the isolated perfused heart was 48 mL (38-108 mL). This protocol was reproducible for coronary artery imaging and did not impair myocardial viability during NESP.

Author Spotlight: Innovative Technique for Coronary Angiography in Marginal Donors

Screening for coronary artery disease could increase the procurement of donor hearts with extended criteria. A protocol for performing reproducible coronary angiography during ex-situ heart perfusion is described herein in a porcine model.

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the ...