The authors are grateful for the support from the Center for Predicative Medicine Animal Models (Carol Vanover, Ashley Biller and Jennifer Kraenzle) and Microbiology (Daniel Cramer and Julie Sotsky) Core Facilities. This work was supported by funding from the NIH (HHSN272201000033I to M.B.L and J.M.W.).

NOTE: All of the procedures described here were reviewed and approved by the University of Louisville Institutional Biosafety Committee (protocol # 13-056) and Institutional Animal Care and Use Committee (protocol # 13-064).
1. Preparation of Dye
<ol>
	<li>Dilute 0.1% (w/v) Coomassie Brilliant Blue in PBS and filter sterilize using a 0.45 &#181;M syringe filter.</li>
</ol>
2. Preparation of Pseudomonas aeruginosa Culture
<ol>
	<li>15 hrs before instillation, inoculate 3 ml of broth culture with a single bacterial colony.</li>
	<li>Grow the culture 15 hr at 37 &#176;C on a shaker (200 rpm).</li>
	<li>Centrifuge 1 ml of culture in a 1.5 ml microfuge tube 12,000 x g for 30 sec.</li>
	<li>Remove the medium and resuspend the pellet in 1ml of PBS.</li>
	<li>Dilute an aliquot of the bacterial stock suspension 1:10 in PBS and measure the OD600 of the diluted bacterial suspension to determine the bacterial concentration.</li>
	<li>Dilute the bacterial stock suspension in PBS to the desired concentration of bacterial inoculum, using a delivery volume of 50 &#181;l for IMIT inoculation.</li>
</ol>
3. IMIT Instillation
<ol>
	<li>Place a group of mice into isoflurane anesthesia induction chamber and anesthetize using a 2 - 3% isoflurane/oxygen mixture.</li>
	<li>At the initial onset of sedation, scruff the mouse, holding the mouse upright, and administer 10 &#181;l of a 2% lidocaine solution by gavage needle to the back of the throat and allow the solution to drain down to the epiglottis. Return the mouse to the anesthesia chamber.
	<ol>
		<li>Allow a minimum of 5 min to permit the lidocaine to take full effect as a local anesthetic.</li>
	</ol>
	</li>
	<li>When mice have achieved desired level of sedation (breathing rate of ~60 bpm), reduce the isoflurane to 2% to maintain sedation.</li>
	<li>Preload dye or bacterial inoculum into a 250 &#181;l gas-tight precision syringe fit with a 22 G long blunt needle.
	<ol>
		<li>First draw up 150 &#181;l of air, measured by the Teflon plunger of the syringe. Next, draw up 50 &#181;l of inoculum by advancing the Teflon plunger from the 150 &#181;l mark to the 200 &#181;l mark on the syringe body. 
		NOTE: When the sample is ejected into an intubated mouse, the 50 &#181;l suspension will be delivered first, followed by a 150 &#181;l air cushion which will distribute the inoculum throughout the lung.</li>
	</ol>
	</li>
	<li>Remove one mouse from the induction chamber and lay supine on an intubation platform. Secure the mouse to the platform by hooking its incisors with an O-ring attached to Velcro strip, and securing the Velcro to the platform. Raise the mouse to a 45&#176; incline.</li>
	<li>Using a micro cotton applicator, retract the tongue with a rolling motion, using the dominant hand.</li>
	<li>With the nondominant hand, use an operating otoscope fit with an intubation specula to both maintain tongue retraction and visualize the glottis.</li>
	<li>With the dominant hand, use a guide wire threaded through a 20 G catheter to intubate the mouse, seating the catheter to a 10 mm depth into the mouse trachea (catheter is fit with a silicon sleeve with 10 mm of exposed catheter). Remove the otoscope/specula.</li>
	<li>Confirm that the mouse has been correctly intubated by securing the catheter with the nondominant hand while briefly attaching a Luer connected length of 1/16&#8221; clear tubing containing a colored dye. 
	NOTE: The dye will rapidly migrate back and forth in response to breathing.</li>
	<li>Do not proceed with subsequent steps if confirmation of intubation has not been established at this point. If the intubation attempted failed, reset the catheter and guide wire for one additional attempt at intubation. 
	NOTE: It is unadvisable to attempt more than two intubations of a mouse in one session without causing trauma to the mouse.</li>
	<li>Continue to secure the catheter with the nondominant hand while inserting the precision syringe/blunt needle containing the liquid suspension/air cushion.</li>
	<li>Dispense the liquid/air directly into the lung in a single fluid motion and immediately remove the needle/catheter from the mouse.</li>
	<li>Return the mouse to a cage and allow recovery from anesthesia.</li>
</ol>
4. Characterization of IMT Delivery
<ol>
	<li>After IMIT instillation, euthanize the mouse by CO2 asphyxia at an appropriate time post-inoculation.</li>
	<li>Secure the euthanized mouse on a dissection board and soak the chest and abdomen with 70% EtOH using a squirt bottle.</li>
