We would like to thank Duke Large Animal Surgical Core and Duke Perfusion Services for their assistance during these procedures. We would also like to thank Paul Lezberg and TransMedics, Inc. for support.

NOTE: Two female Yucatan pigs are selected, with one designated to be the cardiac graft donor and the other the recipient. Pigs aged 6-8 months, weighing approximately 30 kg, and having compatible blood types are recommended. The overview of the protocol is demonstrated in Figure 1. Housing and the treatment procedures for the pigs are performed in accordance with the guidelines of the Animal Care and Use Committee of Duke University Medical Center.
1. </str...

<ol>
	<li>Virani, S. 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=Heart+disease+and+stroke+statistics-2021+update:+A+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2021 update: A report from the American Heart Association.</a> Circulation. 143 (8), 254 (2021).</li><li>Stehlik, J., Kobashigawa, J., Hunt, S. A., Reichenspurner, H., Kirklin, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honoring+50+years+of+clinical+heart+transplantation+in+circulation:+in-depth+state-of-the-art+review.">Honoring 50 years of clinical heart transplantation in circulation: in-depth state-of-the-art review.</a> Circulation. 137 (1), 71-87 (2018).</li><li>Kadner, A., Chen, R. H., Adams, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation:+experimental+development+and+clinical+experience.">Heterotopic heart transplantation: experimental development and clinical experience.</a> European Journal of Cardiothorac Surgery. 17 (4), 474-481 (2000).</li><li>Hastings, C. 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=Drug+and+cell+delivery+for+cardiac+regeneration.">Drug and cell delivery for cardiac regeneration.</a> Advanced Drug Delivery Reviews. 84, 85-106 (2015).</li><li>Sahoo, S., Kariya, T., Ishikawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+therapeutic+agents+to+the+heart.">Targeted delivery of therapeutic agents to the heart.</a> Nature Reviews. Cardiology. 18 (6), 389-399 (2021).</li><li>Stamp, N. 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=Successful+heart+transplant+after+ten+hours+out-of-body+time+using+the+TransMedics+Organ+Care+System.">Successful heart transplant after ten hours out-of-body time using the TransMedics Organ Care System.</a> Heart, Lung &amp; Circulation. 24 (6), 611-613 (2015).</li><li>Ragalie, W. S., Ardehali, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+normothermic+ex-vivo+perfusion+of+cardiac+allografts.">Current status of normothermic ex-vivo perfusion of cardiac allografts.</a> Current Opinion in Organ Transplantation. 25 (3), 237-240 (2020).</li><li>Koerner, M. 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=Normothermic+ex+vivo+allograft+blood+perfusion+in+clinical+heart+transplantation.">Normothermic ex vivo allograft blood perfusion in clinical heart transplantation.</a> Heart Surgery Forum. 17 (3), 141-145 (2014).</li><li>Rosenbaum, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+preservation+versus+static+preservation+for+cardiac+transplantation:+effects+on+myocardial+function+and+metabolism.">Perfusion preservation versus static preservation for cardiac transplantation: effects on myocardial function and metabolism.</a> Journal of Heart and Lung Transplantation. 27 (1), 93-99 (2008).</li><li>Cullen, P. P., Tsui, S. S., Caplice, N. M., Hinchion, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+state-of-the-art+review+of+the+current+role+of+cardioprotective+techniques+in+cardiac+transplantation.">A state-of-the-art review of the current role of cardioprotective techniques in cardiac transplantation.</a> Interactive Cardiovascular and Thoracic Surgery. 32 (5), 683-694 (2021).</li><li>Rurik, J. G., Aghajanian, H., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+and+immunotherapy+for+cardiac+injury+and+repair.">Immune cells and immunotherapy for cardiac injury and repair.</a> Circulation Research. 128 (11), 1766-1779 (2021).</li><li>Rincon, M. Y., VandenDriessche, T., Chuah, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+cardiovascular+disease:+advances+in+vector+development,+targeting,+and+delivery+for+clinical+translation.">Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.</a> Cardiovascular Research. 108 (1), 4-20 (2015).</li><li>Kieserman, J. M., Myers, V. D., Dubey, P., Cheung, J. Y., Feldman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+landscape+of+heart+failure+gene+therapy.">Current landscape of heart failure gene therapy.</a> Journal of the American Heart Association. 8 (10), 012239 (2019).</li><li>Perin, E. C., Perin, E. C., Miller, L. W., Taylor, D. A., Wilkerson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stem cell and gene therapy for cardiovascular disease. , 279-287 (2016).</li><li>Bishawi, 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=A+normothermic+ex+vivo+organ+perfusion+delivery+method+for+cardiac+transplantation+gene+therapy.">A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy.</a> Scientific Reports. 9 (1), 8029 (2019).</li><li>Hulot, J. S., Ishikawa, K., Hajjar, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+the+treatment+of+heart+failure:+promise+postponed.">Gene therapy for the treatment of heart failure: promise postponed.</a> European Heart Journal. 37 (21), 1651-1658 (2016).</li><li>Ho, C. 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=Molecular+characterization+of+swine+leucocyte+antigen+class+I+genes+in+outbred+pig+populations.">Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations.</a> Animal Genetics. 40 (4), 468-478 (2009).</li><li>Ho, C. 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=Molecular+characterization+of+swine+leucocyte+antigen+class+II+genes+in+outbred+pig+populations.">Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations.</a> Animal Genetics. 41 (4), 428-432 (2010).</li></ol>

