Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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, ....

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.......

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

NameCompanyCatalog NumberComments
1,000 mL Glass BeakerPyrex, Chicago, IL
1,500 mL Glass BeakerPyrex, Chicago, IL
Air Trap Compliance ChamberRadnoti130149
BioamplifiersCWE IncBPM-832
ClampsFisher ScientificS02626
DAQ (Data Acquisition)National Instruments, Austin, TXNI USB-6343
Gas MixerCWE Inc, Ardmore, PAGSM-4
Heating CoilRadnoti, Covina, CA158822
Heating PlateThermo Fisher Scientific, Waltham, MA11-100-49SH
HeparinPfizerW63422
LabVIEW Full Development System 2014National Instruments
PentobarbitalDiamondback DrugsG2270-0235-50
pH700 ProbeOAKTON, Vernon Hills, IL EW-35419-10
Polystat Water BathCole-ParmerEW-12121-02
Rodent VentilatorHarvard Apparatus, Holliston, MAModel 683
Roller PumpCole-Parmer, Wertheim, Germany Ismatec REGLO Digital MS 2/8
Sprague Dawley RatCharles River, Wilmington, MAStrain code 001
VetScan i-STATAbraxis, Chicago, ILi-STAT 1

References

  1. Uhlig, S., Taylor, A. E. . Methods in Pulmonary Research. , (1998).
  2. Ghaidan, H., et al. Ten year follow-up of lung transplantations using initially rejected donor lungs after reconditioning using ex vivo lung perfusion.

Explore More Articles

BiologyRatDIY ModelPulmonary Vascular ToneDrug EffectsGas EffectsIsolated LungsLung IsolationVentilationCost effectiveModularAdaptable

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved