Name: normothermic negative pressure ventilation ex situ lung perfusion: evaluation of lung function and metabolism
Uploaded: 2022-02-14T14:00:00
Description: Watch this Scientific Journal Video about Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism at JoVE.com

This research was funded on behalf of The Hospital Research Foundation.

The procedures performed in this manuscript comply with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The institutional animal care committee of the University of Alberta approved the protocols. Female juvenile Yorkshire pigs between 35-50 kg were used exclusively. Proper biosafety training was required by all individuals involved in ESLP procedures. A schematic overview of the entire NPV-ESLP experiment is represented in Figure 1</str...

<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. 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. 19, 404-484 (2019).</li><li>Chambers, D. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liberalization+of+donor+criteria+may+expand+the+donor+pool+without+adverse+consequence+in+lung+transplantation.">Liberalization of donor criteria may expand the donor pool without adverse consequence in lung transplantation.</a> The Journal of Heart and Lung Transplantation. 19 (12), 1199-1204 (2000).</li><li>Snell, G. I., Griffiths, A., Levvey, B. J., Oto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Availability+of+lungs+for+transplantation:+Exploring+the+real+potential+of+the+donor+pool.">Availability of lungs for transplantation: Exploring the real potential of the donor pool.</a> The Journal of Heart and Lung Transplantation. 27 (6), 662-667 (2008).</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. 45 (1), 40-45 (2014).</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 (1), 1200-1206 (2012).</li><li>Buchko, M. T., 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=Total+parenteral+nutrition+in+ex+vivo+lung+perfusion:+Addressing+metabolism+improves+both+inflammation+and+oxygenation.">Total parenteral nutrition in ex vivo lung perfusion: Addressing metabolism improves both inflammation and oxygenation.</a> American Journal of Transplantation. 19 (12), 3390-3397 (2019).</li><li>Andreasson, A. S. I., 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=Profiling+inflammation+and+tissue+injury+markers+in+perfusate+and+bronchoalveolar+lavage+fluid+during+human+ex+vivo+lung+perfusion.">Profiling inflammation and tissue injury markers in perfusate and bronchoalveolar lavage fluid during human ex vivo lung perfusion.</a> European Journal of Cardio-Thoracic Surgery. 51 (3), 577-586 (2017).</li><li>Sadaria, M. R., 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=Cytokine+expression+profile+in+human+lungs+undergoing+normothermic+ex-vivo+lung+perfusion.">Cytokine expression profile in human lungs undergoing normothermic ex-vivo lung perfusion.</a> The Annals of Thoracic Surgery. 92 (2), 478-484 (2011).</li><li>Ricard, J. D., Dreyfuss, D., Saumon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator-induced+lung+injury.">Ventilator-induced lung injury.</a> European Respiratory Journal. 42, 2-9 (2003).</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. 37 (4), 520-530 (2018).</li><li>Lai-Fook, S. J., Rodarte, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleural+pressure+distribution+and+its+relationship+to+lung+volume+and+interstitial+pressure.">Pleural pressure distribution and its relationship to lung volume and interstitial pressure.</a> Journal of Applied Physiology. 70 (3), 967-978 (1991).</li><li>Buchko, M. T., 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+transplantation+using+negative+pressure+ventilation+ex+situ+lung+perfusion+with+extended+criteria+donor+lungs.">Clinical transplantation using negative pressure ventilation ex situ lung perfusion with extended criteria donor lungs.</a> Nature Communications. 11 (1), 5765 (2020).</li><li>Buchko, M. T., 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+low-cost+perfusate+alternative+for+ex+vivo+perfusion.">A low-cost perfusate alternative for ex vivo perfusion.</a> Transplantation Proceedings. 52 (10), 2941-2946 (2020).</li><li>Forgie, K. 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=Left+lung+orthotopic+transplantation+in+a+juvenile+porcine+model+for+ESLP.">Left lung orthotopic transplantation in a juvenile porcine model for ESLP.</a> The Journal of Visualized Experiments. , (2021).</li></ol>

There are several critical surgical steps along with troubleshooting needed to ensure a successful ESLP run. Juvenile porcine lungs are extremely delicate compared to adult human lungs, so the procuring surgeon must be cautious when handling porcine lungs. It is critical to attempt a &#34;no-touch&#34; technique to avoid causing trauma and atelectasis when dissecting out the lungs. &#34;No-touch&#34; means using the bare minimum amount of manual manipulation of the lungs during procurement. Recruitment maneuvers while on...

