Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Published: May 26, 2023

doi:

William J. Cleveland*¹, Josephine E. Hees*^1,2, Claudius Balzer², Hunter F. Douglas, Troy Stevens^4,5, Matthias L. Riess^6,7

¹Anesthesiology,Vanderbilt University Medical Center, ²Anesthesiology,University of Greifswald, ³Department of Physiology and Cell Biology,University of South Alabama, ⁴Department of Medicine,University of South Alabama, ⁵Center for Lung Biology,University of South Alabama, ⁶Anesthesiology,Tennessee Valley Healthcare System Veterans Affairs Medical Center, ⁷Pharmacology,Vanderbilt University

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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's animal care facility, with free a…

Representative Results

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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Support was provided, in part, by a Merit Review Award (101 BX003482) from the U.S. Department of Veteran Affairs Biomedical Laboratory R&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.

Materials

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

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