Portions of this work were supported by a generous contribution by the Fondation Marcel et Rolande Gosselin and Fondation Mr Stefane Foumy. Nicolas Noiseux is scholar of the FRQ-S.
The authors wish to thanks Josh Zhuo Le Huang, Gabrielle Gascon, Sophia Ghiassi, and Catherine Scalabrini for their support in data collection.

All animal care and experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee of the Centre Hospitalier de l&#8217;Universit&#233; de Montr&#233;al Research Center.
1. Preliminary Preparations
<ol>
	<li>Turn on the water bath to heat the cardioplegia delivery system (Figure 1A) and the Langendorff ex vivo perfusion system (Figure 1B)....

<ol>
	<li>Gass, A. 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=Cardiac+Transplantation+in+the+New+Era.">Cardiac Transplantation in the New Era.</a> Cardiology in Review. 23 (4), 182-188 (2015).</li><li>von Dossow, V., Costa, J., D'Ovidio, F., Marczin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+trends+in+heart+and+lung+transplantation:+Guarding+the+most+precious+gift+ever.">Worldwide trends in heart and lung transplantation: Guarding the most precious gift ever.</a> Best Practice &amp; Research. Clinical Anaesthesiology. 31 (2), 141-152 (2017).</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>Manyalich, M., Nelson, H., Delmonico, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+and+opportunity+for+donation+after+circulatory+death+worldwide.">The need and opportunity for donation after circulatory death worldwide.</a> Current Opinion In Organ Transplantation. 23 (1), 136-141 (2018).</li><li>Shemie, S. 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=National+recommendations+for+donation+after+cardiocirculatory+death+in+Canada:+Donation+after+cardiocirculatory+death+in+Canada.">National recommendations for donation after cardiocirculatory death in Canada: Donation after cardiocirculatory death in Canada.</a> CMAJ : Canadian Medical Association Journal. 175 (8), S1 (2006).</li><li>Page, A., Messer, S., Large, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+transplantation+from+donation+after+circulatory+determined+death.">Heart transplantation from donation after circulatory determined death.</a> Annals of Cardiothoracic Surgery. 7 (1), 75-81 (2018).</li><li>Monteagudo Vela, M., Garcia Saez, D., Simon, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+in+retrieval+and+heart+preservation.">Current approaches in retrieval and heart preservation.</a> Annals of Cardiothoracic Surgery. 7 (1), 67-74 (2018).</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>McNally, S. J., Harrison, E. M., Wigmore, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+considerations+in+the+application+of+preconditioning+to+solid+organ+transplantation.">Ethical considerations in the application of preconditioning to solid organ transplantation.</a> Journal of Medical Ethics. 31 (11), 631-634 (2005).</li><li>Rao, V., Feindel, C. M., Weisel, R. D., Boylen, P., Cohen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+blood+perfusion+improves+myocardial+recovery+after+heart+transplantation.">Donor blood perfusion improves myocardial recovery after heart transplantation.</a> The Journal of Heart and Lung Transplantation. 16 (6), 667-673 (1997).</li><li>Ramzy, 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=Cardiac+allograft+preservation+using+donor-shed+blood+supplemented+with+L-arginine.">Cardiac allograft preservation using donor-shed blood supplemented with L-arginine.</a> The Journal of Heart and Lung Transplantation. 24 (10), 1665-1672 (2005).</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>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>Flecknell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia (Fourth Edition). , 77-108 (2016).</li><li>Kearns, M. 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=A+Rodent+Model+of+Cardiac+Donation+After+Circulatory+Death+and+Novel+Biomarkers+of+Cardiac+Viability+During+Ex+vivo+Heart+Perfusion.">A Rodent Model of Cardiac Donation After Circulatory Death and Novel Biomarkers of Cardiac Viability During Ex vivo Heart Perfusion.</a> Transplantation. 101 (8), e231-e239 (2017).</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> The Annals of Thoracic Surgery. 105 (6), 1763-1770 (2018).</li><li>Iyer, 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=Increasing+the+tolerance+of+DCD+hearts+to+warm+ischemia+by+pharmacological+postconditioning.">Increasing the tolerance of DCD hearts to warm ischemia by pharmacological postconditioning.</a> American Journal of Transplantation. 14 (8), 1744-1752 (2014).</li><li>Sanz, M. N., 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=Cardioprotective+reperfusion+strategies+differentially+affect+mitochondria:studies+in+an+isolated+rat+heart+model+of+donation+after+circulatory+death+(DCD).">Cardioprotective reperfusion strategies differentially affect mitochondria:studies in an isolated rat heart model of donation after circulatory death (DCD).