This research is funded on behalf of the University Hospital Foundation.

All the procedures were performed in compliance with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The protocols were approved by the institutional animal care committee of the University of Alberta. This protocol has been applied in female juvenile Yorkshire pigs between 35-50 kg. Pigs are pathogen-free, food-grade specimens. They are purchased from the Swine Research and Technology Centre in Edmonton, AB, Canada (https://srtc.ualberta.ca). All individual...

<ol>
	<li>Chambers, D. 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+international+thoracic+organ+transplant+registry+of+the+international+society+for+heart+and+lung+transplantation:+Thirty-fifth+adult+lung+and+heart-lung+transplant+report-2018;+focus+theme:+Multiorgan+transplantation.">The international thoracic organ transplant registry of the international society for heart and lung transplantation: Thirty-fifth adult lung and heart-lung transplant report-2018; focus theme: Multiorgan transplantation.</a> The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 37 (10), 1169-1183 (2018).</li><li>Valapour, 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=OPTN/SRTR+2017+annual+data+report:+Lung.">OPTN/SRTR 2017 annual data report: Lung.</a> American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 19, 404-484 (2019).</li><li>Klein, A. 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=Organ+donation+and+utilization+in+the+united+states,+1999-2008.">Organ donation and utilization in the united states, 1999-2008.</a> American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 10 (4), 973-986 (2010).</li><li>Kotecha, 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=Continued+successful+evolution+of+extended+criteria+donor+lungs+for+transplantation.">Continued successful evolution of extended criteria donor lungs for transplantation.</a> The Annals of Thoracic Surgery. 104 (5), 1702-1709 (2017).</li><li>Singh, E., 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=Sequence+of+refusals+for+donor+quality,+organ+utilization,+and+survival+after+lung+transplantation.">Sequence of refusals for donor quality, organ utilization, and survival after lung transplantation.</a> The Journal of Heart and Lung Transplantation. 38 (1), 35-42 (2019).</li><li>Cypel, 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+lung+perfusion+in+clinical+lung+transplantation.">Normothermic ex vivo lung perfusion in clinical lung transplantation.</a> The New England Journal of Medicine. 364 (15), 1431-1440 (2011).</li><li>Wallinder, 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=Early+results+in+transplantation+of+initially+rejected+donor+lungs+after+ex+vivo+lung+perfusion:+A+case-control+study.">Early results in transplantation of initially rejected donor lungs after ex vivo lung perfusion: A case-control study.</a> European Journal of Cardio-Thoracic Surgery: Official Journal of the European Association for Cardio-Thoracic Surgery. 45 (1), 40-44 (2014).</li><li>Sage, E., 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=Lung+transplantation+from+initially+rejected+donors+after+ex+vivo+lung+reconditioning:+The+french+experience.">Lung transplantation from initially rejected donors after ex vivo lung reconditioning: The french experience.</a> European Journal of Cardio-Thoracic Surgery: Official Journal of the European Association for Cardio-Thoracic Surgery. 46 (5), 794-799 (2014).</li><li>Valenza, F., 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=Extracorporeal+lung+perfusion+and+ventilation+to+improve+donor+lung+function+and+increase+the+number+of+organs+available+for+transplantation.">Extracorporeal lung perfusion and ventilation to improve donor lung function and increase the number of organs available for transplantation.</a> Transplantation Proceedings. 44 (7), 1826-1829 (2012).</li><li>Fildes, J. E., 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=Clinical+outcome+of+patients+transplanted+with+marginal+donor+lungs+via+ex+vivo+lung+perfusion+compared+to+standard+lung+transplantation.">Clinical outcome of patients transplanted with marginal donor lungs via ex vivo lung perfusion compared to standard lung transplantation.</a> Transplantation. 99 (5), 1078-1083 (2015).</li><li>Cypel, 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=Experience+with+the+first+50+ex+vivo+lung+perfusions+in+clinical+transplantation.">Experience with the first 50 ex vivo lung perfusions in clinical transplantation.</a> The Journal of Thoracic and Cardiovascular Surgery. 144 (5), 1200-1206 (2012).</li><li>Clark, S. 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=A+new+porcine+model+of+reperfusion+injury+after+lung+transplantation.">A new porcine model of reperfusion injury after lung transplantation.</a> Laboratory Animals. 33, 135-142 (1999).</li><li>Karimi, 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=Technical+pearls+for+swine+lung+transplantation.">Technical pearls for swine lung transplantation.</a> Journal of Surgical Research. 171, 107-111 (2011).</li><li>Kruger, 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=Porcine+pulmonary+auto-transplantation+for+ex+vivo+therapy+as+a+model+for+new+treatment+strategies.">Porcine pulmonary auto-transplantation for ex vivo therapy as a model for new treatment strategies.</a> Interactive CardioVascular and Thoracic Surgery. 23, 358-366 (2016).</li><li>Mariscal, 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=Pig+lung+transplant+survival+model.">Pig lung transplant survival model.</a> Nature Protocols. 13, 1814-1828 (2018).</li><li>Aboelnazar, N. 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=Negative+pressure+ventilation+decreases+inflammation+and+lung+edema+during+normothermic+ex+vivo+lung+perfusion.">Negative pressure ventilation decreases inflammation and lung edema during normothermic ex vivo lung perfusion.</a> The Journal of Heart and Lung Transplantation: The Official Publication of the International Society for Heart Transplantation. 37 (4), 520-530 (2018).</li></ol>

