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>
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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 would allow sufficient working time for the operators, especially beginners. Positioning of the animal at a 45&#176; angle is important for proper visualization of vocal cords. Placement of speculum at the right depth helps in retraction of the tongue throughout the procedure which again allows good visualization of the vocal cord. A team of two people can easily coordinate this work. One could coordinate the anesthesia and caging of animals while the other could deal with the instillation. The most technically challenging part of ITI is the correct intubation into the trachea. Success of the technique is confirmed by administration of dye following intubation. It is very important to confirm the initial step of correct intubation, as there is a good chance for the tubing to slip into esophagus resulting in the delivery of the substance into the stomach, rather than the lung.
The only part that one must be careful is the potential trauma associated with misintubation. One also must be very gentle and careful in order to avoid penetrating through the trachea or the tissue surrounding the vocal cords. It is also recommended not to conduct ITI if there have been 2 or 3 misses2.
There are different routes for administration of drugs/biological agents with each one having its own inherent advantages and disadvantages. Selection of a method is based principally on study objectives and nature of the intervention. Both intranasal instillation and aerosolization techniques deliver agents to the upper respiratory tract as well as the lungs. This benefits studies involving upper respiratory tract13,21 however, the delivery of a substance to lungs is unreliable. In addition, swallowing, sneezing and the varying breathing rates may lead to inconsistencies in the doses delivered. However, the physicochemical properties of some substances affect their efficient aerosolization15. Researchers use intratracheal inoculation to get around this problem, which regardless of particle size and viscosity, delivers inoculum/drugs directly into the lungs23.
The two main intratracheal delivery methods include transoral intratracheal14,15 and transtracheal instillation with or without tracheotomy16,17. ITI is a procedure where a wide range of treatment doses can be administered to a large number of animals quickly, once trained18. While transoral intratracheal instillation is routinely used in adult rats, the more invasive technique such as surgical incision was required in neonates16,19,20. Researchers still avoid the use of this transoral ITI technique in pups because of several reasons. The small size of the neonatal rodent makes the visualization of the laryngeal lumen difficult along with poor success in intubation. Also, the traditional metal laryngoscope used for ITI in adults cannot be used in neonate because of the small size of the oral cavity and the fragile mucosal tissues16,18,10.&#160;Smaller speculum and catheters are required to view the laryngeal cavity and deliver the therapeutics/ agents into the lung. The operator must be highly skilled to achieve this. Finally, recovery from anesthesia, hypothermia, maternal rejection, and cannibalism create additional problems for rat neonate recovery and survival21,22. Our study employed the use of gas anesthesia followed by recovery in heating pads and reuniting with lactating dams. This avoids problems associated with hypothermia, maternal rejection, or cannibalism. Many of the non-surgical intervention studies involve a blind intubation of the trachea through the oral cavity. This is especially not acceptable in the case of drug where the effect may be missed if it is wrongly instilled into esophagus. In this study, the tracheal opening is visualized using an otoscope and a slightly bent pipette tip is inserted directly into the trachea to deliver the substance, the dye in this case. Our technique demonstrates an effective way of administering the drug into the trachea of a small rat pup.
The process of ITI, is a reliable method when performed following meticulous training. Once trained it can be done rapidly and effectively as in adult rodents13,24,25. The correct endotracheal instillation can be confirmed by several methods including the dye or liquid movement in a tubing or syringe26,27,28. As it is possible to visualize the tracheal opening in this method, the misses are very less. Apnea was observed in a few pups immediately after ITI which was recovered spontaneously18,29. Using otoscope along with the smallest speculum served as a perfect fit for the small oral cavity of the neonatal rat18. The results of this study indicated that the substance can be consistently delivered to all the lobes of the lung as confirmed by the dye localization. This method would be of great significance in experimental studies in which neonatal rats are required to reliably mimic neonatal lung conditions30,31,32. This technique could also be used to carry out lung function studies33 as well as cell/ stem cell transplantation studies34,35,36 which currently employs surgical interventions and could be distressing to pups.
This technique also contributes to the principles of Refinement and Reduction in animal research. This method serves as an alternative to direct intratracheal injection with a needle which is a blind technique and is invasive as it pierces the trachea causing pain and bleeding. In complete contrast this technique serves to reduce pain while refining the introduction of a drug into the trachea, achieving immediate reduction of pain and suffering, and improvement of welfare of animals involved in research37. In addition, the administration of drug into trachea is directly visualized ensuring efficacy. Though the instillation of drug into trachea is widely practiced in larger animal our refinement to use this in a 5-day old rat pup is the innovation we would like to stress here.
This article offers a simple, minimally invasive, and reproducible method which could be used for administration of injurious agents in order to simulate pathological conditions as well as for local administration of drugs, antioxidants, cells/ stem cells for neonatal therapies.

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><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 within the lobule of the lung. With repeated administration, we have been able to ensure 100% success in delivering this to the lung to both lobes and all lobules. We have ensured that no dye reaches stomach or outside of lungs. This testifies the efficacy of technique as 100% administration into the lungs. The isoflurane anesthesia allowed faster recovery of the pups after the procedure. 
Rat pups from day 5 tolerated this procedure and took less than 5 minutes to carry out following anesthesia. Some animals though developed transient apnea, regained the normal respiratory pattern in a few minutes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61729/61729fig01.jpg" /> 
Figure 1: Otoscope components. (A) power source 2.5 V (B) magnifying lens (C) transilluminator (D) speculum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61729/61729fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61729/61729fig02.jpg" /> 
Figure 2: The positioning of the animal. The positioning of the animals at an angle of 45&#176; provided better visualization of tracheal opening without the interference of the epiglottis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61729/61729fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61729/61729fig03.jpg" /> 
Figure 3: Intratracheal instillation. Visualization of the tracheal opening using otoscope/ throat illuminator system to achieve direct delivery to lungs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61729/61729fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61729/61729fig04.jpg" /> 
Figure 4: ITI instillation and Evans blue staining. (A) ITI instillation delivers the dye throughout the lungs. The dye can be seen distributed to both lobes of the lung as indicated by the black arrow. Absence of dye in the stomach confirms the success of the technique (red arrow). (B) Lungs from rat pups instilled with 50 &#181;L of 0.25% Evans blue dye. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61729/61729fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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5.7K 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. 

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

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

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

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

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

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

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

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

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 ...

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

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 (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

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

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 ...

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.