	<li>If evaluating distribution of an imaging agent throughout the lung, remove the lungs from the animal using sterile technique and display the lungs as appropriate for imaging. 
	NOTE: Lungs may be prepared for additional histological staining techniques through appropriate fixation or cryopreservation.</li>
	<li>If evaluating the bacterial burden of infected lung tissue, remove the lungs from the animal using sterile technique. Place lungs into a sterile, preweighed 1 oz sample bag. Weigh and record the weight of the sample bag + lungs.
	<ol>
		<li>Add 1 ml of sterile 1x PBS to each sample bag+ tissues. Reseal sample bag.</li>
		<li>Homogenize tissues by gentle rolling a 25 ml serological pipette over the sample bag + tissue.</li>
		<li>Generate serial dilutions of the lung homogenate in sterile PBS and plate on agar plate (LB, or as appropriate to the bacterial species being studied):
		<ol>
			<li>Conduct a six fold serial dilution in a U-bottom 96 well plate by multichannel pipettor then plate triplicate samples by multichannel on the agar plate.</li>
		</ol>
		</li>
		<li>Incubate agar plates overnight at 37 &#176;C and enumerate colony forming units the next day.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Reznik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy,+physiology,+and+function+of+the+upper+respiratory+tract.">Comparative anatomy, physiology, and function of the upper respiratory tract.</a> Environ Health Perspect. 85, 171-176 (1990).</li><li>Hoyt, R. F. J., Hawkins, J. V., St. Clair, M. B., Kennet, M. B., Fox, J. G., 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=Chapter+2.">Chapter 2.</a> The Mouse in Biomedical Research, Volume 2, Second Edition: Diseases (American College of Laboratory Animal Medicine). 2, 23-90 (2007).</li><li>Visweswaraiah, A., Novotny, L. A., Hjemdahl-Monsen, E. J., Bakaletz, L. O., Thanavala, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+tissue+distribution+of+marker+dye+following+intranasal+delivery+in+mice+and+chinchillas:+a+multifactorial+analysis+of+parameters+affecting+nasal+retention.">Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention.</a> Vaccine. 20, 3209-3220 (2002).</li><li>Eyles, J. E., Spiers, I. D., Williamson, E. D., Alpar, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+radioactivity+following+intranasal+administration+of+radioactive+microspheres.">Tissue distribution of radioactivity following intranasal administration of radioactive microspheres.</a> J Pharm Pharmacol. 53, 601-607 (2001).</li><li>Southam, D. S., Dolovich, M., O'Byrne, P. M., Inman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+intranasal+instillations+in+mice:+effects+of+volume,+time,+body+position,+and+anesthesia.">Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia.</a> Am J Physiol Lung Cell Mol Physiol. 282, 833-839 (2002).</li><li>Miller, M. 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=Visualization+of+murine+intranasal+dosing+efficiency+using+luminescent+Francisella+tularensis:+effect+of+instillation+volume+and+form+of+anesthesia.">Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.</a> PLoS ONE. 7, (2012).</li><li>Lathem, W. W., Crosby, S. D., Miller, V. L., Goldman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+primary+pneumonic+plague:+a+mouse+model+of+infection,+pathology,+and+bacterial+transcriptional+activity.">Progression of primary pneumonic plague: a mouse model of infection, pathology, and bacterial transcriptional activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17786-17791 (2005).</li><li>Warawa, J. M., Long, D., Rosenke, R., Gardner, D., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+diagnostic+imaging+to+characterize+altered+respiratory+tract+colonization+by+the+Burkholderia+pseudomallei+capsule+mutant.">Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the Burkholderia pseudomallei capsule mutant.</a> Front Microbiol. 2, 133 (2011).</li><li>Gutierrez, M., Pfeffer, T. L., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+3+Secretion+System+cluster+3+is+a+critical+virulence+determinant+for+lung-specific+melioidosis.">Type 3 Secretion System cluster 3 is a critical virulence determinant for lung-specific melioidosis.</a> Submitted. , (2014).</li><li>Fodah, R. 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=Correlation+of+Klebsiella+pneumoniae+comparative+genetic+analyses+with+virulence+profiles+in+a+murine+respiratory+disease+model.">Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model.</a> PLoS ONE. In revision, (2014).</li><li>Soto-Montenegro, M. 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=Assessment+of+airway+distribution+of+transnasal+solutions+in+mice+by+PET/CT+imaging.">Assessment of airway distribution of transnasal solutions in mice by PET/CT imaging.</a> Mol Imaging Biol. 11, 263-268 (2009).</li></ol>

