Name: Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation (Video) | JoVE Journal
Uploaded: 2023-09-29T14:20:00.000+00:00
Duration: 5 min 49 s
Description: Learn to establish a reproducible rat ex vivo lung perfusion model for investigating ischemia-reperfusion injury and inflammatory signaling in transplantation biology. This method enables cost-effective evaluation and preservation of donor lungs.

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Figure 1 was created with BioRender.com (confirmation of publication and licensing rights [Agreement: BT25KGSKWF]).

Animal experiments were performed in compliance with the Animal Use Protocol approved by the Animal Care Committee at the University Health Network. Male Lewis rats (255-330 g) were given ad libitum access to food and water. Then, they were maintained in a controlled environment (18-22 &#176;C) with a 12 h day-night cycle. See the Table of Materials for details related to all materials, solutions, and instruments used in this protocol.
1. Initialization of the&#160;&#160;ex vivo lung perfusion system
<ol>
	<li>Ensure that all transducers are connected to the isolated perfusion organ equipment and the data acquisition system (Figure 1). Then, open the data acquisition software.</li>
	<li>Before perfusion, fill the EVLP circuit with 150 mL of Steen solution supplemented with 1,000 USP units of sodium heparin, 50 mg of cefazolin, and 50 mg of methylprednisolone.</li>
	<li>Set the warm water bath to 20 &#176;C. Start circulating warm water to warm up the entire EVLP system.</li>
</ol>
2. Donor lung procurement procedure
<ol>
	<li>Anesthetize the rat with an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg), or according to local practice, and confirm adequate anesthesia by assessing the toe-pinch reflex. Throughout the procedure, apply ocular lubrication to prevent corneal drying and maintain the animal&#8217;s body temperature using a heating pad or an equivalent system until organ harvest.</li>
	<li>Perform orotracheal intubation (14 G intravenous catheter). After intubation, connect the tracheal tube to the small-animal ventilation system. Ventilate the rat with a tidal volume of 10 mL/kg, rate of 60 breaths/min, inspired oxygen fraction (FiO2) of 0.5, and positive end expiratory pressure (PEEP) of 2 cmH2O.</li>
	<li>Place the rat in the supine position.</li>
	<li>With the help of sterile&#160;scissors and forceps, enter the peritoneal cavity and carry the incision cranially, performing a median laparotomy and injecting sodium heparin (400 USP units) into the portal vein.</li>
	<li>Subsequently, enter the thoracic cavity through the xyphoid process. Carry out the sternotomy with the cranial resection of the sternum and cautious radial opening of the diaphragm, so as not to damage the lung.</li>
	<li>To retrieve the heart and lung block, make an incision in the inferior vena cava (IVC) and along the apex of the left heart.</li>
	<li>Using micro scissors, perform an anterior incision in the right ventricular outflow tract. Then, insert an 18 G intravenous catheter into the pulmonary trunk and flush the lungs with 20 mL of low potassium dextran (LPD) solution containing 10 &#181;g/mL of prostaglandin E1.</li>