</a> American Journal of Transplantation. , (2018).</li><li>Van de Wauwer, 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=The+mode+of+death+in+the+non-heart-beating+donor+has+an+impact+on+lung+graft+quality.">The mode of death in the non-heart-beating donor has an impact on lung graft quality.</a> European Journal of Cardio-Thoracic Surgery. 36 (5), 919-926 (2009).</li><li>Quader, M., 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=Determination+of+Optimal+Coronary+Flow+for+the+Preservation+of+&amp;#34;Donation+after+Circulatory+Death&amp;#34;+in+Murine+Heart+Model.">Determination of Optimal Coronary Flow for the Preservation of &amp;#34;Donation after Circulatory Death&amp;#34; in Murine Heart Model.</a> ASAIO journal (American Society for Artificial Internal Organs : 1992). 64 (2), 225-231 (2018).</li><li>Priebe, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+open-chest+model.">The acute open-chest model.</a> British Journal Of Anaesthesia. 60 (8 Suppl 1), 38-41 (1988).</li><li>Narita, M., 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=Cardiac+effects+of+vecuronium+and+its+interaction+with+autonomic+nervous+system+in+isolated+perfused+canine+hearts.">Cardiac effects of vecuronium and its interaction with autonomic nervous system in isolated perfused canine hearts.</a> Journal of Cardiovascular Pharmacology. 19 (6), 1000-1008 (1992).</li><li>Dhital, K. 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 (London, England). 385 (9987), 2585-2591 (2015).</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. 35 (12), 1443-1452 (2016).</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>Mayr, 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=Cardiac+troponin+T+and+creatine+kinase+predict+mid-term+infarct+size+and+left+ventricular+function+after+acute+myocardial+infarction:+a+cardiac+MR+study.">Cardiac troponin T and creatine kinase predict mid-term infarct size and left ventricular function after acute myocardial infarction: a cardiac MR study.</a> Journal of Magnetic Resonance Imaging. 33 (4), 847-854 (2011).</li><li>Remppis, 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=Intracellular+compartmentation+of+troponin+T:+release+kinetics+after+global+ischemia+and+calcium+paradox+in+the+isolated+perfused+rat+heart.">Intracellular compartmentation of troponin T: release kinetics after global ischemia and calcium paradox in the isolated perfused rat heart.</a> Journal of Molecular and Cellular Cardiology. 27 (2), 793-803 (1995).</li><li>Rossello, X., Hall, A. R., Bell, R. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Langendorff+Perfused+Isolated+Mouse+Heart+Model+of+Global+Ischemia-Reperfusion+Injury:+Impact+of+Ischemia+and+Reperfusion+Length+on+Infarct+Size+and+LDH+Release.">Characterization of the Langendorff Perfused Isolated Mouse Heart Model of Global Ischemia-Reperfusion Injury: Impact of Ischemia and Reperfusion Length on Infarct Size and LDH Release.</a> Journal of Cardiovascular Pharmacology and Therapeutics. 21 (3), 286-295 (2016).</li><li>Dornbierer, M., 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=Early+reperfusion+hemodynamics+predict+recovery+in+rat+hearts:+a+potential+approach+towards+evaluating+cardiac+grafts+from+non-heart-beating+donors.">Early reperfusion hemodynamics predict recovery in rat hearts: a potential approach towards evaluating cardiac grafts from non-heart-beating donors.</a> PloS One. 7 (8), e43642 (2012).</li><li>Henry, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+staircase+effect+in+the+rat+heart.">Positive staircase effect in the rat heart.</a> The American Journal of Physiology. 228 (2), 360-364 (1975).</li><li>Markert, M., 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=Evaluation+of+a+method+to+correct+the+contractility+index+LVdP/dt(max)+for+changes+in+heart+rate.">Evaluation of a method to correct the contractility index LVdP/dt(max) for changes in heart rate.</a> Journal of Pharmacological and Toxicological Methods. 66 (2), 98-105 (2012).</li><li>Azar, T., Sharp, J., Lawson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rates+of+male+and+female+Sprague-Dawley+and+spontaneously+hypertensive+rats+housed+singly+or+in+groups.">Heart rates of male and female Sprague-Dawley and spontaneously hypertensive rats housed singly or in groups.</a> Journal of the American Association for Laboratory Animal Science. 50 (2), 175-184 (2011).</li><li>Bonney, S., Hughes, K., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+cardioprotection:+the+role+of+adenosine.">Anesthetic cardioprotection: the role of adenosine.</a> Current Pharmaceutical Design. 20 (36), 5690-5695 (2014).</li><li>Ali, A. 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=Rat+model+of+veno-arterial+extracorporeal+membrane+oxygenation.">Rat model of veno-arterial extracorporeal membrane oxygenation.</a> Journal of Translational Medicine. 12, 37 (2014).</li></ol>

