Support was provided, in part, by a Merit Review Award (101 BX003482) from the U.S. Department of Veteran Affairs Biomedical Laboratory R&#38;D Service, a NIH grant (5R01 HL123227), a Transformative Project Award (962204) from the American Heart Association, and by institutional funds awarded to Dr. Riess. Dr. Balzer received unrelated funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number BA 6287/1-1. The authors would like to thank Matthew D. Olsen, Chun Zhou, Zhu Li, and Rebecca C. Riess for their valuable contributions to the study.

The in vivo portion of the experiments (from general anesthesia to euthanasia) requires prior approval by the respective Institutional Animal Care and Use Committee (IACUC). All procedures described herein were approved (protocol number M1700168) by the IACUC at Vanderbilt University Medical Center, Nashville, Tennessee, and were performed in compliance with the ARRIVE guidelines14. Prior to experimentation, all the rats were housed in the institute&#39;s animal care facility, with free access to water and food. Including different studies outside the purview of this manuscript, we have used a total of 148 male Sprague Dawley rats, 7-10 weeks old, with a weight between 250 g and 400 g so far.
1. Apparatus construction
NOTE: All parts, including respective manufacturers, are listed in the Table of Materials.
<ol>
	<li>To hold each piece of equipment in place, construct a custom-built lattice to allow for easy configuration and incorporation of the new devices (Figure 1). Attach aluminum poles (1 to 2 ft in length, 1 cm in diameter, available at any hardware store) to each other using cross clamps to create a 3D lattice, and place on a plastic tray (30 in x 21 in) to prevent spillage of fluids.</li>
	<li>Mount pressure transducers at an equal height to the lungs and connect them to the lines for the pulmonary artery (PA), pulmonary vein (PV), and to the ventilation tube. 
	NOTE: These transducers transmit data to their respective bioamplifiers connected to the data acquisition system (DAQ) and its software.</li>
	<li>Rather than using commercially available cannulas, craft custom cannulas (Figure 2) from segments of 2 mm wide, hard plastic tubing that are flared at the end, using an open flame to allow for the secure tying-off of sutures. Bend them into a U-shape to reduce stress on the lungs during hanging and to fit in the lung chamber.</li>
	<li>To make the chamber the lungs are to be ventilated and perfused in, place a 1,000 mL beaker inside a 1,500 mL beaker with a water bath between the two and inside the 1,000 mL beaker, creating a double boiler. Place these beakers on a heating plate set to 48 &#176;C, creating a chamber for the lungs that is both humid and resistant to fluctuations in temperature.</li>
	<li>Keep the buffer for the experiment in a 150 mL volumetric flask placed on a heating plate set to 37 &#176;C. Use a magnetic stirring bar to circulate the buffer inside the beaker. Set the meniscus of the buffer at a height such that it is 4 cm above the lung, to create an innate 4 cmH2O of pressure on the PV. During the surgery, ensure that the animal&#39;s lungs are at the same height as the buffer to reduce hydrostatic edema formation. 
	NOTE: Using a flask minimizes the amount of surface area in contact with room air, which minimizes gas diffusion.</li>
	<li>Use a roller pump to move the buffer through a circuit consisting of a heating coil and an air trap prior to perfusing the lung. Recycle the effluent from the PV back into the volumetric flask. Connect both the heating coil and air trap to a circulating water bath also set to 37 &#176;C. Adjust the temperature of the water bath according to the pump speed, so the perfusate has a constant temperature of 37 &#176;C.</li>
</ol>
2. Procedure
<ol>
	<li>Prior to the start of the experiment, prepare the setup.
	<ol>
		<li>Ensure that the software is running (see below) and properly collecting data.</li>