	<li>Immediately after flushing, clamp the lower third of the trachea at the end of inspiration to preserve the lungs in an inflated state.</li>
	<li>Harvest the heart and lung block and place it in LPD solution for storage. 
	&#8203;NOTE: The lung graft can be stored at temperatures and different intervals of time according to the research purpose. Here, we show representative results of lungs that underwent varying lengths of cold ischemic time (CIT) on ice.</li>
</ol>
3. Ex vivo lung perfusion procedure
<ol>
	<li>Place a 0 silk ligature beneath the main pulmonary artery (PA) and around the ventricles, and pretied to facilitate cannulation.</li>
	<li>Connect the PA cannula in the inflow line of the EVLP system and start the peristaltic pump with 10% of maintenance flow, allowing the removal of any air in the PA cannula.</li>
	<li>Place and secure the inflow cannula into the main PA (OD: 2.0 mm/Head Diameter: 2.5 mm), followed by the drainage cannula from the apex of the heart into the left atrium (LA-Head Diameter: 4.0 mm) through the mitral valve for subsequent perfusion. Use forceps to dilate the mitral valve, facilitating the cannulation.</li>
	<li>Make a small hole in the trachea and insert the tracheal cannula (OD: 2.0 mm/L: 14 mm). Then, connect it to the ventilation line of the system.</li>
	<li>Connect the LA cannula to the outflow line of the system.</li>
	<li>NOTE: We set 10% of maintenance flow as the initiation of perfusion. To avoid the insertion of air bubbles in the lungs, ensure the bubble trap is filled.</li>
	<li>Gradually increase the flow rate perfusion to reach 20% of the cardiac output (CO). 
	NOTE: To perfuse both lungs, we used an estimated CO of 75 mL/min for 250 g rats10,11. The flow rate of perfusion was gradually increased to 1 h to reach the target flow rate according to our clinical EVLP protocol12. The perfusion parameters are summarized in Table 113.</li>
	<li>Remove the tracheal clamp 20 min after the initiation of perfusion and start lung ventilation followed by starting the flow of EVLP gas (8% CO2, 6% O2, 86% N2) to maintain inflow perfusate PCO2 of between 35 and 45 mmHg. 
	&#8203;NOTE: The ventilation parameters are summarized in Table 113.</li>
</ol>
4. Parameters and sample management
<ol>
	<li>Every hour during EVLP, record in real time the dynamic lung compliance and pulmonary vascular resistance (PVR). 
	NOTE: At 5 min before the physiological assessments, the lungs should be expanded to an inflation pressure.</li>
	<li>Take perfusate samples from the sample port every hour and flash freeze in liquid nitrogen for further analyses. 
	NOTE: In addition, we perform a recruitment maneuver (up to 20 cmH2O) 5 min before assessment, after which we take perfusates for blood gas analyses to measure pH, PCO2, PO2, electrolytes, glucose, and lactate.</li>
	<li>Stop perfusion and clamp the trachea to maintain the lungs in an inflated state. Subsequently, isolate lung samples and flash freeze them in liquid nitrogen or place them in fixing solutions for further studies. 
	NOTE: Here, the lungs were perfused for 4 h.</li>
</ol>

