This work was supported in part by R01HD090887-01A1 from NICHD to AH. The authors also acknowledge the facilities provided by Dr. Peter Mc Farlane&#39;s lab such as inhalation anesthesia/ heating pad system. Ms. Catherine Mayer&#39;s valuable assistance in setting up of the system is appreciated. No role was played by the funding body in the design of the study, collection, analysis and interpretation of data or in writing the manuscript.

All experiments were approved by the Institutional Animal Care and Use Committee (protocol # 2020-0035) at the Case Western Reserve University. All animals were treated in accordance with the NIH guidelines for the care and use of laboratory animals.
1. Animals
<ol>
	<li>Commercially obtain pregnant Sprague Dawley rats.</li>
	<li>Maintain animals at an approved veterinary facility with 14 h/10 h light-dark cycle and 45-60% relative humidity.</li>
</ol>
2. Preparation of test compound
<ol>
	<li>Use Evans blue dye as the test compound to assess the efficacy of the intratracheal instillation procedure.</li>
	<li>Prepare a 0.25% (w/v) solution of the dye in phosphate-buffered saline (pH 7.2) and filter sterilize using a 0.45 &#181;m syringe filter.</li>
</ol>
3. Administration of anesthesia
<ol>
	<li>Anesthetize rat pups using gas anesthesia (3% isoflurane in 100% oxygen), using a modified delivery system adapted for small rat neonates.</li>
	<li>Check for the loss of tail and pedal reflexes and shallow breathing to ensure the proper depth of anesthesia for carrying out the procedure.</li>
</ol>
4. Intratracheal instillation (ITI)
<ol>
	<li>Use rat pups at post-natal day 5 (PN 5) for the ITI. Average weight of a PN 5 rat pup is 12 grams.</li>
	<li>Restrain the anesthetized rat pup on an inclined flat platform using laboratory labelling tape. The pup is restrained at an angle of about 45&#176; in the supine position.</li>
	<li>Open the mouth of the neonate, and gently pull the tongue out to one side using a blunt forceps.</li>
	<li>Use a small otoscope speculum of 2 mm diameter connected to the otoscope to hold the tongue gently and for proper visualization of the larynx.</li>
	<li>Use the throat illuminator system i.e., the operating otoscope, and the magnifying lens for proper visualization of vocal cords (Figure 1).</li>
	<li>Position&#160;the animals at an angle of 45&#176; in an inclined plane. The wired bar lids of mouse cages are used (Figure 2). 
	NOTE: Positioning the animal at an angle of 45&#176; provides better visualization of tracheal opening without the interference of the epiglottis.</li>
	<li>Take a long-angled pipette tip which is used for loading western blot gels. Cut the base of the pipette tip using a surgical blade so that it fits well into the tip of 1 cc syringe.</li>
	<li>Use the sterile 1 mL syringe fitted into a long-angled pipette tip to deliver 30-50 &#181;L of the substance into the lung. Invert the syringe and aspirate nearly 0.9 cc air into the 1 mL syringe connected to the pipette tip followed by the dye or the substance to be delivered. This allows the air behind the dye to be pushed into the trachea after the dye is administered as shown in Figure 3. The intratracheal administration is achieved by visualizing the laryngeal lumen (vocal cords) and inserting the pipette tip fitted to a syringe into the tracheal lumen.</li>
	<li>Use the speculum of the otoscope to hold the tongue and expose the vocal cords. Speculum serves the role of the blade of a laryngoscope. Bend the pipette tip to an angle of 30&#176; to facilitate easy introduction of the agent through the cone-shaped speculum into the tracheal opening.</li>
	<li>Introduce the pipette tip into the tracheal opening to the point of about 2 mm beyond the vocal cords. Push the piston of the syringe to administer the dye or the drug through the speculum of the operating otoscope as shown in Figure 3. The introduction of air into the lung soon after the administration of the agent prevents the substance from coming back to the laryngeal cavity.</li>
	<li>After administering the pup with the dye or normal saline, place the pups on an integrated circulating fluid heating pad (38&#176;C) until their respiratory movements are regular. After complete recovery from anesthesia, reunite the pups with the dam.</li>
</ol>
5. Characterization of ITI delivery
<ol>
	<li>After ITI, euthanize the rat pups by giving excessive anesthesia (Ketamine 100 mg/kg and Xylazine 10 mg/kg) / thiopentone followed by exsanguination at an appropriate time post-administration. Euthanasia was performed as part of the experiment to collect lung tissue to demonstrate the efficacy.</li>
	<li>Secure the euthanized rat pup on a dissection board and wipe the chest and abdomen with 70% ethyl alcohol.</li>
	<li>For evaluating the distribution of the dye throughout the lung, remove the lungs from the animal using sterile technique and display the lungs as appropriate for imaging (Figure 4A,B).</li>
</ol>