The protocol presented here introduces a simple, convenient and versatile model of cardiac DCD, offering the opportunity to assess cardiac functional recovery, tissue damage and the use of post-conditioning cardioprotective agents to improve recovery of donor hearts otherwise discarded for transplantation. Ex vivo heart perfusion systems (EVHP) systems have been optimized to provide a platform for evaluating cardiac function and offer a unique opportunity to deliver and test modified solutions supplemented with post-cond...

Solid organ transplantation in general and cardiac transplantation, in particular, are on the rise worldwide1,2. The standard method of organ procurement is donation after brain death (DBD). Given the strict inclusion criteria of DBD, less than 40% of the offered hearts are accepted3, thereby limiting the offer in face of increasing demand and extending the organ waiting list. To address this issue, the use of organs donated after circulatory death (DCD) is considered a potential solution4.
In DCD donors, however, an agonal phase following withdrawal of care and a period of unprotected warm ischemia before resuscitation are inevitable5. The potential organ injury after circulatory death can lead to organ dysfunction, explaining the reluctance to routinely adopt DCD heart transplantations. It is reported that only 4 centers use DCD hearts clinically, with stringent criteria that includes very short warm ischemia times and young donors without chronic pathologies6,7. For ethical and legal reasons, limited or no cardioprotective interventions can be applied in donors prior to circulatory death5,8,9. Thus, any mitigation to alleviate the ischemia-reperfusion (IR) injury is limited to cardioprotective therapies initiated during early reperfusion with cardioplegic solutions, and do not allow for proper functional assessment. Ex vivo heart perfusion (EVHP) and reconditioning of the DCD heart using dedicated platforms has been proposed as an alternative solution and studied by various scholars10,11,12,13. EVHP offers a unique opportunity to deliver post-conditioning agents to DCD hearts to improve functional recovery. However, for efficient clinical translation, many technical and practical issues remain to be addressed, and this is further compounded by a lack of consensus on a range of perfusion and functional criteria to determine transplantability6,8.
Herein we report the development of a reproducible pre-clinical small animal DCD protocol combined with an ex vivo heart perfusion system that can be used to investigate organ post-conditioning initiated at the time of procurement, during initial reperfusion, and/or throughout EVHP.