<ol><li>Chambers, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+International+Thoracic+Organ+Transplant+Registry+of+the+International+Society+for+Heart+and+Lung+Transplantation%3A+Thirty-eighth+adult+lung+transplantation+report+%E2%80%94+2021%3B+Focus+on+recipient+characteristics%5BTitle%5D)+AND+%22The+Journal+of+Heart+and+Lung+Transplantation%22%5BJournal%5D)">The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-eighth adult lung transplantation report — 2021; Focus on recipient characteristics.</a> The Journal of Heart and Lung Transplantation. 40 (10), 1060-1072 (2021).</li>
<li>Meyer, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+lung+transplantation%5BTitle%5D)+AND+%22F1000Research%22%5BJournal%5D)">Recent advances in lung transplantation.</a> F1000Research. 7, 1684(2018).</li>
<li>Valapour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OPTN%2FSRTR+2018+Annual+Data+Report%3A+Lung%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">OPTN/SRTR 2018 Annual Data Report: Lung.</a> American Journal of Transplantation. 20, 427-508 (2020).</li>
<li>Chaney, J., Suzuki, Y., Cantu, E., van Berkel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+donor+selection+criteria%5BTitle%5D)+AND+%22Journal+of+thoracic+disease%22%5BJournal%5D)">Lung donor selection criteria.</a> Journal of thoracic disease. 6 (8), 1032-1038 (2014).</li>
<li>Watanabe, T., Cypel, M., Keshavjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Journal+of+Thoracic+Disease%22%5BJournal%5D)">Ex vivo lung perfusion.</a> Journal of Thoracic Disease. 13 (11), 6602-6617 (2021).</li>
<li>Saddoughi, S. A., Cypel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+the+lung+donor+pool%5BTitle%5D)+AND+%22Clinics+in+Chest+Medicine%22%5BJournal%5D)">Expanding the lung donor pool.</a> Clinics in Chest Medicine. 44 (1), 77-83 (2023).</li>
<li>Wang, A., Ali, A., Keshavjee, S., Liu, M., Cypel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion+for+donor+lung+assessment+and+repair%3A+a+review+of+translational+interspecies+models%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Ex vivo lung perfusion for donor lung assessment and repair: a review of translational interspecies models.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 319 (6), L932-L940 (2020).</li>
<li>Noda, K., Philips, B. J., Atale, N., Sanchez, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+protection+in+lung+grafts+through+heparanase+inhibition+during+ex+vivo+lung+perfusion+in+rats%5BTitle%5D)+AND+%22The+Journal+of+Heart+and+Lung+Transplantation%22%5BJournal%5D)">Endothelial protection in lung grafts through heparanase inhibition during ex vivo lung perfusion in rats.</a> The Journal of Heart and Lung Transplantation. 42 (6), 697-706 (2023).</li>
<li>Nelson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+of+isolated+ex+vivo+lung+perfusion+in+a+rat+model%3A+lessons+learned+from+developing+a+rat+EVLP+program%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Method of isolated ex vivo lung perfusion in a rat model: lessons learned from developing a rat EVLP program.</a> Journal of Visualized Experiments. (96), (2015).</li>
<li>Malik, A. B., Kaplan, J. E., Saba, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reference+sample+method+for+cardiac+output+and+regional+blood+flow+determinations+in+the+rat%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">Reference sample method for cardiac output and regional blood flow determinations in the rat.</a> Journal of Applied Physiology. 40 (3), 472-475 (1976).</li>
<li>Noda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+prolonged+ex+vivo+lung+perfusion+for+graft+preservation+in+rats%5BTitle%5D)+AND+%22European+Journal+of+Cardio-Thoracic+Surgery%22%5BJournal%5D)">Successful prolonged ex vivo lung perfusion for graft preservation in rats.</a> European Journal of Cardio-Thoracic Surgery. 45 (3), e54-e60 (2014).</li>
<li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technique+for+prolonged+normothermic+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22The+Journal+of+Heart+and+Lung+Transplantation%22%5BJournal%5D)">Technique for prolonged normothermic ex vivo lung perfusion.</a> The Journal of Heart and Lung Transplantation. 27 (12), 1319-1325 (2008).</li>
<li>Ohsumi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+translational+rat+ex+vivo+lung+perfusion+experimentation%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">A method for translational rat ex vivo lung perfusion experimentation.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 319 (1), L61-L70 (2020).</li>
<li>Soares, P. R. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+between+perfadex+and+locally+manufactured+low-potassium+dextran+solution+for+pulmonary+preservation+in+an+ex+vivo+isolated+lung+perfusion+model%5BTitle%5D)+AND+%22Transplantation+Proceedings%22%5BJournal%5D)">Comparison between perfadex and locally manufactured low-potassium dextran solution for pulmonary preservation in an ex vivo isolated lung perfusion model.</a> Transplantation Proceedings. 43 (1), 84-88 (2011).</li>
<li>Pêgo-Fernandes, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+model+of+isolated+lung+perfusion+in+rats%3A+first+Brazilian+experience+using+the+IL-2+isolated+perfused+rat+or+guinea+pig+lung+system%5BTitle%5D)+AND+%22Transplantation+Proceedings%22%5BJournal%5D)">Experimental model of isolated lung perfusion in rats: first Brazilian experience using the IL-2 isolated perfused rat or guinea pig lung system.</a> Transplantation Proceedings. 42 (2), 444-447 (2010).</li>
<li>Yamanashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+of+donor+mononuclear+phagocytes+with+clodronate-liposome+during+ex+lung+perfusion+attenuates+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Reduction of donor mononuclear phagocytes with clodronate-liposome during ex lung perfusion attenuates ischemia-reperfusion injury.</a> The Journal of Thoracic and Cardiovascular Surgery. 165 (4), e181-e203 (2023).</li>
</ol>