Delivery of therapeutics during ex vivo perfusion in cardiac transplantation offers a strategy to modify the allograft and potentially improve transplant outcomes. The protocol presented here incorporates the state-of-the-art normothermic ex vivo sanguinous perfusion storage and offers promising potential to test isolated delivery of cell, gene, or immunotherapies to the allograft11,12,13. To date, cardiac deli...

Heart failure is a condition that affects an estimated 6 million adults in the United States and is projected to increase to 8 million adults by the year 20301. Cardiac transplantation is the gold standard treatment for end stage heart failure. However, it is not without its limitations and complications. It remains limited by the number of available donor hearts, primary graft dysfunction, rejection of the heart, and the side effects of long-term immunosuppression2. These limitations are particularly important in young recipients who may experience allograft failure and require subsequent re-transplantation to achieve normal life expectancy.
An ideal intervention to overcome these limitations would treat entire cardiac allografts with therapeutics prior to implantation into the recipient that can improve the viability of the allograft and confer &#34;cardioprotection.&#34; Such interventions would be given prophylactically to minimize the incidence of ischemic insults, allograft rejection, cardiac allograft vasculopathy, and even repair marginal allografts. Translational studies for developing these types of interventions require a large-animal model of cardiac transplantation to allow for the long-term surveillance of the cardiac graft. The porcine, heterotopic heart transplantation model in the intraabdominal position has proven ideal for this purpose. Heart transplantation in this position allows for testing the effects of novel therapies and assessing the immunopathology of graft rejection. Additionally, the heterotopic model is advantageous over the orthotopic model due to better overall survival of the recipient, no requirement for cardiopulmonary bypass, and no requirement of the graft to maintain the recipient&#39;s circulation3.
Effective delivery of therapeutic interventions to the heart, such as gene, cell, or immuno- therapy, is a significant barrier to clinical application4,5. The technology introduced by ex vivo perfusion devices allows grafts to be continually perfused, maintaining them in a nonworking but metabolically active state6,7,8,9. This offers a unique opportunity to treat a whole heart with advanced therapeutics while minimizing the potential side effects of systemic delivery10,11,12,13. Another advantage of utilizing ex vivo perfusion devices for therapeutic delivery is that they allow the administration of medications to the coronary circulation over extended periods that are not feasible using traditional cold static storage methods. This allows for more global delivery of the therapeutics to the graft14. Using the protocol presented here, we successfully delivered the firefly luciferase gene to a whole porcine cardiac graft using adenoviral vectors15. The aim of this protocol is to provide a reproducible and robust approach for achieving delivery of a therapeutic to the entire cardiac allograft prior to transplantation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0 Looped Maxon suture</td>
			<td>Covidien</td>
			<td>GMM-341L</td>
			<td>Used to close fascia of the laparotomy incision</td>
		</tr>
		<tr>
			<td>0 Silk ties</td>
			<td>Medtronic, Inc</td>
			<td>S346</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G Angiocath</td>
			<td>BD</td>
			<td>381144</td>
			<td>Used to de-air the left ventricle of the donor heart after implantation</td>
		</tr>
		<tr>
			<td>20 Fr LV vent</td>
			<td>Medtronic, Inc</td>
			<td>12002</td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 Silk sutures</td>
			<td>Ethicon, Inc.</td>
			<td>SA11G</td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 Silk ties</td>
			<td>Ethicon, Inc.</td>
			<td>SA65H</td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 Vicryl suture</td>
			<td>Ethicon, Inc.</td>
			<td>J259H</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Fr venous cannula</td>
			<td>Medtronic, Inc</td>
			<td>68124</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Prolene sutures</td>
			<td>Ethicon, Inc.</td>
			<td>8522</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Monocryl suture</td>
			<td>Ethicon, Inc.</td>
			<td>Y469G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Prolene sutures</td>
			<td>Ethicon, Inc.</td>
			<td>8521</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal hair cutting clipper</td>
			<td>Wahl</td>
			<td>8786-452</td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic clamp</td>