DHF holds patents on Ex situ organ perfusion technology and methods. DHF and JN are founders and major shareholders of Tevosol, Inc.

Several critical surgical steps are involved in this protocol, and troubleshooting is needed to ensure successful transplantation and lung assessment. Juvenile porcine lungs are incredibly delicate compared to adult human lungs, so the operating surgeon must be cautious when handling porcine lungs. This is especially true after a 12 h run of ESLP as the organ will have taken on fluid volume and be susceptible to injury from excessive manipulation. Any undue pressure will cause atelectasis or trauma to the experimental lu...

Lung transplantation is the preeminent long-term treatment for end-stage lung disease. Over 4,600 lung transplantations are performed worldwide annually1. However, lung transplantation currently has significant limitations. For one, the necessity for organs continues to eclipse available donors. Despite rates of lung transplantation increasing every year since 2012 due to the combined effects of more candidates being listed for transplant, an increase in the number of donors, and improved use of recovered organs, the transplant waitlist mortality has not decreased significantly2. Organ quality concerns represent another major limitation, with reported organ utilization rates as low as 20%-30%3,4,5. Finally, the trends in the post-operative outcomes of lung transplantation are less than satisfactory, with long-term graft and patient outcomes still lagging that of other solid organ transplantations2.
An emerging technology, ex situ lung perfusion (ESLP), has the potential to mitigate these limitations. ESLP has been increasingly applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs6,7,8,9,10. Consequently, ESLP has increased utilization rates in some centers by 15%-20%6,7,8,9,10,11.
Proper ESLP research requires the in vivo validation of in vitro findings; however, there is limited literature on porcine lung transplantation models for ESLP12,13,14,15. Furthermore, available literature provides inadequate details regarding anesthetic management of Yorkshire pigs for lung transplantation, which can be highly unstable hemodynamically12,13,14,15. Establishing an easily reproducible model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions to reduce lung ischemia-reperfusion injury. The objective of the present study is to describe a porcine model of orthotopic left lung allotransplantation for use with ESLP. The protocol includes descriptions of the anesthetic and surgical techniques, a custom surgical checklist, and details regarding the troubleshooting experience and protocol modifications. The limitations and benefits of the left lung porcine transplantation model have also been discussed in this work. This manuscript does not outline the retrieval process of porcine lungs in 35-50 kg Yorkshire pigs, nor does it cover the establishment and termination of ESLP. This protocol exclusively addresses the recipient transplantation operation.

