The authors thank Brian Johnson of the Histology and Imaging Core at the University of Washington for help with the trichrome staining and analysis. This work was supported by NIH grants HL098067 and HL089455.

The Institutional Animal Care and Use Committees (IACUC) at the University of Washington and Cedars-Sinai Medical Center have approved the animal work necessary for these studies.
1. Preparation
<ol>
	<li>Sterilize both the blunt end forceps and the depressor via autoclave.</li>
	<li>Using a biological safety cabinet, prepare a working stock of BLM in PBS from the lyophilized powder.&#160; Sonicate the solution for 10 min at 35 Khz to assure even mixing. 
	Note: A total volume of between 30 and 45 &#181;l i...

<ol>
	<li>Hyde, D. M., Hamid, Q., Irvin, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy,+pathology,+and+physiology+of+the+tracheobronchial+tree:+emphasis+on+the+distal+airways.">Anatomy, pathology, and physiology of the tracheobronchial tree: emphasis on the distal airways.</a> J Allergy Clin Immunol. 124, S72-S77 (2009).</li><li>Rosenthal, N., Brown, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+ascending:+perspectives+for+human-disease+models.">The mouse ascending: perspectives for human-disease models.</a> Nat Cell Biol. 9, 993-999 (2007).</li><li>Peters, L. L., 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=The+mouse+as+a+model+for+human+biology:+a+resource+guide+for+complex+trait+analysis.">The mouse as a model for human biology: a resource guide for complex trait analysis.</a> Nat Rev Genet. 8, 58-69 (2007).</li><li>Baron, R. M., Choi, A. J., Owen, C. A., Choi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+manipulated+mouse+models+of+lung+disease:+potential+and+pitfalls.">Genetically manipulated mouse models of lung disease: potential and pitfalls.</a> Am J Physiol Lung Cell Mol Physiol. 302, 485-497 (2012).</li><li>Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome.">Acute respiratory distress syndrome.</a> BMJ Clin Evid. 2010, (2010).</li><li>Saguil, A., Fargo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome:+diagnosis+and+management.">Acute respiratory distress syndrome: diagnosis and management.</a> Am Fam Physician. 85, 352-358 (2012).</li><li>Wilson, M. S., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+fibrosis:+pathogenesis,+etiology+and+regulation.">Pulmonary fibrosis: pathogenesis, etiology and regulation.</a> Mucosal Immunol. 2, 103-121 (2009).</li><li>Wuyts, W. 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=The+pathogenesis+of+pulmonary+fibrosis:+a+moving+target.">The pathogenesis of pulmonary fibrosis: a moving target.</a> Eur Respir J. 41, 1207-1218 (2013).</li><li>Mouratis, M. A., Aidinis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+pulmonary+fibrosis+with+bleomycin.">Modeling pulmonary fibrosis with bleomycin.</a> Curr Opin Pulm Med. 17, 355-361 (2011).</li><li>Moeller, A., Ask, K., Warburton, D., Gauldie, J., Kolb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bleomycin+animal+model:+a+useful+tool+to+investigate+treatment+options+for+idiopathic+pulmonary+fibrosis?.">The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?.</a> Int J Biochem Cell Biol. 40, 362-382 (2008).</li><li>Myll&#228;rniemi, M., Kaarteenaho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+treatment+of+idiopathic+pulmonary+fibrosis+-+preclinical+and+clinical+studies+of+pirfenidone,+nintedanib,+and+N-acetylcysteine.">Pharmacological treatment of idiopathic pulmonary fibrosis - preclinical and clinical studies of pirfenidone, nintedanib, and N-acetylcysteine.</a> European Clinical Respiratory Journal. 2, (2015).</li><li>Reinert, T., Baldotto, C. S. d. R., Nunes, F. A. P., Scheliga, A. A. d. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin-Induced+Lung+Injury.">Bleomycin-Induced Lung Injury.</a> Journal of Cancer Research. 2013, 1-9 (2013).</li><li>Lakatos, H. F., 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=Oropharyngeal+aspiration+of+a+silica+suspension+produces+a+superior+model+of+silicosis+in+the+mouse+when+compared+to+intratracheal+instillation.">Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation.</a> Exp Lung Res. 32, 181-199 (2006).</li><li>Helms, M. N., Torres-Gonzalez, E., Goodson, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tracheal+instillation+of+solutes+into+mouse+lung.">Direct tracheal instillation of solutes into mouse lung.</a> J Vis Exp. , (2010).</li><li>Osier, M., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+inhalation+vs+intratracheal+instillation:+differences+in+particle+effects.">Intratracheal inhalation vs intratracheal instillation: differences in particle effects.</a> Fundam Appl Toxicol. 40, 220-227 (1997).</li><li>Driscoll, K. E., 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=Intratracheal+instillation+as+an+exposure+technique+for+the+evaluation+of+respiratory+tract+toxicity:+Uses+and+Limitations.">Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: Uses and Limitations.</a> Toxicol Sci. 55, 24-35 (2000).</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 (1985). 106, 984-987 (1985).</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> Lab Anim. 41, 128-135 (2007).</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> J Vis Exp. , e50601 (2013).</li><li>Starcher, B., WIlliams, I. <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+for+intratracheal+instillation+of+endotoxin+into+the+lungs+of+mice.">A method for intratracheal instillation of endotoxin into the lungs of mice.</a> Lab Anim. 23, 234-240 (1989).</li><li>Daubeuf, F., Frossard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+bronchoalveolar+lavage+in+the+mouse.">Performing bronchoalveolar lavage in the mouse.</a> Curr Protoc Mouse Biol. 2, 167-175 (2012).</li><li>Li, Y., 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=Severe+lung+fibrosis+requires+an+invasive+fibroblast+phenotype+regulated+by+hyaluronan+and+CD44.">Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44.</a> J Exp Med. 208, 1459-1471 (2011).</li><li>Grazioli, S., 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=CYR61+(CCN1)+overexpression+induces+lung+injury+in+mice.">CYR61 (CCN1) overexpression induces lung injury in mice.</a> Am J Physiol Lung Cell Mol Physiol. 308, L759-L765 (2015).</li><li>Redente, E. F., 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=Tumor+necrosis+factor-alpha+accelerates+the+resolution+of+established+pulmonary+fibrosis+in+mice+by+targeting+profibrotic+lung+macrophages.">Tumor necrosis factor-alpha accelerates the resolution of established pulmonary fibrosis in mice by targeting profibrotic lung macrophages.</a> Am J Respir Cell Mol Biol. 50, 825-837 (2014).</li><li>Lawrenz, M. B., Fodah, R. A., Gutierrez, M. G., Warawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intubation-mediated+intratracheal+(IMIT)+instillation:+a+noninvasive,+lung-specific+delivery+system.">Intubation-mediated intratracheal (IMIT) instillation: a noninvasive, lung-specific delivery system.</a> J Vis Exp. , e52261 (2014).</li></ol>

