Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

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

doi:10.3791/62215

Inflação do ar dos pulmões murinos com fixação vascular de perfusão

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Neuroscience

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JoVE Journal Neuroscience

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Inflação do ar dos pulmões murinos com fixação vascular de perfusão

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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

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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.

Apresentado é um método de inflação do ar com fixação vascular de perfusão dos pulmões que preserva a localização de células dentro das vias aéreas, alvéolos e interstícios para análises estrutura-função. A pressão constante das vias aéreas é mantida com uma câmara de inflação do ar, enquanto fixação é perfundida através do ventrículo direito. Pulmões são processados para estudos histológicos.

Trata-se de um método barato que combina as vantagens da inflação do ar e da fixação da perfusão vascular para a preservação dos pulmões para análises estruturais e funcionais. A principal vantagem dessa técnica é a preservação da morfologia celular e localização dentro dos espaços aéreos do pulmão. Deve-se tomar cuidado ao colocar o adaptador de isca na traqueia e a agulha de perfusão no ventrículo direito para garantir a inflação e fixação adequadas.

Para configurar o aparelho de inflação do ar, coloque uma seringa para a coluna de água em um suporte de anel e meça uma altura vertical de 25 centímetros da plataforma animal até 25 centímetros na coluna de água. Conecte-se à extremidade do tubo da coluna de água à torneira na câmara de ar e conecte um tubo da isca feminina da câmara de ar à torneira do recipiente de processamento de animais. Confirme que a tampa da câmara de ar e a torneira do lado de fora do recipiente de processamento de animais estão fechadas, e que a torneira na tubulação que leva da coluna de água para a câmara de inflação do ar está aberta.

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