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			<td>ABL 800 FLEX Blood Gas Analyzer</td>
			<td>Radiometer</td>
			<td>989-963</td>
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			<td>Adult-Pediatric Electrostatic Filter HME - Small</td>
			<td>Covidien</td>
			<td>352/5877</td>
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			<td>Allison Lung Retractor</td>
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			<td>SORIN GROUP</td>
			<td>01706/03</td>
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			<td>Backhaus Towel Clamp</td>
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			<td>454300</td>
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			<td>Bovine Serum Albumin</td>
			<td>MP biomedicals</td>
			<td>218057791</td>
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			<td>Biomedicus Pump</td>
			<td>Maquet</td>
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			<td>Bronchoscope</td>
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			<td>Cable Ties &#8211; White 12&#8221;</td>
			<td>HUASU International</td>
			<td>HS4830001</td>
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			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C69-500G</td>
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			<td>Cooley Sternal Retractor</td>
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			<td>341162</td>
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			<td>CUSHING Gutschdressing Forceps</td>
			<td>Pilling</td>
			<td>466200</td>
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			<td>Debakey-Metzenbaum Dissecting</td>
			<td>Pilling</td>
			<td>342202</td>
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			<td>Scissors</td>
			<td>Pilling</td>
			<td>342202</td>
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			<td>DeBakey Peripheral Vascular Clamp</td>
			<td>Pilling</td>
			<td>353535</td>
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			<td>Debakey Straight Vascular Tissue Forceps</td>
			<td>Pilling</td>
			<td>351808</td>
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			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767-500G</td>
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			<td>Drop sucker</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Endotracheal Tube 9.0mm CUFD</td>
			<td>Mallinckrodt</td>
			<td>9590E</td>
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			<td>Flow Transducer</td>
			<td>BIO-PROBE</td>
			<td>TX 40</td>
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			<td>Infusion Pump</td>
			<td>Baxter</td>
			<td>AS50</td>
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			<td>Inspire 7 M Hollow Fiber Membrane Oxygenator</td>
			<td>SORIN GROUP</td>
			<td>K190690</td>
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			<td>Intercept Tubing Connector 3/8&#34; x 1/2&#34;</td>
			<td>Medtronic</td>
			<td>6013</td>
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			<td>Intercept Tubing 1/4&#34; x 1/16&#34; x 8&#39;</td>
			<td>Medtronic</td>
			<td>3108</td>
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			<td>Intercept Tubing 3/8&#34; x 3/32&#34; x 6&#39;</td>
			<td>Medtronic</td>
			<td>3506</td>
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			<td>Laryngoscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made with 10-inch blade</td>
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			<td>Metzenbaum Dissecting Scissors</td>
			<td>Pilling</td>
			<td>460420</td>
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			<td>Medical Carbon Dioxide Tank</td>
			<td>Praxair</td>
			<td>5823115</td>