		<li>Calibrate all pressure transducers daily to ensure they are not drifting.</li>
		<li>Prepare the buffer (see Table 1 for ingredients for a physiological saline solution with 4% bovine serum albumin [BSA]) and ensure that the pH is at 7.4, using HCl to adjust accordingly. Set up the perfusion system with warmed buffer (Table 1) circulating throughout, ensuring that no air bubbles are present. Add BSA at least 30 min prior to the start of the experiment to give it sufficient time to dissolve. In the absence of an oxygenator, bubble gas into the perfusion buffer prior to the addition of BSA with a gas composition of 65% N2, 30% O2, and 5% CO2 to mimic the CO2 levels of in vivo lungs. This prevents the solution from foaming once BSA is added.</li>
		<li>Prepare the operating area with all necessary surgical tools, sutures, and tape. Incline the operating board at a 15&#176; angle, so that the anesthetized rat can be positioned with its head elevated above the rest of the body, and the trachea and heart-lung block can be manipulated easily.</li>
		<li>Wearing proper personal protective equipment, weigh the rat and give an intraperitoneal injection of pentobarbital (65 mg/kg-1). After 10 min, use a toe pinch to ensure that the surgical plane of general anesthesia has been reached. Administer more anesthetic if necessary.</li>
	</ol>
	</li>
	<li>Transfer the rat to the operating area and fixate it to the operating board by taping its front legs separately, followed by the hind legs together, making sure that the front legs are taped loose enough to allow a pain reflex to be visualized should anesthesia not be deep enough (Figure 3A).</li>
	<li>Secure the head of the rat by taping the mouth with a long, thin strip behind the front teeth, without restricting the tongue or airflow for the still spontaneously breathing rat (Figure 3B).</li>
	<li>Perform a tracheostomy by pinching the skin above the trachea using forceps and cutting with surgical scissors. Using curved forceps, bluntly dissect the muscle and tissue to reach the trachea. Ensure that there is no bleeding during this step.
	<ol>
		<li>Pass curved forceps underneath the trachea and open them to allow room to pass 3-0 sutures underneath that can then be pre-tied into a box knot (Figure 3B). Make a small incision between the cartilage rings of the trachea and insert the tracheal cannula (modified 18 G needle with 1 mm diameter tubing glued around the tip to create a notch). Tie off the suture, ensuring that no air can escape from the tracheal incision and that the cannula is not putting any strain on the trachea.</li>
	</ol>
	</li>
	<li>Set the ventilator to run with a gas mixture of 30% O2, 5% CO2, and 65% N2, with a tidal volume (VT) of 10 mL/kg, a positive end expiratory pressure (PEEP) of 0 cmH2O, and a rate of 60 breaths/min.</li>
	<li>Once the tracheal cannula is secured with the suture, begin ventilating the rat with the above settings. 
	NOTE: The surgical method used was adapted from Nelson et al.9 and is described step-by-step.</li>
	<li>Remove the fur from the abdomen of the rat using large surgical scissors and forceps. 
	NOTE: Hair removal ointments can also be used prior to the start of the experiment.</li>
	<li>While holding the xiphoid process with forceps, make a small horizontal incision below the ribs, ensuring that the diaphragm stays intact. Once the internal organs can safely be visualized, widen the horizontal cut to expose the entire diaphragm.</li>
	<li>Taking extreme care to avoid puncturing the lungs, inject heparin (3,000 U/kg-1) into the inferior vena cava (IVC) with a 22 G syringe.</li>
	<li>Again, hold the xiphoid process with forceps and cut cranially along the sternum while constantly visualizing the lungs to prevent cutting them.</li>
	<li>To properly see the heart-lung block, spread the rib cage apart using two large forceps and be sure to avoid breaking the ribs, which can result in a sharp bone fragment puncturing the lung (Figure 3C). 
	NOTE: Once the rib cage has been opened, use a PEEP of 2-3 cmH2O, set by a water lock attached to the expiratory limb of the ventilator, to avoid atelectasis.</li>
	<li>At this time, cut the IVC to euthanize the rat by exsanguination. Carefully ensure that at least 1 min has passed since the injection of heparin (see step 2.9) to give it enough time to circulate and prevent microclots in the lungs.</li>
	<li>When placing the perfusion cannulas, constantly check the pressures to make sure that no sudden changes occur. 
	NOTE: A sudden spike in pressure while the pump is running at a constant speed indicates blockage formation, while a sudden decrease can indicate a leakage.</li>
	<li>Trim any excess thymus to allow for easier visualization of the pulmonary vasculature. Locate the PA, pass small, curved forceps underneath, and again pass a 3-0 suture underneath and pre-tie into a box knot.</li>
	<li>Make a small incision into the right ventricle of the heart and insert the PA cannula (made from 2 mm diameter plastic tubing flared at the end using an open flame to allow for sutures to be tied around), being gentle to avoid rupturing the adjacent artery. Secure the cannula using the suture and perfuse, starting at 1.5 mL/min-1 (Figure 3D).</li>
	<li>Immediately excise the apex of the heart to avoid a pressure buildup in the lungs. Using small, curved forceps, rupture the mitral valve and visually ensure that the forceps are able to enter the left atrium unrestricted.</li>
	<li>Place a pre-tied 3-0 suture around the heart below the atrium (Figure 3E).</li>
	<li>Insert the PV cannula (similar construction to the PA cannula) into the left atrium and ensure that buffer can flow out of it prior to tying off the suture. 
	NOTE: It is critical that the PV cannula is placed past the mitral valve to ensure that it can be properly tied off, while not being placed too deep as to injure the PV or encumber flow (Figure 3E). Take extra care with tying the suture, as tying it too loose results in lost flow, while tying it too tight can damage the heart, weakening the placement of the cannula and potentially causing leakages.</li>
	<li>Trim excess tissue between the thoracic cavity and the tracheostomy incision using blunt-tipped scissors to avoid damaging the trachea. Ensure that the entire trachea below the tracheal cannula and the entire heart-lung block are visible.</li>
	<li>Remove the heart-lung block and trachea by holding the tracheal cannula and excising connective tissue behind the trachea with a pair of curved, blunt-tipped scissors, leaving the esophagus attached for extra structural support (Figure 3F).</li>
	<li>Gradually increase the flow rate to a maximum flow of 40 mL/kg-1/min-1. Allow the first 50 mL of buffer to flow out of the system to remove any inflammatory cytokines that may be released (Figure 3G).</li>
	<li>Gently move the isolated lungs to the double boiler chamber created with the two beakers. Make sure that the PA, PV, or tracheal cannula do not become twisted at any point while moving the lungs or hanging them. 
	&#8203;NOTE: Prevention of atelectasis during lung retrieval and connection to the setup is important, especially when EVLP is used for already damaged lungs or when interventions prior to EVLP are planned. This can be achieved by, for example, a small clip on the trachea after inspiration before transfer to the ventilator15.</li>
</ol>
3. Data acquisition
<ol>
	<li>For data collection, use any commercially available analog-to-digital converter and DAQ system.</li>
	<li>Pay attention that the sampling frequency is sufficient for the given purpose (200 Hz here) and that all relevant dependent parameters from bioamplifiers and other input devices are collected concurrently (airway pressure, PA, and PV pressure chosen for this protocol)16.</li>
	<li>Record independent parameters (e.g., perfusion speed, ventilation rate, ventilated gas composition, buffer electrolyte composition, and lung weight).</li>
</ol>