MC is a shareholder of Traferox Technologies Inc and consultant for Lung Bioengineering Inc. MC receives research support from Beyond Air Inc. and Synklino. The authors declare that there have been no competing interests that could have influenced the outcome reported in this paper.

This study described the necessary steps for establishing a rat EVLP protocol. Here, we show that donor lungs can be perfused for 4 h after cold static preservation of up to 18 h at 4 &#176;C. This was demonstrated by assessing lung compliance, pulmonary vascular resistance, perfusate glucose/lactate concentration, and P/F ratio.
The rise of the EVLP platform as an important tool in the evaluation of lung function and for graft reconditioning has motivated several groups to develop a rat EVLP ...

Lung transplantation has been recognized as a therapeutic strategy for end-stage diseases with improvements in surgical methods and immunosuppression during the last decades. Despite the high demand, the number of acceptable deceased lung donors is lower than for other solid organs, converging to the lower number of lung transplants performed1,2,3. To address the donor pool shortage, the medical community has expanded the criteria for lung donation, turning organs previously considered nonviable into potential organs for transplantation. However, the extended criteria require different efforts for a better understanding and intervention in view of the possible systemic consequences originating from the donated organ. Ex vivo lung perfusion (EVLP) emerged as a technique that provides normothermic lung preservation, assessment of lung functions, and reconditioning of lungs previously considered unfeasible for the donation process4,5,6.
Given the growing number of lung transplants since the establishment of EVLP in large transplant centers, lung preservation and repair strategies have been increasingly investigated. In this sense, faced with ethical and logistical issues, as well as in the study of immunological factors associated with lung transplantation, the development of rodent EVLP models has become important, given their reliability, the possibility of genetic manipulation, and lower costs7,8,9. Here, we describe the necessary steps to establish a rat EVLP model and show the inflammatory profile associated with the perfused lungs.

<table><tbody><tr><td>&amp;nbsp;14 G intravenous catheter</td><td>BD</td><td>381167</td><td>&amp;nbsp;Orotracheal intubation</td></tr><tr><td>16 G intravenous catheter</td><td>BD</td><td>381157</td><td>Catheter to flush the lung</td></tr><tr><td>2-0 Suture Sofsilk</td><td>Covidien</td><td>S-305</td><td>&amp;nbsp;Tracheal tube fixation</td></tr><tr><td>3-0 Suture Sofsilk</td><td>Covidien</td><td>S303</td><td>&amp;nbsp;Fixation of arterial and atrial cannulas</td></tr><tr><td>IPL-2 Core Isolated Perfused Lung System for Rat</td><td>Harvard Apparatus</td><td>734276</td><td>Ex Vivo Lung Perfusion (EVLP) system</td></tr><tr><td>Ketamine (Narketan)</td><td>Vetoquinol</td><td>440894</td><td>Sedation/anesthesia</td></tr><tr><td>Large Pulmonary Artery Cannula&amp;nbsp;</td><td>Harvard Apparatus</td><td>73-0711</td><td></td></tr><tr><td>Left Atrial Cannula&amp;nbsp;</td><td>Harvard Apparatus</td><td>73-0712</td><td></td></tr><tr><td>Low potassium dextran glucose solution (Perfadex)</td><td>XVIVO</td><td>19811</td><td>Preservation solution</td></tr><tr><td>Methylprednisolone sodium succinate (SOLU-MEDROL)</td><td>Pfizer</td><td>14705</td><td>Anti-inflammatory</td></tr><tr><td>Prostaglandin E1 (Prostin VR)</td><td>Pfizer</td><td>RX297945</td><td></td></tr><tr><td>Small Animal Ventilator (Model 683)</td><td>Harvard Apparatus</td><td>55-0000</td><td></td></tr><tr><td>Sodium heparin</td><td>Leo Pharma</td><td>453811</td><td></td></tr><tr><td>Steen Solution</td><td>XVIVO</td><td>19004</td><td>Buffered extracellular solution to perfuse lungs during EVLP</td></tr><tr><td>Sugita Aneur clip curv</td><td>Mizuho</td><td>07-940-86</td><td>Tracheal clamp</td></tr><tr><td>Tracheal Cannula&amp;nbsp;</td><td>Harvard Apparatus</td><td>73-3384</td><td></td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer Healthcare&amp;nbsp;</td><td>2169592</td><td>Sedation/anesthesia</td></tr></tbody></table>

establishment of an ex vivo lung perfusion rat model for translational insights in lung transplantation

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem of a low number of lungs considered viable for the donation process. In recent decades, however, this scenario has been positively changed, given the development of ex vivo lung perfusion (EVLP) as a strategy for evaluating and reconditioning marginal lungs. The establishment of EVLP in large transplant centers has favored an increase in the number of lung transplants, both by increasing the diagnostic accuracy of lung function and by constituting an effective platform for the reconditioning of lung grafts. In this context, faced with ethical and logistical issues, as well as in the study of immunological factors associated with lung transplantation, the development of rodent EVLP models has become important, given their reliability, the possibility of genetic manipulation, and lower costs. This paper describes a protocol for establishing a rat EVLP model and shows the inflammatory profile associated with the perfused lungs. This will help propagate knowledge about the rat EVLP model, promoting our understanding of the biological responses associated with that revolutionary technique.

