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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Published: May 24th, 2021

DOI:

10.3791/62392

1Divisions of Pulmonary Medicine, Boston Children's Hospital

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Advances in the preparation and imaging of lung tissue that preserves cellular components without sacrificing anatomical structure provide a detailed understanding of pulmonary diseases. The ability to identify proteins, RNA, and other biological compounds while maintaining physiological structure offers vital information on the spatial arrangement of cells that can broaden the understanding of the pathophysiology in numerous pulmonary diseases. These detailed images can lead to a better understanding of pulmonary vascular diseases, such as pulmonary artery hypertension, when applied to animal models, potentially leading to improved therapeutic strategies.

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All animal care was in accordance with the guidelines of Boston Children's Hospital and the Institutional Animal Care and Use Committee approved protocols. The mice used in this study are wild type C57/B6 mice and Cdh5-CreERT2 x Ai14 tdTomato crossed mice.

1. Preparation of solutions

  1. Prepare phosphate buffer solution (1x PBS) and 2% agarose solution required during the experiment in advance.
    1. Mix 2 g agarose powder into 100 mL of autoclaved water. Heat it in the micr.......

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When added to cells or tissue, the viability reagent is modified by the reducing environment of viable tissue and turns pink/red, becoming highly fluorescent. The representative color changes detected from day 0-1 and day 9-10 are demonstrated in Figure 3. As noted, the solution started blue and turned pink overnight, demonstrating viability. Color change typically occurs within 1-4 h; however, a longer time may be necessary. To assay for viability, a plate reader was used to determine the a.......

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In this manuscript, an enhanced method to produce high-resolution images of murine lung tissue that preserves the vascular structure and optimizes experimental flexibility is described, specifically using the application of PCLS to obtain microslices of lung tissue that can be viewed in three dimensions with preserved contractility of the vasculature. Using the viability reagent, the protocol demonstrates that carefully prepared and preserved slices can retain viability for more than a week. Preserved viability of the mi.......

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The authors would like to thank Drs. Yuan Hao and Kaifeng Liu for their technical support. This work was supported by an NIH 1R01 HL150106-01A1, the Parker B. Francis Fellowship, and the Pulmonary Hypertension Association Aldrighetti Research Award to Dr. Ke Yuan.

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Name Company Catalog Number Comments
0.5cc of fractionated heparin in syringe BD 100 USP units per mL
1X PBS Corning  21-040-CM
20 1/2 inch gauge blunt end needle for trachea cannulation Cml Supply 90120050D
30cc syringe BD 309650
Anti Anti solution Gibco 15240096
Automated vibrating blade microtome Leica VT1200S
Cell Viability Reagent (alamarBlue) Thermofisher DAL1025
Confocal Zeiss 880
Dulbecco’s Modified Eagle Medium and GLutaMAX, supplemented with 10% FBS, 1% Pen/Strep Gibco 10569-010
Endothelin-1 Sigma E7764
KCl Sigma 7447-40-7
Mortar and Pestle Amazon
RIPA lysis and extraction buffer Thermoscientific 89900
Surgical suture 6/0 FST 18020-60
TRIzol Reagent Invitrogen, Thermofisher 15596026
UltraPure Low Melting Point Agarose Invitrogen 16520050
Vibratome Leica Biosystems VT1200 S
Winged blood collection set (Butterfly needle) 25-30G BD 25-30G

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