<ol><li>Uhlig, S., Taylor, A. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aUhlig%2C+S+%22Methods+in+Pulmonary+Research%22">Methods in Pulmonary Research</a>. , Birkhäuser. (1998).</li>
<li>Ghaidan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ten+year+follow-up+of+lung+transplantations+using+initially+rejected+donor+lungs+after+reconditioning+using+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Journal+of+Cardiothoracic+Surgery%22%5BJournal%5D)">Ten year follow-up of lung transplantations using initially rejected donor lungs after reconditioning using ex vivo lung perfusion.</a> Journal of Cardiothoracic Surgery. 14 (1), 125(2019).</li>
<li>Stone, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+perfusion+with+adenosine+A2A+receptor+agonist+enhances+rehabilitation+of+murine+donor+lungs+after+circulatory+death%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Ex vivo perfusion with adenosine A2A receptor agonist enhances rehabilitation of murine donor lungs after circulatory death.</a> Transplantation. 99 (12), 2494-2503 (2015).</li>
<li>Valenza, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+consumption+of+glucose+during+ex+vivo+lung+perfusion+correlates+with+lung+edema%5BTitle%5D)+AND+%22Transplantation+Proceedings%22%5BJournal%5D)">The consumption of glucose during ex vivo lung perfusion correlates with lung edema.</a> Transplantation Proceedings. 43 (4), 993-996 (2011).</li>
<li>Sayner, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paradoxical+cAMP-induced+lung+endothelial+hyperpermeability+revealed+by+Pseudomonas+aeruginosa+ExoY%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by Pseudomonas aeruginosa ExoY.</a> Circulation Research. 95 (2), 196-203 (2004).</li>
<li>McAuley, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+grade+allogeneic+human+mesenchymal+stem+cells+restore+alveolar+fluid+clearance+in+human+lungs+rejected+for+transplantation%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 306 (9), L809-L815 (2014).</li>
<li>Pego-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>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>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), e52309(2015).</li>
<li>Bassani, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion+in+the+rat%3A+detailed+procedure+and+videos%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Ex vivo lung perfusion in the rat: detailed procedure and videos.</a> PLoS One. 11 (12), e0167898(2016).</li>
<li>Watson, K. E., Segal, G. S., Conhaim, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Negative+pressure+ventilation+enhances+acinar+perfusion+in+isolated+rat+lungs%5BTitle%5D)+AND+%22Pulmonary+Circulation%22%5BJournal%5D)">Negative pressure ventilation enhances acinar perfusion in isolated rat lungs.</a> Pulmonary Circulation. 8 (1), 2045893217753596(2018).</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>Hozain, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiday+maintenance+of+extracorporeal+lungs+using+cross-circulation+with+conscious+swine%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Multiday maintenance of extracorporeal lungs using cross-circulation with conscious swine.</a> The Journal of Thoracic and Cardiovascular Surgery. 159 (4), 1640-1653 (2020).</li>
<li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+bioscience+research+reporting%3A+the+ARRIVE+guidelines+for+reporting+animal+research%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.</a> PLoS Biology. 8 (6), e1000412(2010).</li>
<li>van Zanden, J. E., Leuvenink, H. G. D., Verschuuren, E. A. M., Erasmus, M. E., Hottenrott, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+translational+rat+model+for+ex+vivo+lung+perfusion+of+pre-injured+lungs+after+brain+death%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A translational rat model for ex vivo lung perfusion of pre-injured lungs after brain death.</a> PLoS One. 16 (12), e0260705(2021).</li>
<li>Cleveland, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implementation+of+LabVIEW+as+a+virtual+instrument+in+a+cost-effective+isolated+lung+setup%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Implementation of LabVIEW as a virtual instrument in a cost-effective isolated lung setup.</a> The FASEB Journal. 33 (1), 846(2019).</li>
<li>Jamieson, S. W., Stinson, E. B., Oyer, P. E., Baldwin, J. C., Shumway, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Operative+technique+for+heart-lung+transplantation%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Operative technique for heart-lung transplantation.</a> The Journal of Thoracic and Cardiovascular Surgery. 87 (6), 930-935 (1984).</li>
<li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+of+nitric+oxide+synthase+expression+and+activity+during+rat+lung+transplantation%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Alterations of nitric oxide synthase expression and activity during rat lung transplantation.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 278 (5), L1071-L1081 (2000).</li>
<li>Riess, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+measurements+in+blood-free+balanced+salt+solutions+with+three+devices+%28i-STAT%C2%AE%2C+glucose+test+strips+and+ACCU-CHEK%C2%AE+Aviva%29%5BTitle%5D)+AND+%22Anesthesia+and+Analgesia%22%5BJournal%5D)">Glucose measurements in blood-free balanced salt solutions with three devices (i-STAT®, glucose test strips and ACCU-CHEK® Aviva).</a> Anesthesia and Analgesia. 128 (5), 924(2019).</li>
<li>Menezes, A. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+Celsior+and+Perfadex+lung+preservation+solutions+in+rat+lungs+subjected+to+6+and+12+hours+of+ischemia+using+an+ex-vivo+lung+perfusion+system%5BTitle%5D)+AND+%22Clinics%22%5BJournal%5D)">Comparison of Celsior and Perfadex lung preservation solutions in rat lungs subjected to 6 and 12 hours of ischemia using an ex-vivo lung perfusion system.</a> Clinics. 67 (11), 1309-1314 (2012).</li>
<li>Riess, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrolyte+measurements+in+blood-free+balanced+salt+solutions+-+comparison+of+i-STAT%C2%AE+with+ABL80%5BTitle%5D)+AND+%22Anesthesia+and+Analgesia%22%5BJournal%5D)">Electrolyte measurements in blood-free balanced salt solutions - comparison of i-STAT® with ABL80.</a> Anesthesia and Analgesia. 130 (5), 984(2020).</li>
</ol>