			<td>V. Mueller</td>
			<td>CH6201</td>
			<td></td>
		</tr>
		<tr>
			<td>Army Navy retractor</td>
			<td>V. Mueller</td>
			<td>SU3660</td>
			<td></td>
		</tr>
		<tr>
			<td>ATF 40, Cell saver disposable set</td>
			<td>Fresenius Kabi</td>
			<td>9108494</td>
			<td>Cell saver device insert</td>
		</tr>
		<tr>
			<td>Balfour retractor</td>
			<td>V. Mueller</td>
			<td>SU3042</td>
			<td>Used as an abdominal wall retractor</td>
		</tr>
		<tr>
			<td>C.A.T.S cell saver</td>
			<td>Fresenius Kabi</td>
			<td>ES0019</td>
			<td>Cell saver device used to wash donor blood</td>
		</tr>
		<tr>
			<td>Cardiac defibrillator</td>
			<td>Zoll</td>
			<td>M Series</td>
			<td>Cardiac defibrillator</td>
		</tr>
		<tr>
			<td>Castro needle holder</td>
			<td>V. Mueller</td>
			<td>CH8589</td>
			<td></td>
		</tr>
		<tr>
			<td>CG4 iStat cartridges</td>
			<td>Abbott</td>
			<td>03P85-25</td>
			<td>POC testing</td>
		</tr>
		<tr>
			<td>CG8 iStat cartridges</td>
			<td>Abbott</td>
			<td>03P88-25</td>
			<td>POC testing</td>
		</tr>
		<tr>
			<td>DeBakey forceps</td>
			<td>V. Mueller</td>
			<td>CH5902</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery disposable pencil</td>
			<td>Covidien</td>
			<td>E2450H</td>
			<td></td>
		</tr>
		<tr>
			<td>Gerald forceps</td>
			<td>V. Mueller</td>
			<td>NL1451</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemotherm 400CE Dual Reservoir Cooler/Heater</td>
			<td>Cincinnati Sub-Zero</td>
			<td>86022</td>
			<td>Heater cooler used to regulate perfusion temperature on the ex vivo perfusion device</td>
		</tr>
		<tr>
			<td>iSTAT 1</td>
			<td>Abbott</td>
			<td>04P75-03</td>
			<td>POC testing device</td>
		</tr>
		<tr>
			<td>Kocher clamp</td>
			<td>V. Mueller</td>
			<td>SU2790</td>
			<td></td>
		</tr>
		<tr>
			<td>Large clip applier</td>
			<td>Sklar</td>
			<td>50-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Large clips</td>
			<td>Teleflex</td>
			<td>4200</td>
			<td></td>
		</tr>
		<tr>
			<td>Large soft pledgets</td>
			<td>Covidien</td>
			<td>8886867901</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium clip applier</td>
			<td>Sklar</td>
			<td>50-4335</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium clips</td>
			<td>Teleflex</td>
			<td>2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum scissor</td>
			<td>V. Mueller</td>
			<td>CH2006-001</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 10 scalpel blade</td>
			<td>Swann-Mortan</td>
			<td>301</td>
			<td>Used for skin incision</td>
		</tr>
		<tr>
			<td>No. 11 scalpel blade</td>
			<td>Kiato Plus</td>
			<td>18111</td>
			<td>Used for vascular incision</td>
		</tr>
		<tr>
			<td>OCS device with base</td>
			<td>TransMedics, Inc.</td>
			<td></td>
			<td>Ex vivo perfusion device</td>
		</tr>
		<tr>
			<td>OCS disposable</td>
			<td>TransMedics, Inc.</td>
			<td></td>
			<td>Ex vivo perfusion device insert with perfusion kits</td>
		</tr>
		<tr>
			<td>Pacing cable</td>
			<td>Remington Medical</td>
			<td>FL-601-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Pediatric cardioplegia catheter (4Fr)</td>
			<td>Medtronic, Inc</td>
			<td>10218</td>
			<td>Used to deliver cardioplegia to the donor aortic root</td>
		</tr>
		<tr>
			<td>Pediatric Foley catheter</td>
			<td>Teleflex</td>
			<td>RSH170003080</td>
			<td>Placed pre-op to decompress the recipient&#39;s bladder</td>
		</tr>
		<tr>
			<td>Potts scissors</td>
			<td>V. Mueller</td>
			<td>CH13038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure bag x2 (1,000 mL)</td>
			<td>Novaplus</td>
			<td>V4010H</td>
			<td>Used to deliver cardioplegia at a set pressure</td>
		</tr>
		<tr>
			<td>Satinsky clamp</td>
			<td>V. Mueller</td>
			<td>CH7305</td>
			<td>Vascular clamp used for creating anastomoses between donor heart and recipient vessels</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Felco</td>
			<td>FELCO 200A-50</td>
			<td>Used to perform sternotomy</td>
		</tr>
		<tr>
			<td>Small hard pledgets</td>
			<td>Covidien</td>
			<td>8886867701</td>
			<td></td>
		</tr>
		<tr>
			<td>Sternal retractor</td>
			<td>V. Mueller</td>
			<td>CH6950-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Temporary cardiac pacing wires</td>
			<td>Ethicon, Inc.</td>
			<td>TPW32</td>
			<td></td>
		</tr>
		<tr>
			<td>Temporary dual chamber pacemaker</td>
			<td>Medtronic, Inc</td>
			<td>5388</td>
			<td>Cardiac pacing device</td>
		</tr>
		<tr>
			<td>Tourniquet kit</td>
			<td>Medtronic, Inc</td>
			<td>79005</td>
			<td>Rummel tourniquets</td>
		</tr>
		<tr>
			<td>Umbilical tape</td>
			<td>Covidien</td>
			<td>8886861903</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel loops</td>
			<td>Covidien</td>
			<td>31145686</td>
			<td></td>
		</tr>
	</tbody>
</table>

