Georges Lopez Institute, Lissieu, 69380, France
Claudia Lacerda, General Electric Healthcare, Buc, France

The present protocol was approved by the local ethics committee on animal experiments and by the Institutional Committee on Animal Welfare (APAFIS#30483-2021031811339219 v1, Animals Ethics Committee of the University of Paris Saclay, France). Animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals developed by the National Institute of Health and with the Principles of Laboratory Animal Care developed by the National Society for Medical Research.
NOTE: Surgical procedures were performed under strict sterility using the same techniques used for a human being. Experimental procedures included large white piglets (45-60 kg) and were performed under general anesthesia.
1. Animal conditioning and anesthesia protocol
<ol>
	<li>Allow the animals to acclimate for 7 days, with congeners and environmental enrichment, to ensure animal welfare.</li>
	<li>Do not feed the animals 12 h before their inclusion in the experimental protocol.</li>
	<li>Perform a premedication 30 min before the procedure with an intramuscular injection of an equimolar mixture of tiletamine and zolazepam (10 mg/kg) in the neck muscles.</li>
	<li>Once the animal is sedated, insert a catheter into the ear vein, and induce general anesthesia with an intravenous bolus of propofol (2 mg/kg) combined with administration of atracurium (2 mg/kg).</li>
	<li>Intubate the animal with a 7.5 mm orotracheal probe.</li>
	<li>Monitor the animal with continuous EKG, expiratory CO2, and oximetry.</li>
	<li>Maintain general anesthesia with inhaled isoflurane (2%) mixed with 40% oxygen supplement.</li>
</ol>
2. In situ hemodynamic and echocardiographic assessment of the heart
NOTE: Hemodynamic assessment is performed with a Swan Ganz Catheter, while baseline functional assessment of the heart is performed by transthoracic echocardiography.
<ol>
	<li>Insert percutaneously an 8 French (Fr) sheath into the brachiocephalic venous trunk using the Seldinger technique8.</li>
	<li>After de-airing the catheter and setting the 0 pressure, insert the Swan Ganz catheter into the 8 Fr sheath until a pulmonary pressure profile is observed on the monitoring screen.</li>
	<li>Obtain the pulmonary arterial occlusion pressure by pushing the Sawn-Ganz catheter in the pulmonary circulation while the balloon is inflated.</li>
	<li>Assess the cardiac output using the thermodilution approach by infusion of 10 mL of cold (4 &#176;C) saline solution in the proximal line of the Swan Ganz catheter. Repeat the measurement three times.</li>
	<li>Assess the left ventricular ejection fraction (LVEF) using the biplane Simpson technique9.</li>
	<li>Explore the aortic valve and aortic root to identify any structural disorder or aortic regurgitation above grade 2 that could compromise ex situ&#160;perfusion of the heart through the ascending aorta (Figure 1).</li>
</ol>
3. Description and priming of the normothermic ex situ perfusion (NESP) machine
NOTE: A modified NESP module is used to alternatively perform Langendorff perfusion and working mode perfusion. Briefly, connect the aortic line of the circuit to a compliance chamber via a Y-connector. Add a pediatric oxygenator and a cardiotomy reservoir (70-80 cm height above the aortic connector of the module) to provide a left ventricle afterload of approximately 70 mmHg during the working mode. Connect another cardiotomy reservoir (7-10 cm height above the aortic connector of the module) to the main inflow line using a Y-connector to provide a left atrium preload of approximately 10 mmHg during the working mode (Figure 2). Coronary flow is assessed with a flow sensor connected to the pulmonary cannula. A centrifugal pump, a membrane oxygenator, and a heater-cooler machine are connected to the circuit (Figure 2). For solution descriptions, refer to Table 1.
<ol>
	<li>Prime the perfusion circuit with the priming solution (Table 1).</li>
	<li>Set the pump output at 1500 mL/min.</li>
	<li>Add the blood retrieved from the donor pig (1200-1500 mL) in the circuit.</li>
	<li>Set the gas mixer to achieve an oxygen partial pressure &#62;250 mmHg.</li>
	<li>Connect maintenance solution and adrenaline solution (Table 1) to the circuit and set the initial output respectively at 5 mL/h and 0.1 mL/h.</li>
	<li>Set the temperature of the perfusate at room temperature (RT) before placement of the heart in the perfusion module.</li>
	<li>During working mode, connect a syringe of dobutamine with a concentration of 2.5 mg/mL (output between 0.04-0.12 mg/h).</li>
</ol>
4. Heart procurement and instrumentation for normothermic ex situ heart perfusion
<ol>
	<li>Heart procurement
	<ol>
		<li>Place the animal in the supine position and continue to maintain general anesthesia.</li>
		<li>Perform a median sternotomy and open the pericardium.</li>
		<li>Suspend the pericardium with four stay sutures.</li>
		<li>Place 4-0 polypropylene sutures on the right atrium and on the ascending aorta to secure cannulations with tourniquets.</li>
		<li>After heparin infusion (300 UI/kg) and careful dissection of the aortic root, insert a double-staged venous cannula in the right atrium for blood collection and a single-lumen cannula in the ascending aorta for cardioplegia infusion.</li>
		<li>Isolate the superior and the inferior vena cava with Silastic tourniquets.</li>