The authors declare no conflicts of interest, financial or otherwise.

More than 100 experiments have been successfully performed in our lab using this setup. The modular design of this customized setup gave great flexibility to potential changes in experimental requirements. While other setups utilize a deoxygenator18 to mimic constant oxygen consumption and CO2 production by end organs, this simplified model did not employ this feature, due to the focus on studying the effects of different gas compositions on pulmonary vascular tone. This approach, in wh...

Ex vivo lung perfusion (EVLP) techniques1 have seen a rise in usage in the past decade as a means of studying lung transplantations2, ischemia/reperfusion3, lung metabolism4, and immune responses5. Isolated, but intact, ventilated and perfused lungs offer the critically important ability to directly assess the response of the lungs, including the pulmonary vasculature, to potential interventions and/or therapeutics without potential confounders, such as neuronal and hormonal input or changing hemodynamics in vivo. At the same time, they maintain the physiological interplay of ventilation and perfusion, in contrast to in vitro conditions. A proposal looking at immune responses in lungs5, for example, needs the same quality of data as a study focused on increasing the donor pool size6 for lung transplantations. EVLP can be used across a variety of species, including mice3, rats7,8,9,10,11,12, pigs13, and humans2. Therefore, it is necessary to establish a model that can produce reliable data from a variety of different experimental parameters. Clinical relevance will be generated in subsequent studies using the EVLP model as a tool.
While commercial setups are available for purchase for most species, they can often be cost-prohibitive and confine researchers to a specific brand of equipment and proprietary software. Any deviation from the out-of-the-box setup (e.g., going from one species to another) requires foresight and working around the provided setup, which may prove to be difficult or impossible. In the following, a DIY (do it yourself) setup for rat isolated lungs that is both modular and cost-effective, as well as the surgical procedure for isolating the lungs, are described.

