Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Summary

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

Abstract

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg–300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

Introduction

For a number of reasons, lung cancer has not been widely studied in the mouse. One reason for this is that access to the lung is very difficult in vivo, and quantitative analysis of fixed lungs is not commonly done. The methods described in this paper are designed to remedy this situation. The goals herein are to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. While none of these approaches are entirely new, they have not been presented together as stand-alone methods in the simplified manner as described here.

There have been a number of manuscripts that....

Protocol

The following protocol describes a system that works well in 20–35 g mice. The method could easily be adaptable to larger or smaller mice simply by changing the catheter size. All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee.

1. Lung Instillation

1. Select a commercial one-inch long 20 g intravenous cannula to use for the intubation.
2. Modify the catheter tip manually bending it to generate a slight curvature at the tip as illustrated in Figure 1.
3. Anesthetize the mouse with a mixture of ketamine (100 mg/kg) and xylazine (15 mg/kg) injected IP, ....

Results

The procedure describe in the first protocol does not by itself lead to any generalized results. It only describes a very reliable means to instill substances directly into the trachea. Figure 7 shows an example of a lung in which trypan blue was instilled with the method described here. There is widespread distribution of the dye, similar to what has been seen with other dyes or tracers given directly into the trachea or mice^11,12. We have also used this method to deliver either bleomycin or .......

Discussion

The procedures described here have several advantages. First the required equipment is simple and inexpensive. Second, the intubation can be quickly done with few errors. Third, the ability to fix the lungs at a constant pressure, and then measure the fixed lung volume allows proper quantification of structures or cells in the lung¹⁰.

One possible downside to the intubation is the minor surgery. This may limit the ability to repeat the procedure if a 2^nd instillation is r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Laboratory Balance	Ohaus	Adventurer Pro Model AV 313	Other balances can be used if they have a range of 1-300 g
20 g Luer intravenous catheter	Insylte		Several other possible vendors, e.g., Jelco Optiva
500 ml laboratory bottle	Various		Several other possible vendors