<ol>
	<li>Pulmonary Cell Biology Lab: Neonatal Lung Disease. Mayo Clinic Available from: <a target="_blank" href="https://www.mayo.edu/research/labs/pulmonary-cell-biology/projects/neonatal=lung-disease">https://www.mayo.edu/research/labs/pulmonary-cell-biology/projects/neonatal=lung-disease</a> (2020)</li><li>Mart&#237;nez-Burnes, J., L&#243;pez, A., Lemke, K., Dobbin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transoral+intratracheal+inoculation+method+for+use+with+neonatal+rats.">Transoral intratracheal inoculation method for use with neonatal rats.</a> Comparative Medicine. 51 (2), 134-137 (2001).</li><li>Pinkerton, K. E., Crapo, J. D., Witschi, H., Brain, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometry+of+the+alveolar+region+of+the+lung.">Morphometry of the alveolar region of the lung.</a> Toxicology of Inhaled Materials Handbook of Experimental Pharmacology. 95, 259-285 (1985).</li><li>Nardiello, C., Mi&#382;&#237;kov&#225;, I., Morty, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+ahead:+where+to+next+for+animal+models+of+bronchopulmonary+dysplasia.">Looking ahead: where to next for animal models of bronchopulmonary dysplasia.</a> Cell and Tissue Research. 367 (3), 457-468 (2017).</li><li>Sugimoto, M., Ando, M., Senba, H., Tokuomi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+defenses+in+neonates:+Effects+of+bronchial+lavage+fluids+from+adult+and+neonatal+rabbits+on+superoxide+production+by+their+alveolar+macrophages.">Lung defenses in neonates: Effects of bronchial lavage fluids from adult and neonatal rabbits on superoxide production by their alveolar macrophages.</a> Journal of the Reticuloendothelial Society. 27 (6), 595-606 (1980).</li><li>Grayson, M. H., 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=Controls+for+lung+dendritic+cell+maturation+and+migration+during+respiratory+viral+infection.">Controls for lung dendritic cell maturation and migration during respiratory viral infection.</a> Journal of Immunology. 179 (3), 1438-1448 (2007).</li><li>Moreira, 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=Intranasal+delivery+of+human+umbilical+cord+Wharton's+jelly+mesenchymal+stromal+cells+restores+lung+alveolarization+and+vascularization+in+experimental+bronchopulmonary+dysplasia.">Intranasal delivery of human umbilical cord Wharton's jelly mesenchymal stromal cells restores lung alveolarization and vascularization in experimental bronchopulmonary dysplasia.</a> Stem Cells Translational Medicine. 9 (2), 221-234 (2020).</li><li>Bar-Haim, 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=Interrelationship+between+dendritic+cell+trafficking+and+Francisella+tularensis+dissemination+following+airway+infection.">Interrelationship between dendritic cell trafficking and Francisella tularensis dissemination following airway infection.</a> PLoS Pathogens. 4 (11), 1000211 (2008).</li><li>Linderholm, A. L., Franzi, L. M., Bein, K. J., Pinkerton, K. E., Last, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+comparison+of+intranasal+and+intratracheal+administration+of+coarse+PM+in+the+mouse.">A quantitative comparison of intranasal and intratracheal administration of coarse PM in the mouse.</a> Integrative Pharmacology, Toxicology and Genotoxicology. 1 (1), 5-10 (2015).</li><li>Guerra, K., 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=Intra-tracheal+administration+of+a+naked+plasmid+expressing+stromal+derived+factor-1+improves+lung+structure+in+rodents+with+experimental+bronchopulmonary+dysplasia.">Intra-tracheal administration of a naked plasmid expressing stromal derived factor-1 improves lung structure in rodents with experimental bronchopulmonary dysplasia.</a> Respiratory Research. 20 (1), 255 (2019).</li><li>Thomas, R., 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=Influence+of+particle+size+on+the+pathology+and+efficacy+of+vaccination+in+a+murine+model+of+inhalational+anthrax.">Influence of particle size on the pathology and efficacy of vaccination in a murine model of inhalational anthrax.</a> Journal of Medical Microbiology. 