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 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

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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JoVE Journal

Medicine

28.1K Views.  Johns Hopkins University.  Цель данной работы является описание простых методов, которые значительно помочь в настройке и анализа легких мышей с раком легких или других патологий. Мы представляем 3 протоколы просто и надежно выполнять инстилляций легких, фиксация и измерения объема л?...

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

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 ...