<table><tbody><tr><td>1,000 mL Glass Beaker</td><td>Pyrex, Chicago, IL</td><td></td><td></td></tr><tr><td>1,500 mL Glass Beaker</td><td>Pyrex, Chicago, IL</td><td></td><td></td></tr><tr><td>Air Trap Compliance Chamber</td><td>Radnoti</td><td>130149</td><td></td></tr><tr><td>Bioamplifiers</td><td>CWE Inc</td><td>BPM-832</td><td></td></tr><tr><td>Clamps</td><td>Fisher Scientific</td><td>S02626</td><td></td></tr><tr><td>DAQ (Data Acquisition)</td><td>National Instruments, Austin, TX</td><td>NI USB-6343</td><td></td></tr><tr><td>Gas Mixer</td><td>CWE Inc, Ardmore, PA</td><td>GSM-4</td><td></td></tr><tr><td>Heating Coil</td><td>Radnoti, Covina, CA</td><td>158822</td><td></td></tr><tr><td>Heating Plate</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>11-100-49SH</td><td></td></tr><tr><td>Heparin</td><td>Pfizer</td><td>W63422</td><td></td></tr><tr><td>LabVIEW Full Development System 2014</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Pentobarbital</td><td>Diamondback Drugs</td><td>G2270-0235-50</td><td></td></tr><tr><td>pH700 Probe</td><td>OAKTON, Vernon Hills, IL&amp;nbsp;</td><td>EW-35419-10</td><td></td></tr><tr><td>Polystat Water Bath</td><td>Cole-Parmer</td><td>EW-12121-02</td><td></td></tr><tr><td>Rodent Ventilator</td><td>Harvard Apparatus, Holliston, MA</td><td>Model 683</td><td></td></tr><tr><td>Roller Pump</td><td>Cole-Parmer, Wertheim, Germany&amp;nbsp;</td><td>Ismatec REGLO Digital MS 2/8</td><td></td></tr><tr><td>Sprague Dawley Rat</td><td>Charles River, Wilmington, MA</td><td>Strain code 001</td><td></td></tr><tr><td>VetScan i-STAT</td><td>Abraxis, Chicago, IL</td><td>i-STAT 1</td><td></td></tr></tbody></table>

design and implementation of a rat ex vivo lung perfusion model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro models. Laboratories wishing to use isolated lungs need to be aware of important steps and inherent challenges to establish a setup that is affordable, reliable, and that can be easily adapted to fit the topic of interest. This paper describes a DIY (do it yourself) model for ex vivo rat lung ventilation and perfusion to study drug and gas effects on pulmonary vascular tone, independent of changes in cardiac output. Creating this model includes a) the design and construction of the apparatus, and b) the lung isolation procedure. This model results in a setup that is more cost-effective than commercial alternatives and yet modular enough to adapt to changes in specific research questions. Various obstacles had to be resolved to ensure a consistent model that is capable of being used for a variety of different research topics. Once established, this model has proven to be highly adaptable to different questions and can easily be altered for different fields of study.

Following 10 min of stabilization and baseline readings, we randomized a first set of 10 male Sprague Dawley rats into five small groups: global no-flow ischemia for 5, 7.5, 8, 9, or 10 min (n = 2 per group) followed by reperfusion; these limited preliminary dose-finding experiments were conducted to identify the longest possible ischemia time to still allow sufficient ventilation and reperfusion before the eventual development of a precipitous and irreversible increase in airway pressure and edema formation. Importantly...

Watch this Scientific Journal Video about Design and Implementation of a Rat Ex Vivo Lung Perfusion Model at JoVE.com

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro ...

design-and-implementation-of-a-rat-ex-vivo-lung-perfusion-model

Research

JoVE Journal

Biology

744 Views.  Vanderbilt University Medical Center. Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs. 

Video: Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

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 ...

Medicine

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, ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

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.

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

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 ...

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.

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. ...

Evaluation of Apoptosis and Lung Injury in DCD Donor Lungs Preserved by EVLP

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after cardiac death (DCD). However, a critical challenge remains in maximizing the number of viable lungs that meet stringent criteria for clinical use. Ex vivo lung perfusion (EVLP) emerges as a promising technology to evaluate and potentially improve the function of donated lungs. This paper describes the development of a cost-effective and reproducible small animal experimental platform specifically designed for EVLP research. This platform offers a high degree of customization, enabling researchers to tailor experimental parameters to address specific research questions within the field of EVLP. The team established a rat model to simulate DCD and implemented a two-stage preservation approach. Lungs were subjected to static cold storage using a perfusate solution at 4 &#176;C for 4 h, followed by normothermic EVLP for another 4 h. Researchers evaluated various lung function parameters, including pulmonary vascular resistance, pulmonary dynamic compliance, blood gas measurements of the perfusate, and apoptotic cell death in lung tissues. The study demonstrates that EVLP could significantly improve the functional performance of DCD lungs compared to simple cold storage at 4 &#176;C, suggesting that EVLP has the potential to mitigate ischemia-reperfusion injury and enhance graft function following circulatory death, thereby increasing the pool of transplantable lungs.