59, 1415-1427 (2010).</li><li>Sakurai, R., 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+combination+of+the+aerosolized+PPAR-&#947;+agonist+pioglitazone+and+a+synthetic+surfactant+protein+B+peptide+mimic+prevents+hyperoxia-induced+neonatal+lung+injury+in+rats.">A combination of the aerosolized PPAR-&#947; agonist pioglitazone and a synthetic surfactant protein B peptide mimic prevents hyperoxia-induced neonatal lung injury in rats.</a> Neonatology. 113 (4), 296-304 (2018).</li><li>Rayamajhi, M., 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=Non-surgical+intratracheal+instillation+of+mice+with+analysis+of+lungs+and+lung+draining+lymph+nodes+by+flow+cytometry.">Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry.</a> Journal of Visualized Experiments. (51), e2702 (2011).</li><li>Brain, J. D., Knudson, D. E., Sorokin, S. P., Davis, M. 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+distribution+of+particles+given+by+intratracheal+instillation+or+by+aerosol+inhalation.">Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation.</a> Environmental Research. 11 (1), 13-33 (1976).</li><li>Pritchard, J. 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=The+distribution+of+dust+in+the+rat+lung+following+administration+by+inhalation+and+by+single+intratracheal+instillation.">The distribution of dust in the rat lung following administration by inhalation and by single intratracheal instillation.</a> Environmental Research. 36 (2), 268-297 (1985).</li><li>Ruzinski, J. T., Skerrett, S. J., Chi, E. Y., Martin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+of+particles+in+the+lungs+of+infant+and+adult+rats+after+direct+intratracheal+administration.">Deposition of particles in the lungs of infant and adult rats after direct intratracheal administration.</a> Laboratory Animal Science. 45 (2), 205-210 (1995).</li><li>Sun, B., Curstedt, T., Song, G. W., Robertson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+improves+lung+function+and+morphology+in+newborn+rabbits+with+meconium+aspiration.">Surfactant improves lung function and morphology in newborn rabbits with meconium aspiration.</a> Biology of the Neonate. 63 (2), 96-104 (1993).</li><li>Nicholson, J. W., Kinkead, 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=A+simple+device+for+intratracheal+injections+in+rats.">A simple device for intratracheal injections in rats.</a> Laboratory Animal Science. 32 (5), 509-510 (1982).</li><li>Carlon, M., 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=Efficient+gene+transfer+into+the+mouse+lung+by+fetal+intratracheal+injection+of+rAAV2/6.2.">Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6.2.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 18 (12), 2130-2138 (2010).</li><li>Chen, C. -. M., Chen, Y. -. J., Huang, Z. -. H. <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+of+Stem+Cells+in+Term+Neonatal+Rats.">Intratracheal Instillation of Stem Cells in Term Neonatal Rats.</a> Journal of Visualized Experiments. (159), e61117 (2020).</li><li>Reynolds, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+maternal+cannibalism+in+rats.">Preventing maternal cannibalism in rats.</a> Science. 213 (4512), 1146 (1981).</li><li>Park, C. M., Clegg, K. E., Harvey-Clark, C. J., Hollenberg, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+techniques+for+successful+neonatal+rat+surgery.">Improved techniques for successful neonatal rat surgery.</a> Laboratory Animal Science. 42 (5), 508-513 (1992).</li><li>Cleary, G. M., 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=Exudative+lung+injury+is+associated+with+decreased+levels+of+surfactant+proteins+in+a+rat+model+of+meconium+aspiration.">Exudative lung injury is associated with decreased levels of surfactant proteins in a rat model of meconium aspiration.</a> Pediatrics. 100 (6), 998-1003 (1997).</li><li>Das, S., MacDonald, K., Chang, H. -. Y. 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+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> Journal of Visualized Experiments. (73), e50318 (2013).