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>

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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 which CO2 is controlled only by the inspired gas concentration, also allowed for differential regulation of CO2 independent from minute ventilation.
A closed circuit (Figure 1) was used to recirculate the modified Krebs buffer (Table 1) to conserve BSA waste, an important cost-limiting step. Perfusion with constant flow mimicking a constant cardiac output was utilized to allow for the study of different doses of pharmacological agents on pulmonary vascular tone, independent of concomitant cardiac output changes in vivo. This can easily be modified to a constant-pressure perfusion by integrating an adjustable height pressure column in the circuit and adjusting the pump speed accordingly; for this, an additional flow probe is required, since flow, now becomes the dependent variable instead of pressure.
The method of ventilation was with positive rather than more physiological negative pressure, mimicking an unconscious or anesthetized, intubated, and ventilated rather than a spontaneously breathing subject. Changing this to negative pressure ventilation would require the use of a sealed chamber for hanging the lungs and making corresponding changes to setting up the ventilator11, but is still possible to incorporate into the apparatus with minimal effort.
The buffer was maintained at a constant temperature by a heating plate, and passed through a heating coil and heated air trap to ensure no dissolved gasses escaped the solution, causing the lung to be perfused with air emboli. To perfuse the lungs, a peristaltic pump was used. The roller pump we used had the great advantage of having both manual controls and voltage inputs and outputs for easy connection to the DAQ. This made it possible to not only record the pump speed, but also to control the pump electronically. One important aspect of in vivo lungs that is absent in the isolated model is pulsatile flow from the heart. By using sinusoidal, square, or triangle waves generated by the software as voltage outputs to the pump, the setup is able to mimic the pulsatile blood flow coming from an in vivo heart. This effect was slightly dampened by the Windkessel effect from tubing between the buffer and the lungs, but was still seen in both the PA and PV pressure recordings. Non-compliant tubing was used to minimize the Windkessel effect. A three-way stopcock placed in the circuit coming from the PV allowed for the collection of effluent for the analysis of injury markers (e.g., lactate dehydrogenase).
A concern was maintaining a consistent metabolic environment - glucose concentration - in the isolated lungs similar to what would be seen in vivo. The compositions of the buffer, as well as ventilated gasses, play an important role. Valenza et al. showed in a porcine EVLP model that glucose consumption in the lungs correlated with lung function4. As a means to keep the glucose concentration constant in the recirculated buffer despite ongoing glucose metabolism, the glucose level of the solution was checked hourly and glucose added back into the buffer to keep the composition as close to the target of 5.5 mM as possible. A modified Krebs buffer (Table 1) with an electrolyte composition that mimicked blood was used for the perfusion. However, this presented problems when trying to measure the glucose concentration; a standard blood glucose measurement device (ACCU-CHEK, Aviva) was unable to read the glucose levels of the buffer, so other options needed to be explored. Glucose test strips did not prove accurate enough to measure the glucose of the solution when compared to a more clinically relevant device (VetScan i-STAT 1)19, which worked reliably.
While other EVLP setups utilize additives such as Perfadex and Celsior20, this setup used BSA due to prior success with previous experiments and its relative affordability if recirculated. This addition was necessary to maintain a physiological oncotic pressure in the lungs, as experiments performed without BSA deteriorated much more rapidly, typically within 30 min of extraction, due to pulmonary edema. Others have also reported success when using higher concentrations of BSA and whole blood. This has the added benefit of allowing the blood to be fractionated to suit the experiment. Lungs that were extracted by a skilled technician in a timely manner were able to survive for up to 6 h under control conditions before edema formation became a problem. This was apparent when observing changes in the lungs, such as gradual changes in color from white to pink, an increase in size due to edema formation, and sharp pressure increases in the pulmonary vasculature. During the procedure, it is important to measure the electrolyte composition of the buffer after the addition of BSA; measured Ca2+, for example, decreased in the buffer following the addition of BSA, implying that BSA was chelating Ca2+, reducing the amount that was available for the lungs21.
In summary, several commercial isolated lung kits are available for purchase that require significantly less time starting up. However, these can be limited in their scope as to what they can do and might not offer the ability to adapt to different experimental parameters. Taking the DIY approach offers a system that is on par with the commercial setups, while also allowing for flexible adaption to a variety of research topics. Once the initial barrier of entry is overcome, the system can be run with minimal oversight and teaching required.

