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 illustrated in Figure 1.</li>
	<li>Anesthetize the mouse with a mixture of ketamine (100 mg/kg) and xylazine (15 mg/kg) injected IP, and confirm anesthesia by the absence of reflex motion. Apply veterinary ointment on the eyes immediately after anesthesia.&#160;Immediately after anesthesia apply veterinary ointment on the eyes and give carprofen (5-10 mg/kg SQ) for post surgical and instillation analgesia.</li>
	<li>Place the mouse supine on a sloped platform. As shown in Figure 2, a large office binder with suture loops taped on works perfectly well.</li>
	<li>Shave the ventral part of the neck and clean and disinfect the neck area with 70% alcohol.&#160; With new latex- and powder- free gloves, use surgical instruments disinfected with 70% alcohol.</li>
	<li>Using sharp scissors make a small surgical incision in the neck approximately 12 mm below the lower incisor.</li>
	<li>With a forceps gently pull the skin in the neck caudally until the ventral wall of the trachea can be seen.</li>
	<li>Gently retract the tongue and insert the cannula with the bent tip tilted toward the ventral surface of the mouse. As in 1.4, pull gently on the skin in the neck, and insert the cannula into the trachea. 
	NOTE: With a little practice, the catheter will be visible moving down the trachea. If it goes in the esophagus, then there will be no visual sighting of the catheter&#8217;s movement. No incisions are made in the trachea.</li>
	<li>Once the catheter is seen in the trachea in the neck, advanced it about 5 mm to be reliably passed the vocal cords but still well above the carina.</li>
	<li>Prepare to instill up to 50 &#181;l of liquid by injecting through the catheter with a gel loading pipette tip. Place the tip in the luer hub, but before injecting look carefully to observe movement of the fluid in the tip synchronous with the mouse&#8217;s breathing. Then inject the instillate.</li>
	<li>With a 1 ml&#160;syringe, immediately do a relatively rapid inflation of 0.6 ml&#160;of air into the lungs through the catheter to help distribute the liquid deep in the lungs. Remove the cannula.</li>
	<li>Remove the cannula.&#160;</li>
	<li>Use a small amount of cyanoacrylate adhesive to close the small surgical wound as per package insert instructions for VetBond. Place mice in individual cages and visually monitor them until they wake up and behave normally without any indication of discomfort.</li>
</ol>
2. Lung Fixation
NOTE: Once all experimental procedures are done in a mouse, the lungs can be readied for histologic processing by fixation with formaldehyde (or any other desired fixative).
<ol>
	<li>Sacrifice the mouse with an IACUC acceptable procedure. For the representative mouse shown in the video, cervical dislocation of an anesthetized mouse is used.</li>
	<li>Perform a tracheostomy (if not already done) by surgically exposing the ventral side of trachea, making a small cut, and inserting an 18 G&#160;stub needle tip into the trachea, and tying it with thread.</li>
	<li>Carefully open the thorax with a midline incision, cut away the diaphragm, and remove the lateral chest walls to expose the lungs.</li>
	<li>Connect the luer end of the needle to a reservoir on a ring stand containing formaldehyde. See Figure 3.</li>
	<li>Set the top surface of the formaldehyde 25 cm above the level of the mouse. See Figure 3.&#160; Next make sure there is no air in the fixation tubing by running fluid out the end of a stopcock. Connect the luer end of the tracheal cannula to the reservoir tubing. Open the stopcock to inflate the lungs with the formaldehyde. Leave the lungs under pressure for at least 20 min.</li>
	<li>Open the stopcock to inflate the lungs with the formaldehyde. Leave the lungs under pressure for at least 20 min.</li>
	<li>Next, tie off the trachea beyond the end of the stub needle.&#160; It may help to pull back slowly on the needle to expose more of the uncannulated trachea.&#160; When tied securely, remove the stopcock.</li>
	<li>Carefully dissect out the lungs.</li>
	<li>Place the lungs in formaldehyde overnight.&#160; Longer times are fine, and some stains or procedures may specify specific times.&#160; Also any other liquid fixatives, such as z-fix can be used for the instillation and immersion.</li>
	<li>Before further histologic processing, measure the fixed lung volume as described next.</li>
</ol>
3. Measurement of the Fixed Lung Volume
<ol>
	<li>Measure the lung volume using Archimedes principle as illustrated in Figure 4. Remove the fixed lung from the formaldehyde and dissect the heart and any other non-lung tissue.</li>
	<li>Use a previously constructed simple homemade wire support device that is used to keep the lungs fully under water. 
	NOTE: This device needs to be made compatible with whichever balance is being used. A typical device shown in Figure 5 is made from plastic pipettes and thin (20 G) wire. This system works well with the balance used in the video, but could easily be adapted to most laboratory balances.</li>
	<li>Place a beaker with &#8776;200 ml&#160;of water on the balance and tare with the supporting cage in place in the water. See Figure 6. Remove the metal cage; place the lung on the water surface and press under water with the cage.</li>
	<li>Record the weight on the balance. This number reflects the volume of water displaced and is thus a direct measure of the lung volume. Be careful to make sure the lung or suture or any part of the wire cage does not touch the sides or bottom of the beaker.</li>
	<li>For accuracy, repeat this measurement. Remove the lung from the water, and dry on a tissue. Tare the beaker with cage in place again and repeat the lung volume measurement. The two volume measurements should then be averaged. 
	NOTE: If the lungs are left in the formalin for more than about a week, the air in the lungs will be dissolved in the liquid. When this happens, the lung will sink, so it is no longer necessary to use any device as in Figure 5 to keep the lung submerged. In such case the volume can be measured by simply by holding the lung by one of the suture strings until it is completely submerged as illustrated in Figure 4.</li>
</ol>

