Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Stacey  M. Thomas; Joseph Bednarek; William J. Janssen; Patrick  S. Hume

doi:10.3791/62215

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

JoVE Journal
Neuroscience

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

JoVE Journal Neuroscience

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Full Text

8,477 Views

07:19 min

February 2, 2021

DOI: 10.3791/62215-v

Stacey M. Thomas¹, Joseph Bednarek², William J. Janssen¹, Patrick S. Hume¹

¹Division of Pulmonary, Sleep and Critical Care Medicine, Department of Medicine,National Jewish Health, ²Section of Pediatric Gastroenterology, Hepatology, and Nutrition,Children's Hospital Colorado

Overview

This article presents a method for preserving lung structures through an innovative air-inflation with vascular perfusion-fixation technique. This method allows researchers to maintain cell morphology and location within airways, alveoli, and interstitium for detailed structure-function analyses. The preserved samples are suitable for subsequent histologic studies.

Key Study Components

Area of Science

Neuroscience
Biology
Histological Analysis

Background

Traditional methods often compromise cell morphology during lung fixation.
This technique combines air inflation with vascular perfusion fixation to improve sample integrity.
Maintaining location of cells within lung architecture is critical for accurate analyses.

Purpose of Study

To develop a method preserving both structure and function of lung tissues.
To facilitate detailed histologic examination of lungs post-fixation.
To investigate inflammatory responses in lung tissues effectively.

Methods Used

The method involves air inflation of lungs combined with vascular perfusion fixation.
The biological model used is the euthanized mouse lung.
This technique involves precise surgical incisions and careful administration of fluids.
Key steps include setting up an air-inflation apparatus and accurately controlling pressure during fixation.
Importance is placed on the timing and order of inflation and fixative perfusion to prevent damage.

Main Results

The technique successfully preserves cell morphology and location in lung tissues.
Unlike traditional methods, few immune cells are lost in the airway lumen, allowing for more accurate assessments.
The method enhances visibility of inflammatory responses within perfused lung tissues.
Histological analysis demonstrates the viability of the samples for various imaging techniques.

Conclusions

This study enables better preservation of lung structures for detailed analyses.
It has significant implications for understanding lung pathology and responses to injury or infection.
Overall, the method enhances histological examination capabilities in lung research.

Frequently Asked Questions

What are the advantages of this air inflation and fixation method?

This method preserves cell morphology and positioning in lung structures better than traditional methods, allowing for more accurate functional analyses.

How is the mouse lung prepared for inflation and fixation?

Lungs are prepared by making incisions to access the trachea and heart, and through precise placement of a perfusion needle into the right ventricle.

What types of data can be obtained using this technique?

This method allows for histological analysis, including staining and immunohistochemistry to evaluate lung structure and immune response.

How can this method be applied to other studies?

This air-inflation and perfusion fixation technique can be adapted for other tissues where preservation of structural details is critical for analysis.

What are key considerations when using this technique?

Careful attention must be paid during the surgical preparation and the perfusion steps to avoid damaging the lung tissue.

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

This is an inexpensive method that combines the advantages of air inflation and vascular perfusion fixation for preserving lungs for structural and functional analyses. The main advantage of this technique is the preservation of both cell morphology and location within the airspaces of the lung. Care should be taken while placing the lure stub adapter into the trachea and the perfusion needle into the right ventricle to ensure adequate inflation and fixation.

To set up the air-inflation apparatus, place a syringe for the water column into a ring holder and measure a vertical height of 25 centimeters from the animal platform to 25 centimeters on the water column. Attach to the end of the water column tube to the stopcock on the air chamber and attach a tube from the female lure of the air chamber to the stopcock on the animal processing container. Confirm that the cap to the air chamber and the stopcock on the outside of the animal processing container are closed, and that the stopcock on the tubing leading from the water column to the air inflation chamber is open.

View the full transcript and gain access to thousands of scientific videos