a porcine heterotopic heart transplantation protocol for delivery of therapeutics to a cardiac allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

This group has successfully survived 9 pigs between 5 and 35 days following the protocol as presented here, depending on the study design. Out of 10 pigs that have undergone this protocol, only 1 died prematurely from surgical complications, yielding a 90% survival rate. Demonstrated in Figure 2 is a diagram of the configuration of a heterotopic heart transplanted in the intraabdominal position in a pig. When determining the site for anastomosis of the allograft, select a site that minimizes...

Watch this Scientific Journal Video about A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft at JoVE.com

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

This protocol is the first to describe how therapeutics can be effectively administered to a whole cardiac allograft in the large animal model of cardiac transplantation. The main advantage of this protocol is that it allows investigators to test the effects of a therapeutic on a whole cardiac allograft over extended periods of time following transplantation. This technique can be used to investigate interventions that prevent cardiac allograft rejection, primary graft dysfunction, minimize the need for postoperative immunosuppression or extend the lifetime of a cardiac allograft.Optimizing exposure is the most challenging part of the transplant procedure and requires multiple team members to complete. Similarly, experienced perfusionists are needed to coordinate and monitor ex vivo perfusion. To begin, make a 20 to 30 centimeter long incision using the number 10 blade from the manubrium down to the xiphoid, depending on the size of the pig.Use electrocautery to divide the pectoralis major down from the sternum to the xiphoid being careful to divide along the midline of the sternum. Once down to the sternum, score the midline and begin the sternotomy from the xiphoid by dividing it with heavy scissors. Then extend the sternotomy cephalad after each cut.Bluntly separate the heart from the sternum using finger sweeps and complete the sternotomy through the manubrium. After optimizing the surgical field exposure using a sternal retractor, identify and remove the thymus with electrocautery by entering the pericardium longitudinally from the diaphragm to the aorta and creating a pericardial cradle using silk sutures. Fully divide the tissue between the aorta and pulmonary artery and visualize the location of the aortic arch and the brachycephalic trunk to facilitate proper placement of the aortic cross clamp.Using scissors and blunt dissection, circumferentially free the superior vena cava and tie two silk sutures of size zero around the superior vena cava. Apply size 4-0 polypropylene suture to the ascending aorta in a U-stitch manner and to the right atrium in a purse-string manner. Insert a pediatric aortic root cannula secured by the previously placed U-stitch.And then after de-airing the cannula, secure in place with a Rummel tourniquet. Connect the aortic root cannula to the cardioplegia tubing after the tubing has been flushed with del Nido cardioplegia. Create a right atriotomy within the previously placed purse-string.Insert a venous cannula into the right atrium and secure with a Rummel tourniquet. Connect the venous cannula to a sterile suction line connected to the cell saver cardiotomy and collect approximately 1 to 1.3 liters of blood. Then apply the aortic cross clamp, carefully ensuring that the clamp completely occludes the ascending aorta.After placing sterile ice slush on the heart, divide the inferior and superior vena cava just proximal to the azygos vein. The aorta at the level of the arch just distal to the innominate artery and the main pulmonary artery at the bifurcation. Identify and ligate the pulmonary veins, then remove the heart from the chest and place it in a container with sterile ice.To prepare the aorta for placement of the aortic connector, place four pledgeted size 4-0 polypropylene sutures in a simple horizontal mattress fashion around the inside of the distal aorta at a depth of five millimeters below the cut edge, and then tie them down. While holding up the pledgeted aortic sutures, insert the aortic connector into the aorta and tie an umbilical tape around