Name: instillation and fixation methods useful in mouse lung cancer research
Uploaded: 2015-08-31T14:00:00
Description: Watch this Scientific Journal Video about Instillation and Fixation Methods Useful in Mouse Lung Cancer Research at JoVE.com

The following protocol describes a system that works well in 20&#8211;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
<ol>
	<li>Select a commercial one-inch long 20 g intravenous cannula to use for the intubation.</li>
	<li>Modify the catheter tip manually bending it to generate a slight curvature at the tip as ...

<ol>
	<li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J Appl Physiol. 87, 2362-2365 (1999).</li><li>Walters, D. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+endotracheal+intubation+of+mice.">A novel and simple method for endotracheal intubation of mice.</a> Laboratory. 41, 128-135 (2007).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+retrograde+intubation+in+mice.">A technique for retrograde intubation in mice.</a> Lab animal. 35, 39-42 (2006).</li><li>Rivera, B., Miller, S., Brown, E., Price, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+endotracheal+intubation+of+mice+and+rats+used+in+imaging+studies.">A novel method for endotracheal intubation of mice and rats used in imaging studies.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 44, 52-55 (2005).</li><li>Vergari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+orotracheal+intubation+in+mice.">A new method of orotracheal intubation in mice.</a> European review for medical and pharmacological sciences. 8, 103-106 (2004).</li><li>Vergari, A., Polito, A., Musumeci, M., Palazzesi, S., Marano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-assisted+orotracheal+intubation+in+mice.">Video-assisted orotracheal intubation in mice.</a> Laboratory animals. 37, 204-206 (2003).</li><li>Hastings, R. H., Summers-Torres, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Laryngoscopy+in+Mice.">Direct Laryngoscopy in Mice.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 38, 33-35 (1999).</li><li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> Am J Respir Crit Care Med. 181, 394-418 (2010).</li><li>Foster, W. M., Walters, D. M., Longphre, M., Macri, K., Miller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+the+measurement+of+mucociliary+function+in+the+mouse+by+scintigraphy.">Methodology for the measurement of mucociliary function in the mouse by scintigraphy.</a> J Appl Physiol. 90, 1111-1117 (2001).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> Journal of visualized experiments : JoVE. , e50601 (2013).</li><li>Das, S., Macdonald, K., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> Journal of visualized experiments : JoVE. , (2013).</li><li>MacDonald, K. D., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+simple+method+of+mouse+lung+intubation.">An improved simple method of mouse lung intubation.</a> J Appl Physiol. 106, 984-987 (2009).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage.</a> American Reveiw of Respiratory Diseases. 132, 1078-1083 (1985).</li><li>Bishai, J., Fields, M. J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Mouse+Lung+Volumes+Inflated+with+Air+and+Instilled+Fixatives.">Comparison of Mouse Lung Volumes Inflated with Air and Instilled Fixatives.</a> Proc Am Thorac Soc. 3, A549 (2006).</li><li>Mitzner, W., Brown, R., Lee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+lung+volumes+in+mice.">In vivo measurement of lung volumes in mice.</a> Physiol Genomics. 4, 215-221 (2001).</li></ol>

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 2nd instillation is r...

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 have described methods for intubation of the mouse lung primarily for the purpose of doing repeat pulmonary function or bronchoalveolar lavage in individual mice in longitudinal studies. Since that original paper, there have been several other papers that have described different approaches to mouse intubation1-9. While all of these methods can be used successfully, they usually require considerable training, and are often not without a nontrivial failure rate. In addition, in order to carry out pulmonary function measurements, the cannula needs to fit the trachea tightly enough so that there is no air leakage. However, another practical use for intubation is to deliver specific agents (cancer cells or other insults) or therapeutic drugs directly to the lung. Such a procedure does not require a tight fitting cannula nor any sophisticated pulmonary function equipment. The novel feature of this method shown here involves a minor surgical procedure that allows the intubation without any possibility of the cannula entering the esophagus. This simple approach enables successful intubation with relatively little training or experience. As many as 30 mice/hr can be treated using this approach with a failure rate approaching zero.
Once the mice are ready to be sacrificed, the injured or cancerous lungs can then be removed for histologic and pathologic analysis. However, in order to properly quantify any histologic variables for comparison with other lungs, it is essential to standardize the fixation procedures and properly quantify the fixed lung volume10. This paper describes in detail the simple procedures that allow standardized fixation procedures as well as a way to measure the fixed lung volume. The volume is an essential metric in the quantification of the histology, since without such a volume determination, only relative densities can be measured10. Once the lung volume is known, however, absolute measurements of cells and other structural measurements in the lung can then be quantified.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Laboratory Balance</td>
			<td>Ohaus</td>
			<td>Adventurer Pro Model AV 313&#160;</td>
			<td>Other balances can be used if they have a range of 1 - 300g</td>
		</tr>
		<tr>
			<td>20 g Luer intravenous catheter</td>
			<td>Insylte</td>
			<td></td>
			<td>Several other possible vendors, e.g., Jelco Optiva</td>
		</tr>
		<tr>
			<td>500 mL laboratory bottle</td>
			<td>Various</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
	</tbody>
</table>

instillation and fixation methods useful in mouse lung cancer research

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&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

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 mice11,12. We have also used this method to deliver either bleomycin or ...

Watch this Scientific Journal Video about Instillation and Fixation Methods Useful in Mouse Lung Cancer Research at JoVE.com

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research (Video) | JoVE

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.

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

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 ...

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