Lung transplantation (LTx) remains the standard of care for end-stage lung disease1; however, LTx has significant limitations including inadequate donor organ utilization2 and a waitlist mortality of 40%3, which is higher than any other solid organ transplant4,5. Donor organ utilization rates are low (20-30%) due to organ quality concerns. Excessive geographic transportation distance compounded by stringent donor organ acceptance criteria exacerbates these quality concerns. LTx also trails other solid organ transplants in terms of long-term graft and patient outcomes2. Primary graft dysfunction (PGD), most often caused by ischemic reperfusion injury (IRI), represents the leading cause of 30-day mortality and morbidity post-LTx and increases the risk for chronic graft dysfunction6,7. Efforts to decrease IRI and extend safe transport times are paramount to improve patient outcomes.
Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. ESLP facilitates the preservation, assessment, and reconditioning of donor lungs before transplantation. It has exhibited satisfactory short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs, contributing to an increase in the number of suitable donor lungs for LTx, with organ utilization rates increasing by 20% in some centres8,9,10. Compared to the current clinical standard for LTx, cold static preservation (CSP), ESLP offers several advantages: organ preservation time is not limited to 6 h, evaluation of organ function is possible before implantation, and due to continuous organ perfusion, modifications can be made to the perfusate that optimizes organ function11.
The vast majority of current ESLP devices designed for human use utilize positive pressure ventilation (PPV); however, recent literature has indicated that this ventilation strategy is inferior to negative pressure ventilation (NPV) ESLP, with PPV inducing more significant ventilator-induced lung injury12,13,14,15. In both human and porcine lungs, NPV-ESLP exhibits superior organ function when compared to positive pressure ex situ lung perfusion (PPV-ESLP) across various physiological domains, including pro-inflammatory cytokine production, pulmonary edema, and bullae formation15. The homogenous distribution of intrathoracic pressure across the entire lung surface in NPV-ESLP has been suggested as a significant factor underlying this advantage15,16. In addition to its pre-clinical benefits, the clinical safety and feasibility of NPV-ESLP have been demonstrated in a recent clinical trial17. Utilizing a novel NPV-ESLP device, twelve extended criteria donor human lungs were successfully preserved, evaluated, and subsequently transplanted with 100% 30-day and 1-year survival.
The objective of the present manuscript is to demonstrate a working protocol of our lab&#39;s NPV-ESLP device using juvenile porcine lungs under normothermic conditions for 12 h of duration. The surgical retrieval is covered in detail, and our custom software platform&#39;s initiation, management, and termination are also described. The strategy for tissue collection and the management of the samples is also explained.