All lungs with CIT ranging from 20 min to 18 h could be perfused for 4 h (Figure 2)13. Compliance was stable in most groups with the exception of 18 h CIT, which gradually decreased over the 4 h perfusion period (Figure 2A). Despite this, no significant differences in vascular resistance, lung graft oxygenation, and glucose levels were observed for the groups (Figure 2B-D). L...

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem ...

This study established a haproducible rat EVOP model to investigate ischemia hyperfusion injury, inflammatory signally, and donor graft function, aiming to evaluate, preserve and repair lungs ex vivo for improved transplantation outcomes. The most recent developments include using EVOP to access, recondition, and even treat donor lungs before transplantation. Our protocol addresses the need for cost-effective reproducible models for translational lung transplant research.Our findings provide a reliable small MOEVOP model to test new therapies and improve donor lung evaluation and preservation. To begin, check that all transducers are connected to the isolated perfusion organ equipment and the data acquisition system, then launch the data acquisition software. Prior to perfusion, fill the ex vivo lung perfusion or EVLP circuit with 150 milliliters of supplemented steam solution.Set the warm water bath to 20 degrees Celsius and circulate the Perfusate solution to warm up the EVLP system. Orotracheal intubate an anesthetized rat. Next, connect the tracheal tube to the small animal ventilation system to ventilate the rat.Place the rat in a supine position. With a pair of scissors and forceps. Enter the peritoneal cavity and carry the incision cranially to perform a median laparotomy.Now inject sodium heparin into the inferior vena cava. Enter the thoracic cavity through the xiphoid process and cautiously open the diaphragm radially without damaging the lung. Then cranially resect the sternum.To retrieve the heart and lung block, make an incision on the inferior vena cava along the apex of the left heart. With a pair of micro scissors, make an anterior incision in the right ventricular outflow tract. Then insert an 18 gauge intravenous catheter into the pulmonary trunk and flush the lungs with 20 milliliters of low potassium dextran or LPD solution.Immediately after flushing, clamp the lower third of the trachea at the end of inspiration to preserve the lungs in an inflated state. Then harvest the lung and place it in LPD solution for storage. To begin lung perfusion, first, secure a pre-tied zero silk ligature underneath the main pulmonary artery and around the ventricles.Connect the pulmonary artery cannula to the inflow line of the EVLP system. Start the peristaltic pump at 10%of maintenance flow, ensuring the removal of any air in the cannula. Secure the inflow cannula into the main pulmonary artery.Then insert the drainage cannula from the apex of the heart into the left atrium. Connect the left atrial cannula to the outflow line of the system. Now make a small hole in the trachea and insert the tracheal cannula.Connect the tracheal cannula to the ventilation line of the system. Gradually increase the perfusion flow rate to attain 20%of the cardiac output. Release the tracheal clamp 20 minutes after perfusion initiation, then start lung ventilation followed by EVLP gas flow.Record the dynamic lung compliance and pulmonary vascular resistance every hour during EVLP. Take Perfusate samples from the sample port every hour. Flash, freeze the samples in liquid nitrogen for further analysis.After ceasing perfusion, clamp the trachea to maintain the lungs in an inflated state. Then isolate lung samples and tubes, flash freeze the samples in liquid nitrogen. All lungs with CIT ranging from 20 minutes to 18 hours could be perfused for four hours.Compliance gradually decreased in the 18 hour CIT group over the perfusion period. No significant differences in vascular resistance lung graft oxygenation, and glucose levels were observed for the groups. Lactate values tended to increase over time with higher values observed at longer CIT.All groups showed similar values of perfusion electrolytes, and level of edema formation with the 24 hour group showing severe edema.

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