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			<td>Medical Oxygen Tank</td>
			<td>Praxair</td>
			<td>2014408</td>
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			<td>Medical Nitrogen Tank</td>
			<td>Praxair</td>
			<td>NI M-K</td>
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			<td>Mosquito Clamp</td>
			<td>Pilling</td>
			<td>181816</td>
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			<td>Harken Auricle Clamp</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Organ Chamber</td>
			<td>Tevosol</td>
			<td></td>
			<td></td>
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		<tr>
			<td>PlasmaLyte A</td>
			<td>Baxter</td>
			<td>TB2544</td>
			<td></td>
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		<tr>
			<td>Poole Suction Tube</td>
			<td>Pilling</td>
			<td>162212</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate</td>
			<td>Fischer Scientific</td>
			<td>P285-500G</td>
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			<td>PERFADEX Plus</td>
			<td>XVIVO</td>
			<td>19811</td>
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			<td>Satinsky Clamp</td>
			<td>Pilling</td>
			<td>354002</td>
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			<td>Scale</td>
			<td>TANITA</td>
			<td>KD4063611</td>
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			<td>Silicon Support Membrane</td>
			<td>Tevosol</td>
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			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>792519-1KG</td>
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			<td>Sodium Chloride 0.9%</td>
			<td>Baxter</td>
			<td>JB1324</td>
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			<td>Sorin XTRA Cell Saver</td>
			<td>SORIN GROUP</td>
			<td>75221</td>
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			<td>Sternal Saw</td>
			<td>Stryker</td>
			<td>6207</td>
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			<td>Surgical Electrocautery Device</td>
			<td>Kls Martin</td>
			<td>ME411</td>
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			<td>TruWave Pressure Transducer</td>
			<td>Edwards</td>
			<td>VSYPX272</td>
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			<td>Two-Lumen Central Venous Catheter 7fr X2</td>
			<td>Arrowg+ard</td>
			<td>CS-12702-E</td>
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			<td>Vorse Tubing Clamp</td>
			<td>Pilling</td>
			<td>351377</td>
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			<td>Willauer-Deaver Retractor</td>
			<td>Pilling</td>
			<td>341720</td>
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			<td>Yankauer Suction Tube</td>
			<td>Pilling</td>
			<td>162300</td>
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			<td>0 ETHIBOND Green 1X36&#34; Endo Loop 0</td>
			<td>ETHICON</td>
			<td>D8573</td>
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			<td>0 PDS II CP-1 2x27&#8221;</td>
			<td>ETHICON</td>
			<td>Z467H</td>
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		<tr>
			<td>1 VICRYL MO-4 1x18&#8221;</td>
			<td>ETHICON</td>
			<td>J702D</td>
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			<td>2-0 SILK Black 12&#34; x 18&#34; Strands</td>
			<td>ETHICON</td>
			<td>SA77G</td>
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			<td>4-0 PROLENE Blue TF 1x24&#8221;</td>
			<td>ETHICON</td>
			<td>8204H</td>
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			<td>6-0 PROLENE Blue BV 2x30&#8221;</td>
			<td>ETHICON</td>
			<td>M8776</td>
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		<tr>
			<td>21-Gauge Needle</td>
			<td></td>
			<td></td>
			<td></td>
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</table>