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			<td>ABL 800 FLEX Blood Gas Analyzer</td>
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			<td>Adult-Pediatric Electrostatic Filter HME - Small</td>
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			<td>454300</td>
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			<td>Maquet</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>Fisher Scientific</td>
			<td>C69-500G</td>
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			<td>Pilling</td>
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			<td>Pilling</td>
			<td>466200</td>
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			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767-500G</td>
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			<td>Pilling</td>
			<td>481826</td>
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			<td>Pilling</td>
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			<td>Debakey-Metzenbaum Dissecting</td>
			<td>Pilling</td>
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			<td>Pilling</td>
			<td>342202</td>
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			<td>Endotracheal Tube 9.0mm CUFD</td>
			<td>Mallinckrodt</td>
			<td>9590E</td>
			<td>Cuff removed for ESLP apparatus</td>
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			<td>Flow Transducer</td>
			<td>BIO-PROBE</td>
			<td>TX 40</td>
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			<td>Human Albumin Serum</td>
			<td>Grifols Therapeutics</td>
			<td>2223708</td>
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			<td>Infusion Pump</td>
			<td>Baxter</td>
			<td>AS50</td>
			<td></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 1/4&#34; x 1/16&#34; x 8&#39;</td>
			<td>Medtronic</td>
			<td>3108</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercept Tubing 3/8&#34; x 3/32&#34; x 6&#39;</td>
			<td>Medtronic</td>
			<td>3506</td>
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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>MAYO 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>Praxair</td>
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			<td>Medical Oxygen Tank</td>
			<td>Praxair</td>
			<td>2014408</td>
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			<td>Tevosol</td>
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			<td>Baxter</td>
			<td>TB2544</td>
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			<td>Fischer Scientific</td>
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			<td>TANITA</td>
			<td>KD4063611</td>
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			<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 GROUP</td>
			<td>75221</td>
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			<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>Temperature Sensor probe</td>
			<td>Omniacell Tertia Srl</td>
			<td>1777288F</td>
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			<td>THAM Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15504020</td>
			<td>made from UltraPureTM Tris</td>
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			<td>TruWave Pressure Transducer</td>
			<td>Edwards</td>
			<td>VSYPX272</td>
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			<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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normothermic negative pressure ventilation ex situ lung perfusion: evaluation of lung function and metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

At the beginning of lung perfusion and ventilation (preservation mode), the lungs will generally have a low pulmonary artery pressure (&#60; 10 mmHg) and low dynamic compliance (&#60; 10 mL/mmHg) as the perfusate warms to normothermia. Yorkshire pigs weighing 35-50 kg typically results in lungs weighing 350-500 g. During the first hour of NPV-ESLP, the measured expiratory tidal volumes (TVe) are 0-2 mL/kg, and the inspiratory tidal volumes (TVi) are 100-200 mL. TVe generally reaches 4-6 mL/kg within 3-6 h, and after that...

Watch this Scientific Journal Video about Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism at JoVE.com