the aorta to secure the connector. Place a size 4-0 polypropylene purse-string around the distal cut edge of the main pulmonary artery.Insert the pulmonary artery cannula and tie down the ends of the purse-string to secure the cannula. Then connect to the pulmonary artery cannula to the pulmonary artery connector on the device and secure it with the tie. Take the prepared graft from the back table to the ex vivo perfusion device and connect the aortic connector to the device place.The left ventricle vent drain through the untied pulmonary vein into the left atrium and across the mitral valve into the left ventricle. Using the number 10 blade, make a 20 to 30 centimeter long skin incision and then continue dissection down to fascia using electrocautery. After lifting the fascia and peritoneum with two Kocher clamps, carefully make a small incision into the peritoneal cavity using Metzenbaum scissors.Extend the peritoneal opening for the full length of the incision using electrocautery. Placing a finger underneath to protect the underlying viscera. Place a Balfour retractor to optimize exposure and retract to the small bowel cranially using a wet towel.Carry the dissection down to the abdominal aorta in inferior vena cava, then ligate the lymphatics with medium and large clips. On the back table, when the pulmonary vein left atriotomy where the left ventricle ventricle vent had been inserted is oversewed, trim the distal aspect of the aorta and pulmonary artery where attachment to the cannulas may have crushed the tissue. Place a Satinsky clamp on the inferior vena cava and create a longitudinal venotomy measuring approximately 1.5 centimeters using the number 11 blade and Pott's scissors.Anastomose the pulmonary artery graft to the recipient's infrarenal inferior vena cava in an end-to-side fashion using a running size 4-0 polypropylene suture. Then anastomose the aorta graft to the recipient's infrarenal aorta in end-to-side fashion using a running size 4-0 polypropylene suture. Carefully place the heart into the right retroperitoneal space, preventing tension on the anastomosis and kinking of the vessels.Then replace the small bowel. Close the fascia with loop size zero Maxon suture in a running fashion starting from both ends of the incision and tying in the middle, avoiding any injury to the bowel. Close the deep dermal layer with size 2-0 Vicryl in a running fashion and the skin with size 4-0 Monocryl in a running fashion.Several different perfusion parameters were acquired. Circulatory flow rates were measured from the pulmonary artery, the aorta, and the coronary arteries. The aortic pressure including mean pressure, systolic pressure, diastolic pressure were also measured.During ex vivo perfusion, the heart rate and temperature of the cardiac allograft were also measured. During the perfusion period, mixed venous oxygen saturation and the hematocrit values were measured from the perfusate. The viability of the intrabdominal heterotopic heart in situ 35 days indicate successful transplantation using this protocol.The most important thing to remember is to select a site for the allograft that minimizes the potential for tension and kinking of the anastomoses. By following this procedure, investigator will be able to test several different types of therapeutics to determine their effects on immunologic responses against the allograft, as well as determine the longevity of a therapeutics effect on the allograft. After developing this protocol, our group has been able to test different viral vector vehicles for gene delivery, as well as a model for acute allograph rejection to gain an understanding of the pathophysiology of rejection and efficacy of gene delivery using viral vectors.

a-porcine-heterotopic-heart-transplantation-protocol-for-delivery

Research

JoVE Journal

Medicine

2.4K vistas.  Duke University. Este protocolo es el primero en describir cómo la terapéutica puede administrarse eficazmente a un aloinjerto cardíaco completo en el modelo animal grande de trasplante cardíaco. La principal ventaja de este protocolo es que permite a los investigadores probar los efectos de una terapia en un aloinjerto cardíaco completo durante largos períodos de tiempo después del trasplante. Esta técnica se puede utilizar para investigar intervenciones que prevengan el rechazo del aloinjerto cardí...