Perfusion and Inflation of the Mouse Lung for Tumor Histology

Summary

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

Abstract

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

Introduction

A variety of mouse models of lung oncogenesis and cancer metastasis to the lung exist ranging from complex GEMMs to carcinogen-induced models to syngeneic and xenograft models, where cancer cells are injected via intracardiac, intrathoracic, the tail vein, or other methods to establish tumors within the lung. All these models share the common need for histological evaluation of lung histology and pathology. Thus, it is necessary to have a robust yet rapid method to perform necropsies of mice while perfusing the lungs to remove excess blood, and inflating and fixing the lungs to clearly visualize lung architecture. Speed is a critical component of this procedure as it ....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham.

1. Experimental protocol

1. Sacrifice the mouse using an approved IACUC method. Here, we used cervical dislocation of a mouse anesthetized with 5% isoflurane. Use an appropriate mouse for the study; here, we use an 8-week old FVB mouse
2. Using surgical scissors, make a 3.5-5 mm horizontal incision in the middle of lower abdomen. Next, insert surgical scissors into the small hole created from incision and cut vertically up the center midline to just below the neck of the mouse.<....

Results

The above protocol allows for quick perfusion, inflation, and fixation of mouse lungs. The figures shown below represent the importance of each step. Figure 1 depicts H&E stained lungs that have been perfused with PBS and lungs in which the perfusion step has been skipped or the lungs failed to perfuse correctly. As shown, excess blood in the poorly perfused lungs creates less than ideal histology and can make it challenging to fully observe lung architecture. Figure.......

Discussion

The procedure described above for the perfusion, inflation, and fixation of mouse lungs is ideal for quick and efficient preparation of mouse lungs for lung tumor histology and pathology analysis. The procedure does not require any special equipment and can be performed in less than 6 minutes per mouse. The procedure does not require a fixed volume for inflation nor constant fluid pressure. Because this procedure is not standardized, it is not recommended for those wishing to perform stereological or morphometric analyse.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% buffered formalin	Fisher	23-245685
22 G Needle	BD	305155
3 mL syringe	BD	309656
70% Ethanol	Decon	2405
Forceps	Harvard Apparatus	72-8595
Heparin	Fisher	H19
Phosphate Buffered Saline (PBS)	Corning	21-030-CV
Surgical scissors	Harvard Apparatus	72-8428