The authors have nothing to disclose.

IMIT instillation offers key improvements to existing respiratory disease models in the ability to reproducibly instill reagents directly into the lung. It is a rapid approach which is ideally situated for a team of two researchers, one of which manages the logistics of anesthesia and caging, and the other who performs the IMIT technique. Large studies may be conducted using IMIT with an average time commitment of 2 - 3 min per mouse. Because the approach makes use of isoflurane as an anesthetic, mice recover rapidly from the anesthesia, reducing the husbandry time of monitoring animals through recovery.
The most technically challenging aspect of the IMIT method is the initial step of intubating mice. Individuals learning to perform IMIT are able to focus on this first step of catheter placement and ensuring that intubation has been achieved through the visual confirmation of dye movement. The benefit of the approach is that lung-specific instillation is guaranteed through use of the confirmation of intubation, which increases the confidence of both the new researcher as well as the expert attempting to intubate a difficult animal. The key elements of optimizing the likelihood of a successful intubation are: i) achieving a deep sedation to allow sufficient working time, ii) correct placement of the specula in the mouth to allow good visualization of the epiglottis, iii) good depth placement of the specula so that the tongue remains retracted throughout the procedure, and iv) use of the tilting platform to support the researcher&#8217;s hands so that the procedure is conducted relaxed and with a steady approach.
One of the limitations of the IMIT procedure is related to frequency of IMIT instillation events. Due to the potential trauma associated with a missed intubation, it is not recommended that more than two intubation attempts be conducted in a single session (up to two misses). IMIT has an excellent potential in its ability to be used to deliver therapeutics into the murine lung, however therapeutic regimens which make use of very frequent delivery of reagent into the lung may not be suitable for IMIT. It may be possible that IMIT could be used daily to deliver reagents into a murine lung without causing significant trauma, but only when conducted by a highly skilled researcher, as the majority of trauma associated with intubation is thought to be associated with a missed intubation event. Such high-frequency IMIT should be discussed with local veterinarians and IACUC.
An additional potential limitation of IMIT is the size of the mouse which is being intubated. The IMIT procedure described above was developed using mice of approximately 17 - 22 g, where a 20 G catheter was found to be a suitable size for the trachea of mice in this size range. Larger catheters have been successfully used in older mice; the initial development of IMIT made use of an 18 G catheter in BALB/c mice which are &#62;20 g. Importantly, if alternate catheter sizes are used, blunt needles should be sourced which fit the lumen of the catheter and are trimmed to a length that extends just 1mm beyond the catheter tip. Intubation of mice smaller than 17 g may be possible but is not recommended due to the expertise required, and would require use of smaller catheters and specula than are described above.
We have used IMIT for the delivery of several respiratory pathogens in addition to P. aeruginosa, including B. pseudomallei9 and Klebsiella pneumoniae10. The IMIT model has made important advances to our studies of B. pseudomallei respiratory disease, having identified that intranasal inoculation causes an early, URT-related morbidity of mice rather than the systemic disease endpoint observed in human disease9. B. pseudomallei is a Tier 1 select agent of biodefense impact, and as such, respiratory disease models are being developed for aerosol exposure which models a potential biodefense related route of entry for weaponized pathogens. Because current aerosol models result in infection of both the URT and LRT, the same potential early morbidity phenotypes we have identified for the intranasal model of B. pseudomallei respiratory disease may apply to the aerosol model. A future adaptation of the IMIT model could be an intubation-mediated aerosol delivery (IMAD), in which mice are intubated for target aerosol delivery only into the lung. Mechanical ventilators are currently available to maintain isoflurane anesthesia, which could be adapted to deliver an aerosolized, rather than liquid based, pathogen challenge.
IMIT was developed initially as an approach to optimize the delivery of bacteria to the lung, but also has application for the delivery of other reagents into the mouse lung. As discussed above, intranasal delivery of compounds into mice results in a low efficiency, highly variable delivery of reagents into the target organ of the lung. Intranasal delivery of Positron Emission Tomography (PET) imaging reagents to the murine lung yielded a 40% delivery efficiency11, whereas we have demonstrated that IMIT offers an excellent alternative to other lung delivery approaches with its &#62;98% delivery efficacy and multilobar distribution. This improvement in targeted delivery to the lung has the potential to increase the reproducibility of therapeutic delivery for treatment of pulmonary disease. IMIT could similarly offer benefits to studies of: i) the impact of environmental pulmonary irritants, ii) lung cancer phenotypic studies, iii) lung-specific siRNA knock-down.