In instances where aerosolization is impractical due to limited reagent availability, safety, or cost, direct tracheal administration is a superior method for delivery of exogenous agents into the lungs.16 Transtracheal instillation has been widely used to accomplish this; however, as with all surgical intervention, it also carries with it the potential for complications caused by the procedure itself, and not necessarily the agent being instilled.13 For these reasons, it has become...

In spite of some anatomical and physiological differences,1 murine models continue to be invaluable for modeling human biology and disease pathogenesis.2 From a husbandry standpoint, mice are easy to handle, have a low breeding time, an accelerated lifespan, and are relatively inexpensive to house. With the development of diverse genetic strains and strategies (e.g., conditional knock-outs, reporter mice, lineage-tracing approaches, etc.), as well as the wide range of available reagents (e.g., antibodies, recombinant proteins, inhibitors, etc.), mice have become an essential model vertebrate organism to uncover human homeostasis and disease processes.3
Mice have been especially valuable for studying pulmonary conditions, including acute lung injury (ALI) and pulmonary fibrosis.4 ALI in humans can be caused by trauma, injury, or sepsis and is characterized by epithelial and endothelial leak (i.e., edema), inflammation, and nascent fibrosis. In many patients, ALI progresses to its severe form, acute respiratory distress syndrome (ARDS), which often results in fibrosis and death due to respiratory failure.5,6 Pulmonary fibrosis is a progressive, fatal pathology characterized by the excess deposition of extracellular matrix, most notably type I collagen, leading to impaired lung function.7,8 Administration of bleomycin (BLM) is the most widely used and best characterized model for inducing ALI and fibrosis in experimental animals.9 Although BLM-induced pulmonary fibrosis in rodents does not recapitulate fully the human fibrotic phenotypes,10 mouse studies with this model have led to the discovery of many important factors influencing the onset and progression of disease.11
While the exact mechanism(s) behind BLM-induced fibrogenesis are unknown, the initiating injury is thought to arise from contact-dependent DNA strand breaks in the epithelial cells lining the conducting airways and alveoli, and in particular, type 1 pneumocytes.12 The need for direct contact between BLM and the pulmonary epithelium highlights the importance of a robust delivery route, and these concerns are also germane to a broad range of treatments targeted to the distal airways, including recombinant proteins, antibodies, siRNA, virus, bacteria, particulates, and more. Oropharyngeal aspiration (OPA) has been widely used for this purpose13, but a major a shortcoming of OPA is that some portion of the delivered agent may be swallowed into the gastrointestinal tract, thereby leading to imprecision in the administered dose. Another widely used approach is transtracheal instillation, which involves tracheostomy under strong anesthesia to expose the trachea and instillation of an agent directly into the respiratory tract.14 However, not only may such a procedure be undesirable due to its invasivity, but it is also time consuming, requires a fair bit of training, and causes a potent injury to the respiratory tract.15,16 Several protocols have been developed that involve the direct administration of agents into the trachea without the need for surgical intervention,16,17,18,19,20 but these methods involve extended recovery times caused by powerful anesthetics, the use of expensive equipment (i.e., otoscope/laryngoscope, commercially available procedure boards, fiber-optic wires, etc.), an excess of manipulation in the oral cavity, and uncertainty regarding the dosage.
This paper describes a relatively easy method of administration via intubation that allows a researcher to quickly, inexpensively, and reliably instill a reagent into the murine lung with limited risk of residual damage to the surrounding tissues.