		<li>Connect the venous cannula to a blood collecting bag containing 10,000 IU of unfractionated heparin.</li>
		<li>Place the piglet body in the Trendelenburg position to improve blood drainage into the collecting bag.</li>
		<li>After blood collection is completed, cross-clamp the ascending aorta, infuse Del Nido cardioplegia in the aortic root (Table 1), and check that the ascending aorta is under pressure (no aortic regurgitation).</li>
		<li>Unload the right and left atrium by opening the inferior vena cava and right pulmonary vein, respectively, while the superior vena cava is clamped by a tourniquet.</li>
		<li>Once cardioplegia infusion is completed, ligate the left hemiazygos vein with two stiches of 4-0 polypropylene.</li>
		<li>Proceed to heart procurement, keeping 2 cm of the pulmonary trunk along with the left atrium posterior wall.</li>
		<li>Verify that there is no patent foramen ovale by inspecting the atrial septum and close it if necessary using 4-0 polypropylene sutures.</li>
	</ol>
	</li>
	<li>Instrumentation of the heart before NESP
	<ol>
		<li>Place the heart in a 4 &#176;C saline solution and separate the ascending aorta from the pulmonary trunk. Verify that the aortic valve and the coronary ostia are not injured.</li>
		<li>Insert four pledgeted stitches (4-0 polypropylene) 5 mm below the distal section of the ascending aorta and insert the infusion cannula into the aorta. Tight a hose clamp around the aorta to secure the cannula.</li>
		<li>Insert a drainage cannula into the pulmonary trunk and secure with a 3-0 polypropylene running suture.</li>
		<li>Close the inferior and superior vena cava with 5-0 polypropylene running sutures.</li>
		<li>Close the left atrium posterior wall with a 4-0 polypropylene running suture.</li>
		<li>Insert a left vent cannula through the posterior wall of the left atrium wall and snare a tourniquet around.</li>
		<li>Insert a preload cannula into the left atrial appendage and snare a tourniquet around.</li>
	</ol>
	</li>
</ol>
5. Connection to the NESP machine and resuscitation of the heart
NOTE: Before instrumentation of the heart, ensure that the materials necessary for resuscitation are available next to the perfusion circuit, especially a defibrillator with internal probes and an external pacemaker with epicardial electrodes. Ensure that the pressure line is connected to the aortic line, and that output sensor is placed on the coronary flow line. The afterload line must be clamped, as well as the preload line of the working mode circuit.
<ol>
	<li>Decrease the pump flow to 200 mL/min.</li>
	<li>Connect the heart to the aortic connector after de-airing the connector. Ensure that the heart is appropriately connected to the perfusion module so that the inferior ventricular walls and left and right atrium are in front of the operator. Avoid twisting the ascending aorta to prevent aortic regurgitation.</li>
	<li>Adjust the aortic pressure to 30 mmHg at RT.</li>
	<li>During resuscitation, perform a smooth cardiac massage until a sinus rhythm is restored.</li>
	<li>Slowly increase the pump flow within 15-25 min by steps of 50 mL/min to achieve an aortic pressure of 65 mmHg. At the same time, increase the perfusate temperature by steps of 2-4 &#176;C to reach 37 &#176;C.</li>
	<li>Once the aortic pressure is at 65 mmHg, and perfusate temperature is at 37 &#176;C, deliver an electric shock at 5 J if needed, and repeat until the sinus rhythm is restored.</li>
	<li>Secure an epicardial electrode on the right ventricular posterior wall and connect to an external pacemaker. Pace the heart at 80 BPM to overdrive spontaneous rhythm.</li>
	<li>Connect the pulmonary cannula to the coronary flow line.</li>
	<li>Perform arterial and venous blood samples for gas and biochemical analyses of the perfusate. Record the initial lactate concentration and correct biochemical disorders to achieve the following objectives: glucose &#62;1 g/L, K+ 3.5-5.5 mmol/L, Ca2+ 1.0-1.20 mmol/L, pH 7.35-7.45, Na+ 135-145 mmol/L, and HCO3- 20-24 mmol/L.</li>
	<li>Adjust pump flow to reach a mean aortic pressure of 65-75 mmHg and coronary flow of 650-850 mL/min.</li>
	<li>Perform arteriovenous blood gas analysis every 15 min to ensure that myocardial extraction of lactate is effective. If venous lactate is higher than arterial lactate, then increase mean aortic pressure to 80 mmHg by decreasing maintenance solution, and check the lactate concentration 15 min after. If arteriovenous lactate clearance is still impaired, then increase coronary flow to &#62;850 mL and check lactate concentration 15 min later.</li>
</ol>
6. Working mode procedure
NOTE: Efficient arteriovenous clearance of lactate is usually achieved within 30 min after initiation of Langendorff perfusion. Working mode can then be initiated by connecting the preload cannula to the preload reservoir (this line was previously clamped during Langendorff mode). Similarly, the afterload line is connected to the aortic line (Figure 2). Set the flow sensor on the afterload line to measure cardiac output.
<ol>
	<li>Open the preload line and adjust the pump flow to ensure stable filling of the preload reservoir. During this period, the left atrium and the left ventricle are progressively filled with blood.</li>