</li><li>Oka, 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=A+reliable+method+for+intratracheal+instillation+of+materials+to+the+entire+lung+in+rats.">A reliable method for intratracheal instillation of materials to the entire lung in rats.</a> Journal of Toxicologic Pathology. 19 (2), 107-109 (2006).</li><li>Watanabe, A., Hashimoto, Y., Ochiai, E., Sato, A., Kamei, K. <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+for+confirming+correct+endotracheal+intubation+in+mice.">A simple method for confirming correct endotracheal intubation in mice.</a> Laboratory Animals. 43 (4), 399-401 (2009).</li><li>Kim, J. -. 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=An+automatic+video+instillator+for+intratracheal+instillation+in+the+rat.">An automatic video instillator for intratracheal instillation in the rat.</a> Laboratory Animals. 44 (1), 20-24 (2010).</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> Journal of Visualized Experiments. (93), e52261 (2014).</li><li>Ordodi, V. L., Mic, F. A., Mic, A. A., Sandesc, D., Paunescu, V. <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+device+for+intubation+of+rats.">A simple device for intubation of rats.</a> Lab Animal. 34 (8), 37-39 (2005).</li><li>Cleary, G. M., Wiswell, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meconium-stained+amniotic+fluid+and+the+meconium+aspiration+syndrome.+An+update.">Meconium-stained amniotic fluid and the meconium aspiration syndrome. An update.</a> Pediatric Clinics of North America. 45 (3), 511-529 (1998).</li><li>Vandivort, T. C., An, D., Parks, W. C. <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+method+for+rapid+intubation+of+the+trachea+in+mice.">An improved method for rapid intubation of the trachea in mice.</a> Journal of Visualized Experiments. (108), e53771 (2016).</li><li>Litvin, D. G., Dick, T. E., Smith, C. B., Jacono, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung-injury+depresses+glutamatergic+synaptic+transmission+in+the+nucleus+tractus+solitarii+via+discrete+age-dependent+mechanisms+in+neonatal+rats.">Lung-injury depresses glutamatergic synaptic transmission in the nucleus tractus solitarii via discrete age-dependent mechanisms in neonatal rats.</a> Brain, Behavior, and Immunity. 70, 398-422 (2018).</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 Animals. 41 (1), 128-135 (2007).</li><li>Kim, Y. 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+transplantation+of+mesenchymal+stem+cells+simultaneously+attenuates+both+lung+and+brain+injuries+in+hyperoxic+newborn+rats.">Intratracheal transplantation of mesenchymal stem cells simultaneously attenuates both lung and brain injuries in hyperoxic newborn rats.</a> Pediatric Research. 80 (3), 415-424 (2016).</li><li>Chang, Y. 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=Intratracheal+transplantation+of+human+umbilical+cord+blood-derived+mesenchymal+stem+cells+dose-dependently+attenuate+hyperoxia-induced+lung+injury+in+neonatal+rats.">Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells dose-dependently attenuate hyperoxia-induced lung injury in neonatal rats.</a> Cell Transplantation. 20 (11-12), 1843-1854 (2011).</li><li>Chang, Y. 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=Timing+of+umbilical+cord+blood+derived+mesenchymal+stem+cells+transplantation+determines+therapeutic+efficacy+in+the+neonatal+hyperoxic+lung+injury.">Timing of umbilical cord blood derived mesenchymal stem cells transplantation determines therapeutic efficacy in the neonatal hyperoxic lung injury.</a> PloS One. 8 (1), 52419 (2013).</li><li>Mowat, V., Alexander, D. J., Pilling, 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=A+comparison+of+rodent+and+nonrodent+laryngeal+and+tracheal+bifurcation+sensitivities+in+inhalation+toxicity+studies+and+their+relevance+for+human+exposure.">A comparison of rodent and nonrodent laryngeal and tracheal bifurcation sensitivities in inhalation toxicity studies and their relevance for human exposure.</a> Toxicologic Pathology. 45 (1), 216-222 (2017).</li></ol>