<table border="0" cellpadding="0" cellspacing="0" width="1528">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Bleomycin For Injection, 30 units/vial</td>
			<td style="width:304px;">APP Pharmaceuticals, LLC</td>
			<td style="width:139px;">103720</td>
			<td style="width:604px;">For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt End Forceps</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4&#34;)&#160;</td>
			<td style="width:304px;">Covidien</td>
			<td>E1551G</td>
			<td>Before use, create a 45 degree bend 1.5 cm&#160; from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exel Safelet Catheter 22G X 1&#34;</td>
			<td style="width:304px;">Exel International</td>
			<td>26746</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)</td>
			<td style="width:304px;">BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2ml Pipettor and Filter Tips</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator</td>
			<td style="width:304px;">Dolan Jenner Industries, Inc.</td>
			<td style="width:139px;">181-1</td>
			<td>Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intubation Board</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>See Diagram 1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colored Label Tape: 0.5&#160;in. Wide</td>
			<td style="width:304px;">Fisherbrand</td>
			<td>15-901-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen&#160;</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 1X</td>
			<td style="width:304px;">Corning</td>
			<td>21-040-CV</td>
			<td>Product should be sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0</td>
			<td style="width:304px;">Harvard Apparatus</td>
			<td>517698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table Top Anesthesia Machine Isoflurane</td>
			<td style="width:304px;">Highland Medical Equipment</td>
			<td>N/A</td>
			<td>http://www.highlandmedical.net/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide Top Induction Mouse Isoflurane Chamber</td>
			<td>MIP / Anesthesia Technologies</td>
			<td>AS-01-0530-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FORANE (isoflurane, USP) Liquid For Inhalation 100 mL&#160;</td>
			<td style="width:304px;">Baxter</td>
			<td>1001936040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Nanozoomer Digital Pathology system</td>
			<td style="width:304px;">Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgM ELISA Quantification Kit&#160;</td>
			<td style="width:304px;">Bethyl Laboratories</td>
			<td style="width:139px;">E90-101</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved method for rapid intubation of the trachea in mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

Intubated mice were monitored daily for weight loss and distress, and sacrificed 4, 10 or 17 days later via intraperitoneal injection of 2.5% 2,2,2-tribromoethanol. Bronchoalveolar lavage (BAL) was collected in three washes of PBS as described elsewhere 21, and the right lung was fixed in 10% formalin, paraffin embedded, and stained with Masson&#39;s Trichrome by the University of Washington Histology and Imaging Core 22.
<p class="jove_content"...

Watch this Scientific Journal Video about An Improved Method for Rapid Intubation of the Trachea in Mice at JoVE.com