	<li>Open the aortic afterload line and clamp the main line of the circuit used for Langendorff perfusion. The afterload reservoir is progressively filled-up. Ensure the drainage of the reservoir by an overflow line which brings the perfusate back to the main reservoir of the circuit.</li>
	<li>Initiate the infusion of dobutamine at 0.04 mg/min.</li>
	<li>Perform arterial and venous blood gas sample analysis to ensure that the myocardial extraction of lactate is still effective.</li>
	<li>Once the cardiac output is stable, perform invasive hemodynamic assessment along with epicardial ultrasound measurements.</li>
</ol>
7. Pressure-volume (PV) loop assessment with the conductance method
NOTE: All calibration steps must be performed in working mode.
<ol>
	<li>PV catheter placement into the left ventricle
	<ol>
		<li>Clean the 7 Fr pigtail conductance catheter with saline solution and connect it to the hardware interface.</li>
		<li>Gently push the catheter into the introducer 8 Fr sheath previously inserted through the left atrium roof to be aligned with the mitral valve.</li>
		<li>As soon as the catheter crosses the mitral valve, adjust the appropriate position, considering optimal pressure and volume signals. If there is too much noise, gently move the conductance catheter to improve the quality of the loops.</li>
	</ol>
	</li>
	<li>PV loop catheter calibration
	<ol>
		<li>Pressure calibration
		<ol>
			<li>Once the conductance catheter is appropriately located in the left ventricle, open the calibration interface on the software and calibrate the pressure value using acquisition software for conductance measurements.</li>
			<li>Start recording, select 0 mmHg pressure and 100 mmHg on the control interface, and record for 5 s each.</li>
			<li>Then, stop recording and open the pressure calibration interface. Match the corresponding signal to the pressure level.</li>
			<li>Once calibrated, verify that the signal matches the values obtained by invasive blood pressure monitoring.</li>
		</ol>
		</li>
		<li>Volume calibration
		<ol>
			<li>Conductance calibration
			<ol>
				<li>Open the control interface on the software for conductance measurements.</li>
				<li>Start recording, one after the other, select the volumes suggested by the calibration interface.</li>
				<li>Let the interface record for 5 s each, then stop recording.</li>
				<li>Use the data-trace obtained and open the volume calibration interface.</li>
				<li>Match the corresponding trace to the pressure level.</li>
			</ol>
			</li>
			<li>Parallel volume calibration
			<ol>
				<li>Surrounding heart tissue conducts electricity and contributes to the overall volume signal. Remove this parallel volume for accurate volume measurement (post-processing calibration).</li>
				<li>To assess parallel volume in this setup (myocardial wall), inject 10 cc of hypertonic saline solution (4%) into the left atrium line once.</li>
				<li>Do not repeat the operation to avoid hypernatremia.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Field correction factor calibration
	<ol>
		<li>Enter the stroke volume value obtained from the ultrasound measurements. 
		NOTE: Factor alpha will be calculated considering the ratio of stroke volumes obtained either by ultrasound measurements or conductance catheterization.</li>
	</ol>
	</li>
	<li>PV data collection
	<ol>
		<li>Stop epicardial pacing of the heart to avoid interference with the conductance signal. Record data in a steady state when the signal is stabilized (Figure 3)</li>
		<li>Select a series of 10 consecutive loops and open the analysis software. The software will automatically provide stroke work, pre-recruitable stroke work, maximum dP/dt, minimum dP/dt, and tau index.</li>
		<li>To obtain the end-systolic pressure-volume relationship and end-diastolic pressure-volume relationship, record the signal during preload occlusion. Gradually clamp the atrial perfusion line until preload reduction is effective (Figure 4). Then slowly release the clamp.</li>
	</ol>
	</li>
</ol>
8. Epicardial echocardiography assessment of the heart in a working state
<ol>
	<li>Acquisition of ultrasound loops
	<ol>
		<li>Place three EKG epicardial electrodes connected to the echocardiogram machine.</li>
		<li>Apply a sterile drape around the heart and use a transesophagus probe.</li>
		<li>Apply the probe to the upper wall of the left atrium and manually rotate the transducer until a four-chamber view is obtained (Figure 5).</li>
		<li>Start the echocardiographic acquisition software for myocardial performance assessment using the X-plan mode.</li>
		<li>Then, run the ultrasound probe motor to obtain three and two-chamber views. Analysis of these views allows for measurement of left ventricle ejection fraction and global longitudinal strain9.</li>
	</ol>
	</li>
	<li>Assessment of myocardial work index (MWI)
	<ol>
		<li>Proceed to the acquisition of four-, three-, and two-chamber views and record simultaneous arterial pressure (Figure 6).</li>
		<li>Assess the global longitudinal strain using these views and open MWI software. Use the invasive blood pressure detected by the external sensor on the perfusion circuit during loop acquisition.</li>
		<li>Manually notify the software of the exact opening and closing timing of the aortic and mitral valves. 
		NOTE: MWI software will automatically provide global MWI, constructive work, wasted work, and effective work.</li>
	</ol>
	</li>
</ol>

