Name: a minimally invasive method for intratracheal instillation of drugs in neonatal rodents to treat lung disease
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Description: 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

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...

<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. 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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. 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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 ...

This protocol demonstrates that it is possible to instill research material into the trachea of neonatal rat pups, five-days-old, by direct visualization. This method improves the reliability of administration of research material or drug into the trachea by direct visualization. By avoiding injection into the trachea, this method reduces pain, and more importantly, bleeding into the trachea, allowing improved survival.The development of fine skills is required to perform this protocol, including appropriate positioning of the animal, correct use of operating autoscope to visualize the vocal cords, and efficacy of administration into the lungs. Demonstrating the procedure will be Tara Sudhadevi, a postdoctoral fellow from my laboratory, and Alison Ha, a graduate student. Before beginning the experiment, confirm a loss of tail and pedal reflex in an anesthetized five-day-old rat pup and use laboratory labeling tape to restrain the pup in the supine position on a flat platform inclined at 45 degrees.Use blunt forceps to gently pull the tongue to one side, and with the help of a two-millimeter diameter otoscope speculum, gently hold the tongue in place so that the larynx is visible. Using the operating otoscope with the correct sized speculum, locate the vocal cords through the magnifying lens of the otoscope. For intratracheal instillation, load a one-milliliter syringe fitted with a long-angled, gel-loading pipette tip with 0.9 cubic centimeters of air, followed by 30 to 50 microliters of the substance of interest.Bend the pipette tip to a 30-degree angle and insert the loaded pipette tip through the speculum to about two millimeters beyond the vocal cords. Push the piston of the syringe to administer the substance of interest and the air through the speculum into the vocal cords. When all the substance has been delivered, place the pup on a 38-degree-Celsius integrated circulating fluid heating pad with monitoring until regular respiration, before reuniting the pup with the dam.To evaluate the distribution of the substance within a lung tissue sample, wipe the chest and abdomen of the euthanized rat pups with 70%ethyl alcohol and use sterile technique to remove the lungs according to standard protocols for their visual assessment. In this study, the efficacy of the intra-tracheal instillation technique was assessed by visual examination of the lung tissue collected from a five-day-old rat pup instilled with Evans blue dye. The instillation was nearly 100%efficient, as the dye spread into the lung immediately upon administration and distributed across all the lobes of the lung tissue.The dye was not observed within any tissues outside of the lungs, confirming the ability of the technique to be used for the selective delivery of a therapeutic of interest to the lungs. Placing the animal initially at the correct angle over a firm surface is essential for the success of the next step of visualizing the vocal chords using the operating autoscope.

Research

JoVE Journal

Medicine

A Simple Method of Mouse Lung Intubation

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

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

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 ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...