An Improved Method for Rapid Intubation of the Trachea in Mice

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

The overall goal of this procedure, is to provide quick, easy and reliable administration of reagents into the murine lungs via intubation. The method also limits the risk of residual damage to the surrounding tissues and is inexpensive. This method can help answer key questions in the basic lung biology and mechanisms of disease fields.Such as acute lung injury and fibrogenesis. The main advantages of this technique are that is highly re-produceable, easy to master, does not require specialized equipment or lengthy recovery times. The procedure must be performed using sterilized tools.So autoclave both the blunt end forceps and the depressor. In a sterilized hood, prepare a working stock of re-agent to administer via the intubation. A bolus volume between 30 and 45 microliters is recommended.Sonicate the solution for 10 minutes at 35 kilohertz to assure even mixing. Now, prepare the workspace. Designate at least a square meter of space for the workspace itself.As well as distinct locations for the animals both before and after the procedure. Now, prepare a procedure board. This customized model is cut from an aluminium sheet and reinforced by a T bracket.Similar commercial boards are also available. Using a few strips of tape, fix the base of the procedure board to the bench, so it will be directly in front of the experimenter. Then tie a length of 4.0 suture between the two positioning screws of the procedure board.Next, build a spirometer. Remove the plungers from three one-milliliter syringes. Then load up to 60 microliters of PBS into the top of each syringe barrel.Loosely secure one of these three syringes to the hub of a catheter. When in use, air emanating from the trachea, will cause the PBS in the barrel to rise and fall. Place this next to the procedure board.Place the other two additional syringes within reach to serve as backups. Next, into a fourth syringe, aspirate 300 microliters of air, and place it to one side of the board. Also prepare a few six inch lengths of tape to secure the animal to the board.Then set up an isoflurane chamber if needed. Attach the oxygen, isoflurane and vacuum lines to the appropriate ports on the chamber and the clearance vacuum. Begin with anesthetizing the mouse.Importantly because factors such as the age, weight, and health of the animal can all modify the response the anesthetics, each lab should optimize the isoflurane exposure to meet their own needs. While waiting, load the bolus of reagent into a pipette tip, and set it aside. When the mouse is sedated, suspend it by it's upper incisors from the thread on the procedure platform, with the animals dorsum lying flat against the platform surface.Now, being careful not to restrict ventilation, loosely tape the lower portion of the the thoracic cavity, just above the diaphragm, to the platform, to keep the animal aligned during the procedure. Next, set the gooseneck illuminator to 80 to 100%Position the light one to two centimeters from the surface of the skin near the solar plexus and regularly check that it isn't over heating the skin. From behind the platform use the blunt end forceps to locate the tongue.Then being careful to avoid the lower incisors, gently grip and draw the tongue out of the oral cavity. While securing the tongue, flatten it with the depressor In the non-dominant hand, allowing the forceps to release. Now, guide the gooseneck approximately from the solar plexus to the main stem bronchi to illuminate the trachea.The trachea can also be easily distinguished by the action of respiration, which will cause the light to fluctuate in intensity as it leaves the trachea. When correctly positioned, the trachea can be seen as a central pin of light, with minimal ambient light in the oral cavity itself. The catheter should not enter the oral cavity until the view of the trachea is ideal.The trachea cannot be brought into view through manipulation of either the light source or the depressor. Researchers should release the tongue and re-attempt the procedure. Next, angle the syringe to follow the natural path of the trachea.And lower the 22 gauge catheter tip, with the attached syringe containing the PBS, straight into the lumen. Now, feed the catheter in an additional five millimeters and remove the tongue depressor. If correctly placed the PBS bubble in the barrel will begin to rise and fall with each breath.Be patient when looking for this indicator as both heavy and light sedation can modify the frequency of respiration. Then shift the syringe to the non dominant hand. While gripping the hub, gently remove the syringe leaving the catheter in place.From the dominant hand, deposit the prepared bolus of reagent directly into the center of the catheter hub. Then attach the second syringe with 300 microliters of air and eject the air into the hub. This will help ensure that the bolus is well distributed in the lungs.Now, remove the syringe and replace it with a syringe containing the bubble of PBS. The bubble will continue to rise and fall if the procedure has been performed successfully. Now, remove the catheter and tape.And place the animal in a dry warm place until it regains consciousness. Typically within a couple of minutes. Intubated mice were monitored daily for weight loss.Mice were collected at four, 10, and 17 days post intratracheal intubation with bleomycin. Bronchoalveolar lavage was collected from these lungs and analyzed. Elevated levels of IgM demonstrated a significant time dependent increase in lung permeability indicative of epithelial and/or endothelial barrier dysfunction.The right lungs were fixed and stained with Masson's trichrome. The fibrogenic response, a well established marker of total collagen content, was quantified. The histology sections evidenced treatment dependent thickening of the pulmonary interstitium, and increases in fibrotic lesions.Once mastered this technique can be done in under a minute if properly performed. While attempting this procedure it's important to remember that manipulation of the soft tissues surrounding the trachea is not necessary or recommended. And it can compromise both the health of the animal, and the integrity of the experiment.

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24.8K 次观看.  University of Washington. The overall goal of this procedure, is to provide quick, easy and reliable administration of reagents into the murine lungs via intubation. The method also limits the risk of residual damage to the surrounding tissues and is inexpensive. This method can help answer key questions in the basic lung biology and mechanisms of disease fields.Such as acute lung injury and fibrogenesis. The main advantages of this technique are that is highly re-produceable, easy to master, does not require s...