Mice have been used to model numerous human disease manifestations, including a myriad of respiratory diseases. Surrogate disease models are often unable to recapitulate all aspects of a modeled disease, typically due to important physiologic or immune differences in the two host models. Thus, a goal of improving model systems is to develop approaches that allow surrogates to more closely mirror a disease process or host response as observed in the original host system. There are several key physiologic differences between mice and humans in the mechanism by which they inspire air. Included in these differences are significant ratiometric differences in size between the URT and LRT. It has been estimated that mice possess &#62;100 fold the URT surface area relative to humans, normalized against total lung capacity1,2. Thus, the nasal turbinates of the mouse allow for more extensive filtering of inspired air to facilitate a much greater rate of breathing, which may have a significant impact on studies of pneumonia if infection of the nasal cavity plays a significant role in disease progression.
Several different approaches have been employed to instill bacteria into the lungs of mice to study human-like respiratory disease. The most common of these approaches is intranasal inoculation, in which a liquid suspension is applied at one or both nares of a mouse. While relatively simple, caveats such as instillation volume and type of anesthesia used can impact the efficiency of instillation into the LRT via intranasal inoculation3-5. Specifically, Miller et al. have shown that intranasal instillation of Francisella tularensis in volumes less than 50 &#181;l did not result in instillation of the bacteria into the LRT6. They further observed better LRT instillation when using inhaled isoflurane as opposed to injected ketamine/xylazine for anesthesia. However, our experience with Yersinia pestis intranasal inoculation indicates more consistent inoculation can be achieved using ketamine/xylazine as compared to isoflurane (MBL, unpublished data). These differences could be attributed to pathogen used or to variation in laboratory procedures, but importantly highlight the potential variability in this technique. Furthermore, lungs harvested shortly after intranasal instillation show that a relatively low percentage of the initial bacterial inoculum reaches the lung (in the case of Y. pestis, only 10% were recovered 1 hr after instillation7), suggesting that a large number of bacteria could be retained in the URT (or swallowed into the GI tract). In certain disease models, this significant deposition of bacteria on the URT mucosa may confound our understanding of the disease progression if the organism is capable of colonizing the murine nasal cavity in a manner inconsistent with the human disease. For example, using in vivo imaging, it has been observed that Burkholderia pseudomallei, which does not colonize the human URT, causes an overwhelming opportunistic infection of the murine nasal cavity when delivered by the intranasal instillation method8.
Other methods for instilling bacteria into the lungs of mice have also been employed in infectious disease research. However, compared to intranasal instillation these methods tend to require more technical expertise and/or expensive equipment without eliminating the potential for infection initiation at multiple sites (e.g., aerosol [URT and LRT]; transoral [digestive tract and LRT]; and surgical intratracheal [LRT and blood stream]). Given the potential complications that could be associated with secondary sites of infection, we sought to develop an intratracheal approach which bypasses the URT and delivers pathogen directly into the lungs of anesthetized mice, but also limits inadvertent inoculation into the blood stream or GI tract. Towards this end, intubation-mediated intratracheal (IMIT) instillation was developed as a nonsurgical procedure which guarantees LRT instillation of inoculum by including an intermediate step to verify proper catheter placement prior to instillation. This method is described using dye instillation to visually demonstrate broad distribution of the inoculum throughout the lung, and P. aeruginosa instillation to demonstrate the highly efficacious delivery (&#62;98% of the inoculum) of this method to the lung. Importantly, while originally developed for bacterial delivery, IMIT also offers an effective tool for: i) instillation of various molecules for the study of other respiratory disease models, ii) lung-specific therapeutic delivery, and iii) basic lung function studies, including targeted siRNA delivery to the lung.