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, during ischemia, we chose not to stop the ventilation, but rather reduce its rate from 60 to 20 breaths/min-1 to prevent deflation and irreversible atelectasis of the ex situ lungs. Applying a constant PEEP likely achieves the same goal, in analogy to the tracheas of to-be-transplanted lungs being clamped after inspiration in clinical practice17. Figure 4 suggests that, under these conditions, the longest ischemia time to still allow 150 min of reperfusion was 8 min.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64740/64740fig1v4.jpg" /> 
Figure 1: A custom-built lattice to allow for easy configuration and incorporation of new devices. (A) Photograph of the custom-made lattice used to house all the equipment and devices. Not shown are the pressure overflow, gas mixer, gas supply, DAQ, or computer. (B) Schematic of the closed circuit used to perfuse the lung and each device involved in the setup. 1) The buffer is stored in a volumetric flask to reduce the volume and surface area. 2) Heating plates are used to heat both the buffer and the lung chamber. A digital thermometer is used to monitor the temperature inside the lung chamber. 3) A circulating water bath heats the heating coil and air trap to maintain a constant temperature. 4) In the constant-flow setup described here, the roller pump is used to control lung buffer perfusion. 5) Buffer is passed through a heating coil to maintain its temperature. 6) The air trap ensures no air bubbles reach the lung and helps maintain the temperature. 7) A custom double boiler is used to create a humid environment for the lung and maintain its temperature. 8) A pressure overflow is placed between the ventilator and the lung to prevent over-ventilating the lung. 9) A volume-controlled ventilator is used for reliable tidal volumes. 10) The gas mixer can be used to create different gas compositions. 11) Various gas tanks, such as O2, CO2, and N2, are needed for ventilating different compositions of gas. 12) A simple water column is connected to the ventilator to adjust the PEEP on the lung. 13) The DAQ system is responsible for collecting all the data and sending it to the computer. 14) Data are collected and visualized live on the computer. Abbreviations: DAQ = data acquisition system; PEEP = positive end expiratory pressure; AWP = airway pressure. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64740/64740fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64740/64740fig02.jpg" /> 
Figure 2: Custom-made cannulas. (A) Tracheal cannula (made from an 18 G needle). (B) Pulmonary artery cannula. (C) Pulmonary vein cannula. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64740/64740fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64740/64740fig3v3.jpg" /> 
Figure 3:&#160;Isolation process of the rat lungs. (A) The rat is securely taped down after anesthesia, with the mouth taped as well to ensure minimal movement. (B) Proper isolation and placement of the forceps for performing the tracheostomy. 
(C) Spreading the rib cage to open the surgical area and minimize the risk of a broken rib puncturing the lungs. (D) Placement of the pulmonary artery cannula. (E) Placement of the pulmonary vein cannula after being secured to the heart. (F) Removal of the trachea and heart-lung block from the rat. (G) The rat isolated lungs hanging prior to placement in the chamber. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64740/64740fig3v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64740/64740fig04.jpg" /> 
Figure 4:&#160;Representative results on lung viability after ischemia and reperfusion. Lung viability was defined as survival time until the lungs exhibited a fulminant increase in airway pressure and became fully edematous. Data of limited experiments per group are displayed as mean &#177; standard error of the mean. Statistical tests were not conducted because of the low number of experiments per group. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64740/64740fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>119.0 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&#160; 24.0 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&#160;&#160;&#160; 5.5 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&#160;&#160;&#160; 1.6 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&#160;&#160;&#160; 4.7 mM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&#160; 1.17 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>&#160; 1.18 mM</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (98%)</td>
			<td>4% solution (4.0816&#160;g&#160;/&#160;100&#160;mL)</td>
		</tr>
	</tbody>
</table>
Table 1: Krebs buffer composition.

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

Universidad San Sebastian

Research

JoVE Journal

Biology

702 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 ...

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 ...

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.bdigitaluss.remotexs.co/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 ...

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

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

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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

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

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