<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. M., Wills-Karp, M., 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=Assessment+of+cellular+profile+and+lung+function+with+repeated+bronchoalveolar+lavage+in+individual+mice.">Assessment of cellular profile and lung function with repeated bronchoalveolar lavage in individual mice.</a> Physiol Genomics. 2, 29-36 (2000).</li><li>Hamacher, J., 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=Microscopic+wire+guide-based+orotracheal+mouse+intubation:+description,+evaluation+and+comparison+with+transillumination.">Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.</a> Laboratory animals. 42, 222-230 (2008).</li><li>Spoelstra, E. 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 required. However, with careful surgery and application of the adhesive we have been able to routinely do a second instillation a week or two later. If continuous repeated instillations are required or if repeated pulmonary function tests are needed, another intubation approach may be more desirable13,14.
In doing the instillation, there are several practical issues that should be mentioned. It is important to be as gentle as possible with movement of the tongue in the initial opening of the mouth. If forceps are used, the tips should be covered with rubber tubing, since it is easy to injure the tongue, and this can lead to death of the mouse. Although the methods here were designed for intubation of mice older than 6 weeks, they could easily be adapted to younger mice.
Our instillation procedure was initially designed as an alternative to the method of oropharyngeal aspiration11. While this latter method can be easily learned, the actual volume delivered to the lung remains uncertain, since some of the instillate remains in the pharynx and gets swallowed. With a direct instillation into the trachea and subsequent lung inflation as we demonstrate here, the instillate is directly delivered to the lung. It is worth noting that, although it is possible to use intubation to deliver substances directly to the trachea11,12, such intubation can damage the upper airway or vocal chords, and in general requires a much higher level of training to ensure the 100% success rate that our procedure allows.
The fixation procedure we describe is similar to what many investigators use. There is often some variability in the inflation pressure used for the fixative, but we feel that 25 cm H2O is a reasonable compromise that keeps the lung fully inflated without possible damage from over-inflation. It should be noted, however, that although it may seem that a lung inflated with fluid to 25 cm H2O should be at a volume close to what the total lung capacity might be with air inflation at 35 cm H2O, this is far from truth. In fact, the inflation with fixative generally results in a volume at 70% of the air lung capacity15,16. And with subsequent processing with paraffin embedding, the effective lung volume viewed in histologic sections is likely below functional residual capacity (FRC). The most common fixative is formalin or z-fix, but for immunologic staining, a glutaraldehye mixture is often required. Investigators will need to choose a fixative depending on what is needing to be stained, but further discussion of the optimal fixative is beyond the scope of this methods paper.
In order to do proper quantitative analysis of histologic sections it is essential to have a measurement of lung volume10. Although it is possible to get lung volume from the complete sequence of serial sections (the Cavalieri method), in mouse lungs it is often simpler to simply measure the fixed lung volume as we have described in this document and video. The procedure we describe takes just seconds to do and should be routinely done with all lung fixations. Keep in mind, however, that the volume thus measured does not account for any shrinkage with subsequent processing and embedding, and if this is important, the Cavalieri method should be used. One final note regarding this fixed volume measurement is that the fraction of the lung volume in the left lung in the fixed lungs is significantly smaller than that which has been measured in vivo. CT imaging of the lungs in vivo of two strains of mice at functional residual capacity showed the left lung to be about 40% of the total17, whereas it is generally only 30% in the above measurements from lungs fixed at 25 cm H2O. At the present time, we do not understand why this should be so different, but it should be kept in mind when analyzing changes in quantitative histologic analyses. With regard to studies of lung cancer, having a measurement of lung volume enables an investigator to properly normalize data on specific chemical desnsities or the density of various cell types to absolute numbers in the whole tumor or whole lung.
In summary, the intubation procedure describe here is inexpensive to fabricate and simple to use, and it should enable most investigators and laboratory technicians to quickly learn to successfully instill liquids into mouse lungs with relatively little experience. In addition, the fixation procedures and lung volume measurement for histologic analysis of lungs provide a means for proper repeatable and quantitative analysis of lung cells and lung structure.