<table>
	<tbody>
		<tr>
			<td>0,9% Sodium Chloride. 1L bag</td>
			<td>Baxter</td>
			<td></td>
			<td>Electrolyte solution for flushing in the modified Langendorff system.</td>
		</tr>
		<tr>
			<td>14G 2&#34; I.V catheter</td>
			<td>Jelco</td>
			<td>4098</td>
			<td>To act as endotracheal tube.</td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride</td>
			<td>Milipore-Sigma</td>
			<td>T8877</td>
			<td>Vital coloration</td>
		</tr>
		<tr>
			<td>22G 1&#34; I.V catheter</td>
			<td>BD</td>
			<td>383532</td>
			<td>I.V catheter with extension tube that facilitates manipulation for carotid catheterization</td>
		</tr>
		<tr>
			<td>Adson Dressing Fcp, 4 3/4&#34;, Serr</td>
			<td>Skalar</td>
			<td>50-3147</td>
			<td>Additional forceps for tissue manipulation</td>
		</tr>
		<tr>
			<td>Alm Self-retaining retractor 4x4 Teeth Blunt 2-3/4&#34;</td>
			<td>Skalar</td>
			<td>22-9027</td>
			<td>Tissue retractor used to maintain the chest open.</td>
		</tr>
		<tr>
			<td>Bridge amp</td>
			<td>ADinstruments</td>
			<td>FE221</td>
			<td>Bridge amp for intracarotid blood pressure measurement</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Milipore-Sigma</td>
			<td>C1016</td>
			<td>CaCl2 anhydrous, granular, &#8804;7.0 mm, &#8805;93.0% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Milipore-Sigma</td>
			<td>G8270</td>
			<td>D-Glucose &#8805;99.5% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>DIN(8) to Disposable BP Transducer</td>
			<td>ADinstruments</td>
			<td>MLAC06</td>
			<td>Adapter cable for link between bridge amp and pressure transducer</td>
		</tr>
		<tr>
			<td>Disposable BP Transducer (stopcock)</td>
			<td>ADinstruments</td>
			<td>MLT0670</td>
			<td>Pressure transducer for intracarotid blood pressure measurement</td>
		</tr>
		<tr>
			<td>dPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Electrolyte solution without calcium or magnesium.</td>
		</tr>
		<tr>
			<td>Eye Dressing Fcp, Str, Serr, 4&#34;</td>
			<td>Skalar</td>
			<td>66-2740</td>
			<td>Additional forceps for tissue manipulation</td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Milipore-Sigma</td>
			<td>HT501128</td>
			<td>Fixative solution</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Sunbean</td>
			<td>756-CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium 1000 UI/mL</td>
			<td>Sandoz</td>
			<td></td>
			<td>For systemic anticoagulation</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid 36,5 to 38,0%</td>
			<td>Fisher scientific</td>
			<td>A144-500</td>
			<td>Diluted 1:1 for pH correction</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Bimeda</td>
			<td></td>
			<td>Anesthetic. 100 mg/ml</td>
		</tr>
		<tr>
			<td>LabChart</td>
			<td>ADinstruments</td>
			<td></td>
			<td>Control software for the Powerlab polygraph, allowing off-line analyses. Version 7, with blood pressure and PV loop modules enabled</td>
		</tr>
		<tr>
			<td>Left ventricle pressure balloon</td>
			<td>Radnoti</td>
			<td>170404</td>
			<td>In latex. Size 4.</td>
		</tr>
		<tr>
			<td>Lidocaine HCl 2% solution</td>
			<td>AstraZeneca</td>
			<td></td>
			<td>Antiarrhythmic for the cardioplegic solution</td>
		</tr>
		<tr>
			<td>Magnesium Chloride ACS</td>
			<td>ACP Chemicals</td>
			<td>M-0460</td>
			<td>MgCl2+6H2O &#8805;99.0% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>Micro pressure sensor</td>