<table><tbody><tr><td>Rodent, Tilting WorkStand</td><td>Hallowell EMC</td><td>000A3467</td><td>Base should be detached when working in a BSC</td></tr><tr><td>Operating Otoscope Head</td><td>Welch Allyn</td><td>21700</td><td></td></tr><tr><td>Otoscope 3.5 V Li Battery</td><td>Welch Allyn</td><td>71900</td><td></td></tr><tr><td>Mouse Intubation Specula short, Autoclaveable</td><td>Hallowell EMC</td><td>200A3589S</td><td></td></tr><tr><td>Incisor Loops</td><td>Hallowell EMC</td><td>210A3490A</td><td></td></tr><tr><td>Cotton fine tip applicator</td><td>Puritan</td><td>871-PC DBL&amp;nbsp;</td><td>Used for tongue retraction</td></tr><tr><td>I.V. Catheter, 20 G</td><td>Exel Int</td><td>26741</td><td>Optional: fit a silicon sleeve with 10 mm exposed catheter surface</td></tr><tr><td>Gas tight syringe, 250 ul</td><td>Hamilton</td><td>81120</td><td>Used for delivery of liquid inoculum by IMIT</td></tr><tr><td>Blunt Needle, 22 G</td><td>Hamilton</td><td>91022</td><td>Trim to length to protrude 1 mm from 20 G catheter</td></tr><tr><td>Guide wire (Fiber optic wire, 0.5 mm)</td><td>TheFiberOpticStore.com</td><td>FOF .50</td><td>Cut to 6&quot; length: used as guide wire for intubation</td></tr><tr><td>Tuberculin syringe, 1 ml</td><td>Becton Dickinson</td><td>309659</td><td>Assemble with fiber optic wire as guide wire</td></tr><tr><td>Brilliant Blue R (Coomassie)</td><td>Sigma</td><td>B0149</td><td></td></tr><tr><td>Tygon tubing, 1/16&quot;</td><td>Saint Gobain</td><td>ALC00002&amp;nbsp;</td><td></td></tr><tr><td>Male Luer 1/16&quot; barb</td><td>Cole Parmer</td><td>45503-22</td><td></td></tr><tr><td>Female Luer 1/16&quot; barb</td><td>Cole Parmer</td><td>45500-00</td><td></td></tr><tr><td>Lidocaine, USP</td><td>Spectrum</td><td>LI102</td><td>pH lidocaine into solution at 2%(w/v) pH7.0</td></tr><tr><td>Sample bag, 1 oz</td><td>Whirl-Pak</td><td>B01067</td><td></td></tr><tr><td>U-bottom 96 well plate, sterile</td><td>Greiner</td><td>650161</td><td></td></tr></tbody></table>

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.