left lung orthotopic transplantation in a juvenile porcine model for eslp

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

All of the results are in the context of 4 h of reperfusion following 12 h of NPV-ESLP16. During lung explant, there are several clinical outcomes to anticipate (Figure 3). Typically, the pig will remain hemodynamically stable following a successful left lung explantation but may require a low dose infusion of phenylephrine (dose range: 2-10 mg/h) due to a vasodilatory response to surgery. Heart rate should target approximately 100-120 bpm, respiratory rate (RR) 8-30 ...

Watch this Scientific Journal Video about Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP at JoVE.com

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

This protocol is one of the most straightforward lung transplant protocols for large animal models and provides the most detail in terms of surgical management. The main advantages of this technique are describing the surgery, assessing isolated left lung function post-transplant, and stabilizing the pig during surgery. The method is beneficial in ex-situ lung profusion research to validate ex-situ findings with an in vivo reperfusion transplantation model.The in vivo reperfusion model of lung transplantation is beneficial for testing ex-situ lung profusion findings and could also be used in artificial organ testing, such as bio-engineered or mechanical lungs. Begin by positioning the pig in a right lateral decubitus position for the left anterior lateral thoracotomy. Then identify the tip of the left scapula in the xiphoid process, inferior to the sternum with palpation, and connect the two identified spots to mark the 20 centimeter long thoracotomy incision site.After inducing the anesthesia, use a scalpel to make an incision on the skin and then use electrocautery to dissect the subcutaneous layers and muscle layers of the animal. The latissimus dorsi must be divided. After identifying the rib immediately below the incision, cauterize on top of the rib to expose the intercostal muscles while avoiding the intercostal neurovascular bundle.Use a mosquito hemostat to puncture the intercostal muscles immediately above the rib and then feel inside the chest for adhesions with a finger. While the top edge of the rib is being cauterized, push the lung away using a Yankauer suction to extend the thoracotomy anteriorly until one inch away from the sternum, and posteriorly to the paraspinal muscles. By inserting a Cooley sternal retractor, open the thoracotomy 10 centimeter wide to retract the lung and expose the left hemiazygos vein.Use Metzenbaum scissors and a fine lower to dissect the left hemiazygos vein circumferentially. By circling the vessel with silk ties, ligate and transect the vein. Dissect out the left pulmonary artery, or PA, and left pulmonary veins, or PV, before encircling the veins in the silk ties.After five minutes of heparin administration, clamp the PA with a DeBakey cross clamp, the left anterior PV with a Satinsky clamp and the left bronchus with Spoon Pots clamp. Transect the PA, the left inferior PV and the left bronchus leaving at least 0.5 centimeters of tissue cuff. After dividing the left inferior pulmonary ligament, remove the left lung.For transplantation, insert the donor lung into the recipient's chest, beginning with the lower lobe without forcing the lung into the place. Then perform the bronchial anastomosis using a 4.0 Prolene suture on a TF needle. To assess the bronchial anastomosis in suction secretions, perform left and right lung bronchoscopy by inserting a bronchoscope into the endotracheal tube using an adapter connection, then connect the scope to suction before advancing the bronchoscope into the left bronchus.Observe the lung breathing and inspect the vitals in bronchial anastomosis. Repeat the process on the right side. Next, use a 6.0 Prolene suture on BV-1 Needles to perform end-to-End left atrium or LA anastomosis followed by trimming excess tissue.After incorporating the donor SVPs into the inferior PV and LA anastomosis, complete the PA anastomosis with a 6.0 Prolene suture on BV-1 needles using a running end-to-End anastomosis. Later, confirm heparinization and administer a potassium shift to the animal. Then open the PA clamp partially to de-air and let the blood flow through the suture line.Next, release the LA clamp and let the suture line back bleed to further release any trapped air. Tie the LA and then remove the PA clamp completely. Once done, remove the bronchial clamp and increase tidal volumes to target 10 milliliters per kilogram.After inserting a 20 French malleable chest tube, close the thoracotomy in three layers. Using Metzenbaum scissors, open the left pleura to take blood samples from the left lower lobe. Open the right pleura to dissect out the right pulmonary artery.Then place a clamp to stop right lung perfusion. Direct a 21 gauge needle toward the left pulmonary veins and away from the common left atrium to take a blood sample from the LA anastomosis to assess left lung function. After clamping, take the blood samples from the left PV anastomosis with a 21 gauge needle directed toward the left lung.The representative analysis indicates typical PF ratio changes and edema formation during the lung transplant protocol. At the reperfusion, the PF ratios dropped by approximately 100 millimeters of mercury as the left lung was not immediately effective at oxygenation. In isolated left lung assessment at four hours, the PF ratio was stable.In a successful transplant, the left lung could experience approximately 20%to 50%weight gain due to residual blood in the circulation. After 12 hours of the left lung transplant, the blood gas analysis was performed. It was observed that the potassium level was increased during 60 to 120 minutes of reperfusion.Approximately two to four shifts were required during four hour reperfusion to keep potassium lower than five millimoles per liter. Instead of suture ligating the recipient's superior pulmonary veins, the side biting clamps can be used for vascular control and the vessels can be easily over sewn.

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Research

JoVE Journal

Medicine

1.5K visualizações.  University of Alberta. Este protocolo é um dos mais simples protocolos de transplante pulmonar para modelos de animais de grande porte e fornece o maior detalhe em termos de manejo cirúrgico. As principais vantagens dessa técnica são descrever a cirurgia, avaliar a função pulmonar esquerda isolada pós-transplante e estabilizar o porco durante a cirurgia. O método é benéfico na pesquisa de profusão pulmonar ex-situ para validar achados ex-situ com um modelo de transplante de reperfusão in vivo.O m...