			<td>Radnoti</td>
			<td>159905</td>
			<td>Micro pressure sensor and amplifier connected to the intraventricular balloon</td>
		</tr>
		<tr>
			<td>Pacemaker</td>
			<td>Biotronik</td>
			<td>Reliaty</td>
			<td>Set to generate a pulse each 200 ms for a heart rate of 300 bpm.</td>
		</tr>
		<tr>
			<td>pH bench top meter</td>
			<td>Fisher scientific</td>
			<td>AE150</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological monitor</td>
			<td>Kent Scientific</td>
			<td>Physiosuite</td>
			<td>For continuous monitoring of rodent temperature and saturation during the procedure</td>
		</tr>
		<tr>
			<td>Plasma-Lyte A</td>
			<td>Baxter</td>
			<td></td>
			<td>Electrolyte solution used as base to prepare cardioplegia</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Milipore-Sigma</td>
			<td>P4504</td>
			<td>KCl &#8805;99.0% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>Potassium Chloride 2 meq/ml</td>
			<td>Hospira</td>
			<td></td>
			<td>Part of the cardioplegic solution</td>
		</tr>
		<tr>
			<td>PowerLab 8/30 Polygraph</td>
			<td>ADinstruments</td>
			<td></td>
			<td>Electronic polygraph</td>
		</tr>
		<tr>
			<td>Silk 2-0</td>
			<td>Ethicon</td>
			<td>A305H</td>
			<td>Suture material for Langendorff apparatus</td>
		</tr>
		<tr>
			<td>Silk 5-0</td>
			<td>Ethicon</td>
			<td>A302H</td>
			<td>Suture material for carotid</td>
		</tr>
		<tr>
			<td>Small animal anesthesia workstation</td>
			<td>Hallowell EMC</td>
			<td>000A2770</td>
			<td>Small animal ventilator</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Milipore-Sigma</td>
			<td>S5761</td>
			<td>NaHCO3 &#8805;99,5% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Milipore-Sigma</td>
			<td>S7653</td>
			<td>NaCl &#8805;99.5% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide pellets</td>
			<td>ACP chemicals</td>
			<td>S3700</td>
			<td>Diluted to 5N (10 g in 50 ml) for pH correction</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Milipore-Sigma</td>
			<td>S0751</td>
			<td>NaH2PO4 &#8805;99.0% Part of the Krebs solution</td>
		</tr>
		<tr>
			<td>Stevens Tenotomy Sciss, Str, Delicate, SH/SH, 4 1/2&#34;</td>
			<td>Skalar</td>
			<td>22-1240</td>
			<td>Small scisors for atria and cava vein opening</td>
		</tr>
		<tr>
			<td>Tissue slicer blades</td>
			<td>Thomas scientific</td>
			<td>6727C18</td>
			<td>Straight carbon steel blades for tissue slicing at the end of the protocol</td>
		</tr>
		<tr>
			<td>Tuberculin safety syringe with needle 25G 5/8&#34;</td>
			<td>CardinalHealth</td>
			<td>8881511235</td>
			<td>For heparin injection</td>
		</tr>
		<tr>
			<td>Veterinary General Surgery Set</td>
			<td>Skalar</td>
			<td>98-1275</td>
			<td>Surgery instruments including disection scisors and mosquito clamps</td>
		</tr>
		<tr>
			<td>Veterinary Micro Set</td>
			<td>Skalar</td>
			<td>98-1311</td>
			<td>Surgery instruments with microscisors used for carotid artery opening</td>
		</tr>
		<tr>
			<td>Working Heart Rat/Guinea Pig/Rabbit system</td>
			<td>Radnoti</td>
			<td>120101BEZ</td>
			<td>Modular working heart system modified for the needs of the protocol. Includes all the necesary tubbing, water jacketed reservoirs and valves, including 2 and 3 way stop cock</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td></td>
			<td>Sedative. 20 mg/ml</td>
		</tr>
	</tbody>
</table>