To visualize the distribution of instilled material via the IMIT method, 50 &#181;l of 0.1% Coomassie Brilliant Blue dye was instilled into the lungs of an anesthetized mouse. The mouse was immediately euthanized and the lungs were removed by sterile necropsy. Figure 1 shows that the dye was delivered to all lobes of the lung.
To determine the amount of bacteria delivered to the lungs via the IMIT method, three groups of mice (n = 3) were instilled with three different concentrations of P. aeruginosa (1.21 x 108, 1.21x107, and 1.21 x 106 colony forming units [CFU] per 50 &#181;l). Immediately after IMIT instillation, mice were euthanized, lungs removed, and bacterial numbers were enumerated and compared to the inoculums (Figure 2). Delivery of the inoculum is highly efficient via this method, with &#62;98% of the inoculum recovered from the lungs of instilled animals. Furthermore, IMIT instillation was highly reproducible regardless of the concentration of the inoculum (R2 = 0.9951).
<img alt="Figure 1" src="/files/ftp_upload/52261/52261fig1highres.jpg" /> 
Figure 1: IMIT instillation distributes inoculum throughout the lungs. Lungs from mice instilled with 50 &#181;l of 0.1% Coomassie Brilliant Blue show blue dye distributed in all lobes.
<img alt="Figure 2" src="/files/ftp_upload/52261/52261fig2highres.jpg" /> 
Figure 2: IMIT instillation of bacteria into lungs. Mice were instilled with P. aeruginosa and the number of bacteria instilled into the lungs (Log10 CFU &#8211; recovered) were compared to the estimated inoculum (Log10 CFU &#8211; delivered). Each circle represents the CFU/lung of an individual mouse (n = 3 for each bacterial dose).

Watch this Scientific Journal Video about Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System at JoVE.com

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.

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 ...

intubation-mediated-intratracheal-imit-instillation-noninvasive-lung

Nanyang Technological University

Research

JoVE Journal

Medicine

24.3K Views.  University of Louisville Medical School.  Intratracheale intubatie-gemedieerde (IMIT) instillatie van reagentia is een uitstekend invasieve methode voor het bestuderen ademhalingsziekte, alsmede een werkwijze voor het indruppelen therapeutische reagentia direct in de longen. Het is een snelle en zeer reproduceerbare werkwijze die geschikt is voor preklinische proeven. 

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

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Immunology and Infection

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 ...

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

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 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 ...

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.

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 ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and minimizing systemic drug exposure. The transcutaneous technique is simple and allows for the IT delivery of substances to the lung of prematurely born rabbits shortly after birth. Newborn pups are anesthetized with inhaled Isoflurane before being placed in a supine position with the neck extended. The larynx is identified and stabilized before transcutaneous placement of a 26-gauge (G) catheter into the trachea. Following catheterization of the trachea, a 30 G blunt needle attached to a Hamilton syringe is introduced into the IT catheter and is used for delivering a precise volume into the trachea during spontaneous respiration. After the IT injection is completed, the needle and catheter are withdrawn, and the pup is allowed to recover from anesthesia. Transcutaneous IT injection delivers a large proportion of the injected substance to the lung, with the majority remaining in the lung 3 hours after the intervention. The injections are well tolerated from the day of birth and can be repeated for multiple consecutive days without influencing survival. This technique can be used to investigate the effect of pharmaceutical agents on lung development and in the prevention of neonatal lung injury in preterm rabbits.

Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and ...

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

Here, we propose a protocol for intratracheal lipopolysaccharide (LPS) delivery via noninvasive oropharyngeal endotracheal intubation. This method minimizes the trauma of the surgical procedure for the animal and accurately delivers LPS to the trachea and then to the lungs.

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

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

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