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All authors have no conflicts of interest to disclose.

There are some critical steps to consider in the NESP protocol. In situ preliminary assessment of the heart remained important, especially considering the aortic valve that should not present with significant aortic regurgitation (grade 2 or more); otherwise, the resuscitation of the heart will be compromised during the Langendorff period because of impaired coronary perfusion and myocardial ischemia. The initiation of the WM after Langendorff perfusion was a challenging maneuver, requiring at least two persons ...

Heart transplantation is the gold standard treatment for advanced heart failure, but is limited by current organ shortage1. A growing number of donor hearts with extended criteria (age &gt;45 years, cardiovascular risk factors, prolonged low flow, acute left ventricular dysfunction secondary to catecholaminergic storm) are allocated with an increased risk of primary graft failure2. Moreover, hearts donated after controlled circulatory death (DCD) may be presented with myocardial injury secondary to prolonged warm ischemia3. Therefore, there is a need for a better assessment of these donor hearts before transplantation, especially to evaluate their eligibility for heart transplantation4,5. 
Normothermic ex situ perfusion (NESP) preserves the beating heart using warm oxygenated blood. The only commercially available device for NESP preserves the heart in a non-working state (Langendorff mode). This approach was initially applied to expand the preservation of the graft beyond the critical 4 h period of cold ischemia6. Another major advantage of this technology is to provide continuous assessment of myocardial viability based on lactate concentration in the perfusate6. However, this biochemical assessment has never been correlated with post-transplant outcomes to date. Similarly, Langendorff mode for NESP does not allow for hemodynamic and functional evaluation of the heart prior to transplantation. Some authors have reported the potential benefit of intracardiac catheterization during NESP to predict myocardial recovery after transplantation7.
The present report aims to provide a reproducible methodology to evaluate donor heart performance during NESP. We modified the circuit to allow for working mode perfusion and, therefore, for the acquisition of noninvasive functional variables with epicardial echocardiography. Myocardial work index, a load-independent variable, was recorded using pressure-strain loops. We investigated the relationships between myocardial work and hemodynamic variables obtained from intracardiac conductance catheterization.