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.

Following extubation, blood pressure rapidly drops in a predictable pattern (Figure 3). Expected time to death is less than 5 min.
Figure 4 shows an average pressure/time curve at the start of reconditioning following 0, 10 and 15 min of WIT. Contractile function will improve over time. The use of short periods of WIT will allow for contractility to return to normal, and morphological damage will not be detectable (<strong class="xfig...

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Pre-clinical Model of Cardiac Donation after Circulatory Death

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 ...

This protocol targets the limited transplantation organ supply issue by facilitating the evaluation of different cardioprotective conditioning strategies to allow the use of hearts donated after circulatory death. Place the animal on a heating pad set to medium. After confirming a lack of response to toe pinch in an anesthetized adult rat, insert a rectal temperature probe.Attach a transdermal pulse oximeter sensor to a hind paw, and cover the rat with an absorbent pad to maintain body temperature. Make a three-to four-centimeter midline skin incision in the neck, and use blunt tip curved scissors to blunt dissect the subcutaneous tissue, to expose the right sternohyoid muscle. Using nontraumatic forceps, move the muscle laterally until the right carotid artery, jugular vein, and vagus nerve are identified.Use scissors to carefully separate the vagus nerve from the carotid artery. Inject 2, 000 international units per kilogram of heparin into the right jugular vein, applying pressure to the injection site after needle retraction to avoid blood leakage. Use curved forceps to pass two 5-0 silk sutures around the carotid artery.Firmly attach the distal suture to occlude the carotid artery at the superior aspect of the exposed artery, keeping the proximal suture untied, and place the rat under a stereo microscope. Then use microsurgery scissors to carefully make a one-millimeter incision over the anterior wall of the carotid artery, and insert a 22-gauge, one-inch closed intravenous catheter toward the aortic arch. The use of magnification and microdissection tools is highly recommend for these steps.A clean field can be achieved by tensioning the proximal suture to avoid bleeding. Cardiac donation is necessary to assemble the ex vivo cardioplegia apparatus. To initiate the DCD protocol, first extubate the animal, then use mosquito forceps to clamp the trachea, starting the agonal phase.Begin counting the functional WIT when the peak systolic blood pressure drops below 30 millimeters of mercury or asystole or ventricular fibrillation appears, whichever comes first. At the end of WIT, perform a medial sternotomy, keeping the thorax open with an Alm retractor if necessary. Separate the heart from the pulmonary veins and other thoracic structures to complete the cardiectomy.Immediately submerge the heart in ice-cold Krebs solution for a rapid transportation to the ex vivo system. Use a 2-0 silk suture to tightly fix the aorta to the Langendorff apparatus, and then open the stopcock. Now fully open the flow to the cannula, and start the initial reperfusion and stabilization time.Rotate the heart so the atria is facing the pressure sensor, and dissect the pulmonary veins to widen the left ventricular atrial opening if necessary. Next, insert a latex balloon connected to a pressure sensor, making sure that the balloon is fully positioned inside the ventricle. Slowly fill the balloon with saline until the end diastolic pressure reaches 15 millimeters of mercury.Insert the pacing electrode in the anterior face of the heart without puncturing the coronary vessels. Once spontaneous beating is observed, set the pacing to 300 beats per minute. After 10 minutes of stabilization, initiate the continuous intraventricular pressure measurement recording to begin the reconditioning and assessment phase.After one hour, remove the heart from the Langendorff apparatus and use a straight high-carbon steel blade to remove the atria. With the right ventricle facing down, cut one-to two-millimeter-thick transverse ventricular slices. Excise the right ventricle from the third slice of tissue, and snap freeze the left ventricle for downstream biochemical analyses.Submerge the remaining sections in freshly prepared 5%TTC solution in PBS for 10 minutes at 37 degrees Celsius. The next morning, wash the samples two times in fresh PBS and submerge the samples in fresh PBS for imaging. Viable tissues will appear brick-red in color.Following extubation, the blood pressure rapidly drops in a predictable pattern and the expected time to death is less than five minutes. Here, average pressure versus time curves at the start of reconditioning following zero, 10, and 15 minutes of WIT are shown. The contractile function will improve over time, the use of short periods of WIT will allow contractility to return to normal, and morphological damage will not be detectable.The use of a conditioning agent added with the cardioplegia and at the stabilization phase showed that the damage generated by 15 minutes of WIT in this model are amenable to modulation by cardioprotective agents. This technique allows full control of all the relevant variables and pharmacological and nonpharmacological intervention testing and can be reproduced in larger models, facilitating clinical translation.

pre-clinical-model-of-cardiac-donation-after-circulatory-death

Research

JoVE Journal

Medicine

7.8K vistas.  Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM). Este protocolo se dirige al limitado problema de suministro de órganos de trasplante facilitando la evaluación de diferentes estrategias de acondicionamiento cardioprotector para permitir el uso de corazones donados después de la muerte circulatoria. Coloque el animal en una almohadilla calefactora ajustada a media. Después de confirmar la falta de respuesta al pellizco del dedo del dedo del dedo del dedo en una rata adulta anestesiada, inserte una sonda de temperatura rectal.Conec...