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after ...

Ex vivo Lung Perfusion: A Technique for Pulmonary Research in a Rabbit Model

Source: Pacheco-Baltazar, A., et al., <a href="https://www.jove.com/v/62734">Isolated Lung Perfusion System in the Rabbit Model.</a> J. Vis. Exp. (2021).
This video shows an ex vivo technique for isolated lung perfusion. It is a tool for pulmonary research that will aid studies of various physiological and pathological mechanisms involved in lung transplantation procedures.

Perfuzja płuc ex vivo: technika badań płuc na modelu królika

Source: Pacheco-Baltazar, A., et al., Isolated Lung Perfusion System in the Rabbit Model. J. Vis. Exp. (2021).
This video shows an ex vivo technique for ...

JoVE Encyclopedia of Experiments

Large Animal Models

Leporine

Ex Vivo Lung Perfusion: A Technique for Pulmonary Research in a Rabbit Model

Lift the isolated lungs out of the thorax, and carefully place the lungs over a sterile gauze on a Petri dish. To prevent atelectasis, ventilate the lungs using positive-pressure ventilation, with positive end-expiratory pressure set at 2 centimeters of water column. Cut the ventricles off the heart at the level of the atrioventricular groove.
After opening the two ventricles, introduce the pulmonary artery cannula through the right pulmonary artery, and the left atrium cannula through the mitral valve, into the left atrium. Fix the cannulae with a 4-0 silk suture, including the surrounding tissues in the ligatures of the pulmonary artery and left atrium, to avoid structural distension. Inject 250 milliliters of saline isotonic solution through the arterial cannulae to flush the remaining blood from the vascular bed.
For the perfusion setup, place the isolated lungs carefully into the lung chamber. Attach the trachea to the transductor on the chamber cover, and connect the cannulated vessels to the perfusion system. Then, close and secure the chamber with the rotary lock. Next, attach the chamber lid and switch over the stopcock to shift from positive to negative pressure ventilation. Perfuse the lungs with 200 milliliters of artificial, blood-free perfusate, starting with the flow at 3 mL/min/kg.
Then, slowly, step up the flow over 5 minutes to 5 mL/min/kg, and for the next 5 minutes, allow the flow to reach 8 mL/min/kg, followed by a maximum flux of 10 mL/min/kg for 5 minutes. Ventilate the lungs with humidified air at a frequency of 30 beats per minute, a tidal volume of 10 mL//kg and an end-expiratory pressure of 2 centimeters of water column.
To achieve zone three conditions, wait for 10 to 15 minutes to obtain an equilibrium characterized by an isogravimetric state, ensuring that the venous pressure is stable throughout the registry. Ensure that the weight of the lung remains constant and the arterial and left atrial pressures are stable to achieve zone three conditions to open up a maximum number of pulmonary vessels and maintain the microvascular bed content during the experiment.
For the electronic control and signal processing, ensure that the respiratory flow, weight changes, microvascular pressure, tidal volume, vascular resistance, among others are registered on a multiple central electronics unit, that integrates signals coming from the transducers and displays them on the evaluation system.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Podsumowanie

Przeglądaj więcej filmów

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

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

Isolated Lung Perfusion System in the Rabbit Model

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

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

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

Perfuzja płuc ex vivo: technika badań płuc na modelu królika

Perfuzja płuc ex vivo: technika badań płuc na modelu królika