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 a...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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 ...

We demonstrate a simple method for creating and implementing an ex vivo lung setup from scratch. This allows for an affordable and modular setup that can be implemented for a variety of setups. The technique presented here is affordable, modular, and adaptable to the labs setting up the protocol.The ex vivo operation can be used to complement in vivo models to help elucidate novel mechanisms. This DIY setup is designed to study the lungs in a controlled environment. It aligns with the main research focus of our lab, which is to investigate various approaches to improve the outcome after CPR.However, the setup can be adapted to suit different research aims, making it highly versatile, The surgery requires a basic knowledge of respiratory anatomy and physiology and can quickly be mastered by following the protocol. Nonetheless, it is vital to diligently follow the steps described in our protocol, and most of all, protect the lungs, especially when removing the ribs. To perform a tracheostomy, pinch the skin of an anesthetized rat above the trachea with forceps and cut the skin.Bluntly dissect the muscle and tissue to reach the trachea, ensuring no bleeding. Then pass curved forceps underneath the trachea and open them to allow room to pass 3-0 sutures underneath. Then pre-tie the sutures into a box knot.Make a small incision between the cartilage rings of the trachea and insert the tracheal cannula. Tie off the suture to prevent air from escaping the tracheal incision and to ensure the cannula is not putting strain on the trachea. Once the tracheal cannula is secured, begin ventilating the rat.Using large surgical scissors and forceps, remove the fur from the rat's abdomen. Grasp the xiphoid process with the forceps and make a small horizontal incision below the ribs, taking care to keep the diaphragm intact. Widen the horizontal cut to expose the entire diaphragm.With a 22 gauge syringe, inject heparin into the inferior vena cava, being careful not to puncture the lungs. Grasp the xiphoid process with forceps and cut cranially along the sternum, constantly visualizing the lungs to prevent cutting them. Spread the rib cage apart using two large forceps and cut the inferior vena cava to euthanize the rat by exsanguination.Trim any excess thymus to allow for easier visualization of the pulmonary vasculature. Locate the pulmonary artery and pass small curved forceps underneath. Again, pass a 3-0 suture underneath and pre-tie into a box knot.Then make a small incision into the right ventricle of the heart and insert the PA cannula. Secure the cannula using the suture and begin perfusing at 1.5 milliliters per minute. Immediately excise the apex of the heart to prevent pressure buildup in the lungs.Using small curved forceps, rupture the mitral valve and visually confirm that the forceps can enter the left atrium without obstruction. Tightly wrap a 3-0 suture around the heart below the atrium, and insert the PV cannula into the left atrium, ensuring that buffer can flow out of it before tying off the suture. Next, using blunt tip scissors, trim any excess tissue between the thoracic cavity and the tracheostomy incision to avoid damaging the trachea.Ensure the trachea below the tracheal cannula and the entire heart-lung bloc are visible. To remove the heart-lung bloc and trachea, hold the tracheal cannula and use curved blunt tip scissors to excise the connective tissue behind the trachea. Lung viability after ischemia and reperfusion is presented.The longest ischemia time to allow 150 minutes of reperfusion was eight minutes. It's crucial to properly cannulate the vasculature for the isolated lungs to remain viable throughout the experiment. The lungs remain intact after the procedure, allowing for basic measurements such as wet or dry weight or histology to be conducted post-experiment.

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

Research

JoVE Journal

Biology

637 visualizações.  Vanderbilt University Medical Center. Demonstramos um método simples para criar e implementar uma configuração pulmonar ex vivo do zero. Isso permite uma configuração acessível e modular que pode ser implementada para uma variedade de configurações. A técnica apresentada aqui é acessível, modular e adaptável aos laboratórios que configuram o protocolo.A operação ex vivo pode ser usada para complementar modelos in vivo para ajudar a elucidar novos mecanismos. Esta configuração DIY foi projetada para estudar...