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Dr.Darren Freed created this ESLP device, and it uses patented negative pressure ventilation technology and software, which can't be seen anywhere else in the field of ESLP research. Our lab has previously shown that negative pressure ventilation causes less lung injury during ESLP compared to positive pressure ventilation. This is likely due to the more physiologic mechanism of negative pressure ventilation.This device and protocol are designed to extend the safe preservation time of donor lungs and to recondition marginal lungs to acceptable transplant standards. The device itself is very simple to operate. The greatest challenge is surgically removing the juvenile pig lungs without causing any injury.They're extremely fragile compared to adult human lungs. Begin by maintaining the expiratory tidal volume of 10 milliliters per kilogram by increasing the peak pressures for maximum alveolar recruitment. Perform a midline sternotomy.Use electrocautery to incise the skin and subcutaneous tissue. Bluntly dissect the soft tissue attachments from the posterior table of the inferior sternum. Use a sternal saw to divide the sternum.Insert a sternal retractor and open wide. After performing the cardiectomy, open and remove the mediastinal pleura bilaterally to expose the lungs. Then divide the pleural attachments from the diaphragm toward the left lower lung lobe, using a Deaver retractor to hold the diaphragm upwards.Now, divide the inferior pulmonary ligament on the left and continue up toward the hilum. Next, divide the inferior vena cava and pleural attachments from the diaphragm, and then retract the diaphragm upwards using the Deaver retractor. Divide the inferior pulmonary ligament on the right side and continue up towards the hilum.Then divide the innominate vein and arch vessels to expose the trachea. After bluntly dissecting the tissue surrounding the trachea, clamp the trachea using a tubing clamp at maximal inhalation, ensuring the expiratory tidal volume is 10 milliliters per kilogram. Transect the trachea and lift the clamp portion upwards to provide surgical traction.Then dissect the posterior trachea from the esophagus using blunt dissection with heavy Metzenbaum scissors and a free hand. Divide any remaining pleural attachments. Transect the aorta above and below the left bronchus and remove the lungs from the chest with a segment of descending aorta.After weighing the lungs with the clamp, quickly store them in a cooler full of ice. Begin by adding 500 milliliters of packed red blood cells to the perfusion circuit to reach a final volume of 1.5 liters of perfusate. Take photographs of the lungs for data records.For the lung biopsy, encircle a one cubic centimeter portion with zero silk suture and tie it. Then excise this tied portion, using scissors for tissue analysis as described in the text manuscript, and divide the biopsy segment. Next, to secure the 3/8 half-inch tubing adapter to the main pulmonary artery, grasp the opposite sides of the main pulmonary artery using forceps or snaps.Then, insert the adapter with the half-inch portion into the main pulmonary artery and hold it in place while simultaneously securing the adapter in position using zero silk or Ethibond ties. Now place the lungs in a supine position on the silicone support membrane and connect them to the ESLP device. Place a second tubing clamp on the trachea near the location of the tracheal bronchus.After removing the more distal clamp, intubate the trachea with the endotracheal tube and secure it in position using two zip ties. Then clamp the ventilation line using a tubing clamp before releasing the proximal clamp from the trachea. Connect the pulmonary artery adapter to the pulmonary artery line.Once the main pulmonary artery is de-aired, start the time for perfusion. On the settings page, click start heater and set the temperature to 38 degrees Celsius. Enter the pig's weight to calculate cardiac output flow.On the main page, set the continuous positive airway pressure to 20 centimeters of water and click start continuous positive airway pressure. When ventilation begins, unclamp the ventilation line. After zeroing the arterial pressure sensor, clamp the pulmonary artery line above the pressure sensor with the tubing clamp.Open the sensor, click zero pulmonary artery pressure and zero blood flow on the settings page and confirm the readings are zeroed on the main page. Close the pressure sensor stopcock to read the line pressure. Open the line to the pulmonary artery cannula.Select 10%cardiac output on the main page. Click return to PA Manual and unclamp the pulmonary line. Switch the perfusate solution in the top lid before securing it to the machine to prevent condensation formation during ESLP.Draw 10 milliliters of perfusate for centrifugal analysis and draw a time zero arterial blood gas. Place the sample on ice before centrifugation. Once the lungs have been perfused for 10 minutes, increase the flow to 20%of the cardiac output.When the perfusate temperature reaches 32 degrees Celsius, secure the chamber lid in place with clamps to create an airtight seal. Ensure that the lungs are optimally positioned and the air leaks are repaired. With the lid secure, clamp the ventilation tubing before turning off continuous positive airway pressure.On the settings page, select zero intrathoracic pressure, airway pressure, and airflow. Then confirm readings are zeroed on the main page. Click start CPAP before unclamping the ventilation tubing.Set the ventilation parameters mentioned in the text manuscript and click press to start vent to activate negative pressure ventilation. Attach the sideport ventilation tubing to the chamber. Over the next few breaths, maintain the ventilation parameters as mentioned in the text manuscript.After 10 minutes of perfusion, increase the flow to 20%and after 20 minutes of perfusion, increase the flow to 30%cardiac output. Add dextrose and insulin to the system. Evaluate lung function, using 50%cardiac output and a deoxygenating mixed sweep gas over a five minute duration, as per the manuscript instructions.At every odd hour during preservation mode, draw a 10 milliliters sample of perfusate for future analysis. Draw one milliliter of pre-deoxygenator arterial blood gas sample every hour and from pre and post deoxygenator ports following five minutes of evaluation mode. The trends in mean pulmonary artery pressure, dynamic compliance, and pulmonary vascular resistance over 12 hours of perfusion and ventilation can be affected by the specific ESLP experimental protocol employed.A typical trend for the ratio of partial pressure of arterial oxygen and fraction of inspired oxygen at the evaluation time points of five and 11 hours throughout negative pressure ventilation ex-situ lung perfusion is demonstrated here. Edema formation is another important index of inflammation associated with endothelial permeability, demonstrating a typical weight gain of 30%at the end of 12 hours of negative pressure ventilation ex-situ lung perfusion. The perfusate acts as a surrogate indicator of overall lung status.Therefore, blood gas analysis of the perfusate provides extensive information on the metabolic state of the lungs. During the evaluation model of ESLP, which occurs at three, five, seven, nine, and 11 hours during a 12 hour run, an upward trend in left arterial partial pressure of oxygen is observed. It's very important to be as gentle as possible when surgically removing the lungs and to also move quickly and efficiently.The establishment of this device, protocol, and lab has led to a successful clinical trial and several discoveries that prolong the preservation and reconditioning of donor lungs.