<table><tbody><tr><td>3T Heater Cooler System</td><td>Liva Nova, Ch&amp;acirc;tillon, France</td><td>IM-00727 A</td><td>Extracorporeal Heater Cooler device</td></tr><tr><td>4-0 polypropylene suture</td><td>Peters, bobigny, France</td><td>20S15B</td><td>sutures</td></tr><tr><td>5-0 polypropylene suture</td><td>Peters, bobigny, France</td><td>20S10B</td><td>sutures</td></tr><tr><td>Adenosine</td><td>Efisciens BV, Rotterdam, Netherlands</td><td>9088309</td><td>Drugs for the ex-vivo perfusion</td></tr><tr><td>Adrenaline</td><td>Aguettant, Lyon, France</td><td>600040</td><td>Drugs for the ex-vivo perfusion</td></tr><tr><td>Atracurium</td><td>Pfizer Holding France, Paris, France</td><td>582547</td><td>Drugs for the induction of the anesthesia</td></tr><tr><td>DeltaStream</td><td>Fresenius Medical Care, L&amp;rsquo;Arbresle, France</td><td>MEH2C4024</td><td>Extracorporeal blood pump</td></tr><tr><td>EKG epicardial electrodes</td><td>Cardinal Health LLC, Waukegan, Illinois, USA</td><td>31050522</td><td>EKG detection electrodes</td></tr><tr><td>External pacemaker</td><td>Medtronic Inc. Minneapolis, Minneapolis, USA</td><td>5392</td><td>Pacemaker device</td></tr><tr><td>Glucose 5%</td><td>B.Braun Melsungen AG, Melsungen, Germany</td><td>3400891780017</td><td>Drugs for the priming solution</td></tr><tr><td>Heart Perfusion Set, Organ Care System</td><td>Transmedics, Andover, MA, USA</td><td>Ref#1200</td><td>Normothermic ex-vivo heart perfusion device</td></tr><tr><td>Intellivue MX550</td><td>Philips Healthcare, Suresnes, France</td><td>NA</td><td>Permanent monitoring system</td></tr><tr><td>Istat 1</td><td>Abbott, Chicago, Ill, USA</td><td>714336-03O</td><td>Blood Analyzer machine</td></tr><tr><td>Labchart</td><td>AD Instruments Ltd, Paris, France</td><td>LabChart&amp;nbsp;v8.1.21</td><td>Pressure Volume loops aquisition software</td></tr><tr><td>Magnesium</td><td>Aguettant, Lyon, France</td><td>564 780-6</td><td>Drugs for the cardioplegia</td></tr><tr><td>Magnesium Sulfate</td><td>Aguettant, Lyon, France</td><td>600111</td><td>Drugs for the cardioplegia</td></tr><tr><td>Mannitol 20%</td><td>Macopharma, Mouvoux, France</td><td>3400891694567.00</td><td>Drugs for the cardioplegia</td></tr><tr><td>Methylprednisolone</td><td>Mylan S.A.S, Saint Priest, France</td><td>400005623</td><td>Drugs for the priming solution</td></tr><tr><td>Millar Conductance Catheter</td><td>AD Instruments Ltd, Paris, France</td><td>Ventri-Cath 507</td><td>Pressure Volume loops conductance catheter</td></tr><tr><td>MWI software</td><td>General Electric Healthcare, Chicago, Ill, USA</td><td>NA</td><td>software used for the Ultrasound echocardiographic machine</td></tr><tr><td>Orotracheal probe</td><td>Smiths medical ASD, Inc., Minneapolis, Minneapolis, USA</td><td>100/199/070</td><td>probe for the intubation during anesthesia</td></tr><tr><td>Potassium chloride 10%</td><td>B.Braun Melsungen AG, Melsungen, Germany</td><td>3400892691527.00</td><td>Drugs for the cardioplegia</td></tr><tr><td>Propofol</td><td>Zoetis France, Malakoff, France</td><td>8083511</td><td>Drugs for the induction of the anesthesia</td></tr><tr><td>Quadrox-I small Adult Oxygenator</td><td>Getinge, G&amp;ouml;teborg, Sweden</td><td>BE-HMO 50000</td><td>Extracorporeal blood oxygenator</td></tr><tr><td>Ringer solution</td><td>B.Braun Melsungen AG, Melsungen, Germany</td><td>DKE2323</td><td>Drugs for the cardioplegia</td></tr><tr><td>Sodium Bicarbonate</td><td>Laboratoire Renaudin, itxassou, France</td><td>3701447</td><td>Drugs for the cardioplegia</td></tr><tr><td>Sodium chloride</td><td>Aguettant, Lyon, France</td><td>606726</td><td>Drugs for the priming solution</td></tr><tr><td>Swan Ganz Catheter</td><td>Merit Medical, south jordan, utah, USA</td><td>5041856</td><td>Right pressure and cardiac output probe</td></tr><tr><td>Tiletamine</td><td>Virbac France, Carros, France</td><td>3597132126021.00</td><td>Drugs for the induction of the anesthesia</td></tr><tr><td>Transesophagus probe (3&amp;ndash;8 MHz 6VT)</td><td>General Electric Healthcare, Chicago, Ill, USA</td><td>NA</td><td>Ultrasound echocardiographic transesophagus probe</td></tr><tr><td>Vivid E95 ultraSound Machine</td><td>General Electric Healthcare, Chicago, Ill, USA</td><td>NA</td><td>Ultrasound echocardiographic machine</td></tr><tr><td>Xyloca&amp;iuml;ne 2%</td><td>Aspen, Reuil-malmaison, France</td><td>600550</td><td>Drugs for the cardioplegia</td></tr><tr><td>Zolazepam</td><td>Virbac France, Carros, France</td><td>3597132126021.00</td><td>Drugs for the induction of the anesthesia</td></tr></tbody></table>