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><tbody><tr><td>Laboratory Balance</td><td>Ohaus</td><td>Adventurer Pro Model AV 313&amp;nbsp;</td><td>Other balances can be used if they have a range of 1-300 g</td></tr><tr><td>20 g Luer intravenous catheter</td><td>Insylte</td><td></td><td>Several other possible vendors, &lt;em&gt;e.g.&lt;/em&gt;, 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 elastase to the lung, which results in widespread fibrosis or emphysema, respectively.
The procedures described for quantifying the structural changes in postmortem lungs provide data on the lung volumes fixed at a pressure of 25 cm H2O. Such volume measurements are essential to convert subsequent unbiased histologic measurements of cell or tissue densities into total numbers10. This paper only describes the means to obtain a very accurate lung volume. In nine 10&#8211;12 week old healthy Balb/c mice we measured a mean (&#177; SD) fixed lung volume of 0.82 &#177; 0.09 ml with the left lung volume being about 30% of the total. In Balb/c mice given 3 U of pancreatic elastase (with the method described in this paper) to generate experimental emphysema, the lung fixed volume increased to 1.15 &#177; 0.13 ml, with the left lung fraction remaining at 30%.
<img alt="Figure 1" src="/files/ftp_upload/52964/52964fig1.jpg" /> 
Figure 1: Intravenous catheter with slightly curved tip used for instillation.
<img alt="Figure 2" src="/files/ftp_upload/52964/52964fig2.jpg" /> 
Figure 2: Sloped 3-ring binder used to support mice for the intubation. This binder set up to hold 3 mice.
<img alt="Figure 3" src="/files/ftp_upload/52964/52964fig3.jpg" /> 
Figure 3: Formalin filled reservoir on ring stand connected to mouse, with top of fluid set 25 cm above the mouse. This fixation should normally be done in a fume hood.
<img alt="Figure 4" src="/files/ftp_upload/52964/52964fig4.jpg" /> 
Figure 4: Archimedes Principle.&#160;The weight of water displaced by a submerged object equals the volume of the object. Because the lung normally retains some residual air, a device as in Figure 5 is needed to keep the lung fully submerged.
<img alt="Figure 5" src="/files/ftp_upload/52964/52964fig5.jpg" /> 
Figure 5: Lab made support for keeping lungs completely underwater. Device constructed from plastic pipettes and metal wire.
<img alt="Figure 6" src="/files/ftp_upload/52964/52964fig6.jpg" /> 
Figure 6: Laboratory balance with beaker and tared submersion device ready for lung volume measurement.
<img alt="Figure 7" src="/files/ftp_upload/52964/52964fig7.jpg" /> 
Figure 7: Example of a lung given trypan blue through the intubation catheter.

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

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.

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 ...

instillation-and-fixation-methods-useful-in-mouse-lung-cancer-research

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28.2K Views.  Johns Hopkins University. 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.

Video: Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Perfusion and Inflation of the Mouse Lung for Tumor Histology

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.

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.

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

Cancer Research

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

Biology

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Preserving Fungal Spore Localization in Lungs for Fluorescent Microscopy Analysis

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Immunology and Infection

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...

Adenocarcinoma Engraftment: A Method to Implant Cancer Cells into the Murine Lungs

Source: Stevens, L.E. et al, <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56650/pre-conditioning-airways-mice-with-bleomycin-increases-efficiency">Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment.</a> J. Vis. Exp. (2018).
This video describes a method to engraft lung adenocarcinoma cells in murine lungs by preconditioning the airways with fibrosis-inducing agent&#8211;bleomycin. This method significantly enhances orthotopic engraftment of lung cancer cells into murine lungs

Source: Stevens, L.E. et al, Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment. J. ...

JoVE Encyclopedia of Experiments

Lung Cancer

In vivo study

Lung Instillation and Fixation: Introducing Liquid into the Trachea and Fixing Lungs to Measure the Fixed Lung Volume

Source: Limjunyawong, N. et al,&#160; <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/52964/instillation-and-fixation-methods-useful-in-mouse-lung-cancer-research">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> J. Vis. Exp. (2015).
This video describes a simple and reliable method of introducing liquid into the trachea for lung instillation, fixation, and lung volume measurement.

Source: Limjunyawong, N. et al,&#160; Instillation and Fixation Methods Useful in Mouse Lung Cancer Research. J. Vis. Exp. (2015).
This video describes a ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Summary

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Chapters in this video

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Perfusion and Inflation of the Mouse Lung for Tumor Histology

Direct Tracheal Instillation of Solutes into Mouse Lung

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Adenocarcinoma Engraftment: A Method to Implant Cancer Cells into the Murine Lungs

Lung Instillation and Fixation: Introducing Liquid into the Trachea and Fixing Lungs to Measure the Fixed Lung Volume