normothermic-negative-pressure-ventilation-ex-situ-lung-perfusion

Research

JoVE Journal

Medicine

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

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 ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

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 ...

A Standardized Workflow for Hyperpolarized &lt;sup&gt;129&lt;/sup&gt;Xe MRI to Study Pulmonary Function

Acquiring Hyperpolarized 129Xe Magnetic Resonance Images of Lung Ventilation

Hyperpolarized 129Xe MRI comprises a unique array of structural and functional lung imaging techniques. Technique standardization across sites is increasingly important given the recent FDA approval of 129Xe as an MR contrast agent and as interest in 129Xe MRI increases among research and clinical institutions. Members of the 129Xe MRI Clinical Trials Consortium (Xe MRI CTC) have agreed upon best practices for each of the key aspects of the 129Xe MRI workflow, and these recommendations are summarized in a recent publication. This work provides practical information to develop an end-to-end workflow for collecting 129Xe MR images of lung ventilation according to the Xe MRI CTC recommendations. Preparation and administration of 129Xe for MR studies will be discussed and demonstrated, with specific topics including choice of appropriate gas volumes for entire studies and for individual MR scans, preparation&#160;and delivery of individual 129Xe doses, and best practices for monitoring subject safety and 129Xe tolerability during studies. Key MR technical considerations will also be covered, including pulse sequence types and optimized parameters, calibration of 129Xe flip angle and center frequency, and 129Xe MRI ventilation image analysis.

Author Spotlight: Standardization and Best Practices for Advancing Lung Imaging Using &lt;sup&gt;129&lt;/sup&gt;Xe MRI

Hyperpolarized 129Xe magnetic resonance imaging (MRI) is a method for studying regionally resolved aspects of pulmonary function. This work presents an end-to-end standardized workflow for hyperpolarized 129Xe MRI of lung ventilation, with specific attention to pulse sequence design, 129Xe dose preparation, scan workflow, and best practices for subject safety monitoring.

Hyperpolarized 129Xe MRI comprises a unique array of structural and functional lung imaging techniques. Technique standardization across sites is ...

Advancing Lung Transplantation with PolyhHb-Based Perfusion in Rat EVLP Models

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. However, new and exciting technology, such as ex vivo lung perfusion (EVLP), offers lung transplant providers extended assessment for marginal donor allografts. This dynamic assessment platform has led to an increase in lung transplantation and has allowed providers to use donors that were previously discarded, thus expanding the donor pool. Current perfusion techniques use cellular or acellular perfusates, and both have distinct advantages and disadvantages. Perfusion composition is critical to maintaining a homeostatic environment, providing adequate metabolic support, decreasing inflammation and cellular death, and ultimately improving organ function. Perfusion solutions must contain sufficient protein concentration to maintain appropriate oncotic pressure. However, current perfusion solutions often lead to fluid extravasation through the pulmonary endothelium, resulting in inadvertent pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as the proper protocols to test them in clinically relevant translational transplant models.

Author Spotlight: Utilizing Next-Generation Polymerized Human Hemoglobin for Improved Donor Lung Evaluation and Preservation in Rats

Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat ex vivo lung perfusion.