The authors have nothing to disclose.

Intratracheal instillation is an excellent method that offers several advantages over the existing methods for respiratory disease interventions as well as disease model development. It is a quick method and with experience, can be performed with an average speed of 2-3 minutes per animal. The key considerations for a successful intubation are proper sedation of the animal, it&#39;s correct positioning, especially the head, as well as accurate depth of placement/ size of the specula in the oropharynx. Proper sedation wou...

Neonates born prematurely have poorly developed lungs requiring many interventional therapies such as long-term ventilation. These interventions place the surviving neonates at a high risk of subsequent sequelae1. Experimental animal models serve as an important tool in simulating various disease conditions, studying the pathobiology of diseases, and evaluating therapeutic interventions. Even though a broad range of animal models from mice, rat, and rabbit to pre-term lambs and pigs are available, mice and rat are the most used. 
The primary advantage of using mice and rats are the relatively short gestation period and reduced cost. They are also readily available, easy to maintain in disease-free environments, genetically homogeneous and have relatively less ethical concern2,3. Another major advantage of the rodent model is that at birth the neonatal pup is at late canalicular/early saccular stage of lung development which is morphologically equivalent to the lung of a 24-week preterm neonatal human infant going on to develop bronchopulmonary dysplasia4. In addition, as their lung development rapidly progresses to completion within the first 4 weeks of life, it is feasible to study the post-natal lung maturation in a reasonable time frame4. Despite these advantages, the small size of the mice and rat pups is a source of concern for various interventions, which compels most researchers to use adult animals rather than pups5. Neonatal lungs are in a developmental stage and the response of a neonate to an inciting agent differs from that of an adult. This makes it appropriate to use neonatal animal models to study human neonatal disease conditions.
There are different methods to administer drugs/ biological agents to the lung. This includes intranasal6,7 or intratracheal8,9,10 instillation as well as aerosol inhalation11,12. Each approach has its own technical challenges, advantages, as well as limitations13. Intratracheal route of administration of therapeutic agents is preferred to study the direct therapeutic impact in the organ bypassing the systemic effects. This route could also be used to study lung pathology caused by inciting agents. There are both invasive and minimally invasive techniques to do this and is easy to perform in adults. However, in pups, because of the small size of the animal, there are technical challenges associated with the intubation process. The current study presents a simple, consistent, non-surgical intratracheal instillation (ITI) method in rat pups that could be used to study the efficacy of various neonatal therapeutic interventions as well as to generate animal models simulating neonatal respiratory diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Evans Blue dye</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>314-13-6</td>
			<td>Confirmation of drug administration into lungs</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Hospira. Inc, Lake Forest, IL, USA</td>
			<td>Dispensed from Animal care facility</td>
			<td>For sedation</td>
		</tr>
		<tr>
			<td>Operating Otoscope</td>
			<td>Welch Allyn, Hillrom, Chicago, IL, USA</td>
			<td>21770- 3.5V</td>
			<td>For visualization of vocal cords</td>
		</tr>
		<tr>
			<td>Otoscope Rechargeable Handle</td>
			<td>Welch Allyn, Hillrom, Chicago, IL, USA</td>
			<td>71050-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip (Gel loading)</td>
			<td>Fisherbrand</td>
			<td>02-707-139</td>
			<td>Administering the drug</td>
		</tr>
		<tr>
			<td>Platform for restraining (inclined plane)</td>
			<td>Animal care facility</td>
			<td>Dispensed from Animal care facility</td>
			<td>Wired roof of mice cage can be used</td>
		</tr>
		<tr>
			<td>3M Micropore Surgical White Paper (sticking tape)</td>
			<td>3M, St. Paul, MN, USA</td>
			<td>1530-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock SyringeSyringes (1 ml)</td>
			<td>BD Franklin Lakes, NJ , USA</td>
			<td>NBD2515</td>
			<td>Administering the drug</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Hospira. Inc, Lake Forest, IL, USA</td>
			<td></td>
			<td>For sedation</td>
		</tr>
	</tbody>
</table>

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.

The instillation of Evans blue revealed multifocal distribution of the dye involving all pulmonary lobes (Figure 4A,B). Our result as shown in Figure 4 demonstrates efficacy of distribution to all lobes. The picture is taken immediately after ITI of the dye into the trachea. 100% efficacy was achieved in instilling the dye into the trachea followed by its spread into all the lobes on both sides. It is expected that the dye would spread further w...

Watch this Scientific Journal Video about A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease at JoVE.com

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

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 ...

a-minimally-invasive-method-for-intratracheal-instillation-drugs

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Introduction to Respiratory System Drugs

SCIENTISTS IN ACTION

5.4K Views.  Case Western Reserve University. 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. 