Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

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

doi:10.3791/62215

Inflado al aire de los pulmones murinos con fijación de perfusión vascular

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Inflado al aire de los pulmones murinos con fijación de perfusión vascular

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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.

Se presenta un método para la inflación del aire con fijación de perfusión vascular de los pulmones que preserva la ubicación de las células dentro de las vías respiratorias, los alvéolos y el intersticio para los análisis de estructura-función. La presión constante de las vías respiratorias se mantiene con una cámara de inflado de aire, mientras que el fijador se perfunde a través del ventrículo derecho. Los pulmones se procesan para estudios histológicos.

Este es un método económico que combina las ventajas de la inflación del aire y la fijación de perfusión vascular para preservar los pulmones para análisis estructurales y funcionales. La principal ventaja de esta técnica es la preservación tanto de la morfología celular como de la ubicación dentro de los espacios aéreos del pulmón. Se debe tener cuidado al colocar el adaptador de talón de señuelo en la tráquea y la aguja de perfusión en el ventrículo derecho para garantizar un inflado y una fijación adecuados.

Para configurar el aparato de inflado de aire, coloque una jeringa para la columna de agua en un soporte de anillo y mida una altura vertical de 25 centímetros desde la plataforma del animal hasta 25 centímetros en la columna de agua. Fije al extremo del tubo de la columna de agua a la llave de paso en la cámara de aire y conecte un tubo desde el señuelo hembra de la cámara de aire a la llave de paso en el contenedor de procesamiento de animales. Confirme que la tapa de la cámara de aire y la llave de paso en el exterior del recipiente de procesamiento de animales están cerradas, y que la llave de paso en el tubo que va desde la columna de agua hasta la cámara de inflado de aire está abierta.

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