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.

We herein described a NESP protocol in a monoventricular working state, using a modified heart perfusion module usually employed in clinical practice for Langendorff perfusion of the donor heart before transplantation. This piglet model of NESP using the present custom module was developed in 2019. The modifications of the circuit were minor, as most of the perfusion circuit was re-used for experiments. The cap of the module provided a flexible and waterproof membrane to protect the heart during transportation. It also a...

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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.

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 ...

functional-assessment-donor-heart-during-ex-situ-perfusion-insights

Research

JoVE Journal

Medicine

2.0K Views.  Groupe Hospitalier Paris Saint Joseph. Hemodynamische beoordeling van het hart tijdens geïsoleerde Ex situ perfusie is slecht beschreven. Dit protocol biedt een methode om ex situ cardiale prestaties in werkmodus te evalueren. Onze aanpak vergelijkt de invasieve PV-lustechniek met epicardiale echocardiografie voor functionele beoordeling van hun hart tijdens geïsoleerde Ex situ-perfusie.Deze aanpak vereist een teamervaring in Ex situ hartperfusie, samen met de betrokkenheid van een cardioloog die is opgeleid voor transoeo...

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

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

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 ...

Enhancing CVD Research with Modified Langendorff Perfusion

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.

Author Spotlight: Advancing Cardiovascular Research &#8212; Tailored Langendorff Perfusion Techniques for Improved Experimental Outcomes

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and ...

Bioengineering

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

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 ...

Enhanced Cardiac Assessment in Ex Vivo Perfusion with Left Ventricular Loading

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (&#707;6 h).

Author Spotlight: Enhancing Graft Viability Assessment Through Quantitative Metrics and Innovative Reservoir Systems

The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. ...

Point-of-Care Ultrasound for Peripheral Veno-Arterial Extracorporeal Membrane Oxygenation Without Left Ventricular Venting

Over the past several decades, veno-arterial extracorporeal membrane oxygenation (V-A ECMO) has become a critical tool in the management of patients with severe cardiogenic shock and cardiopulmonary failure. Due to the inherent instability of these patients, their transport away from intensive care units is fraught with risk. As a result, bedside diagnostic tools are essential for their daily care. One such tool is point-of-care ultrasound (POCUS) of the heart, which can non-invasively assess several parameters: left ventricular (LV) performance (size, systolic function, stroke volume, aortic valve opening), right ventricular (RV) performance (size, systolic function), and the presence of intracardiac thrombus. Additionally, POCUS can assist in evaluating readiness for V-A ECMO weaning and eventual decannulation. Despite its potential, the use of POCUS in the context of V-A ECMO remains inconsistent due to variability in provider training. This study aims to address this gap by detailing POCUS image acquisition in V-A ECMO, particularly in the absence of LV venting. It covers key aspects such as patient positioning, transducer selection, probe placement, acquisition sequence, and image optimization.

This protocol outlines the use of point-of-care ultrasound (POCUS) to monitor patients on peripheral veno-arterial extracorporeal membrane oxygenation (V-A ECMO) without left ventricular venting. It evaluates left ventricular distension, intracardiac or aortic root thrombus, and identifies useful cardiac POCUS parameters for weaning from V-A ECMO.

Over the past several decades, veno-arterial extracorporeal membrane oxygenation (V-A ECMO) has become a critical tool in the management of patients with ...

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

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