Name: intratracheal administration of dry powder formulation in mice
Uploaded: 2020-07-25T15:00:00
Description: Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

The authors would like to thank Mr. Ray Lee, Mr. HC Leung and Mr. Wallace So for their kind assistance in making the light source and powder insufflator; and the Faculty Core Facility for the assistance in animal imaging. The work was supported by the Research Grant Council, Hong Kong (17300319).

The experiments conducted in this study have been approved by the Committee on the Use of Live Animals for Teaching and Research (CULATR), The University of Hong Kong. Dry powder formulations prepared by spray freeze drying (SFD) containing 0.5% of luciferase messenger RNA (mRNA), 5% synthetic peptide PEG12KL4 and 94.5% of mannitol (w/w) are used in this study to demonstrate mRNA expression in the lung16. The mass median aerodynamic diameter (MMAD) of SFD powder is 2.4 &#956;m. Spray dr...

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G., Young, E. F., Braunstein, M. S., Welch, J. T., Hickey, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dry+powder+combination+of+pyrazinoic+acid+and+its+n-propyl+ester+for+aerosol+administration+to+animals.">A dry powder combination of pyrazinoic acid and its n-propyl ester for aerosol administration to animals.</a> International Journal of Pharmaceutics. 514 (2), 384-391 (2016).</li><li>Phillips, J. E., Zhang, X., Johnston, 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=Dry+powder+and+nebulized+aerosol+inhalation+of+pharmaceuticals+delivered+to+mice+using+a+nose-only+exposure+system.">Dry powder and nebulized aerosol inhalation of pharmaceuticals delivered to mice using a nose-only exposure system.</a> JoVE (Journal of Visualized Experiments). 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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=Proof-of-Principle+Study+in+a+Murine+Lung+Infection+Model+of+Antipseudomonal+Activity+of+Phage+PEV20+in+a+Dry-Powder+Formulation.">Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation.</a> Antimicrobial Agents and Chemotherapy. 62 (2), (2018).</li><li>Ito, T., Okuda, T., Takayama, R., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+Evaluation+Method+for+Gene+Silencing+by+Serial+Pulmonary+Administration+of+siRNA+and+pDNA+Powders:+Naked+siRNA+Inhalation+Powder+Suppresses+Luciferase+Gene+Expression+in+the+Lung.">Establishment of an Evaluation Method for Gene Silencing by Serial Pulmonary Administration of siRNA and pDNA Powders: Naked siRNA Inhalation Powder Suppresses Luciferase Gene Expression in the Lung.</a> Journal of pharmaceutical sciences. 108 (8), 2661-2667 (2019).</li><li>Patil, J. S., Sarasija, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+drug+delivery+strategies:+A+concise,+systematic+review.">Pulmonary drug delivery strategies: A concise, systematic review.</a> Lung India. 29 (1), 44-49 (2012).</li><li>Ihara, D., 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=Histological+Quantification+of+Gene+Silencing+by+Intratracheal+Administration+of+Dry+Powdered+Small-Interfering+RNA/Chitosan+Complexes+in+the+Murine+Lung.">Histological Quantification of Gene Silencing by Intratracheal Administration of Dry Powdered Small-Interfering RNA/Chitosan Complexes in the Murine Lung.</a> Pharmaceutical Research. 32 (12), 3877-3885 (2015).</li><li>Qiu, 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=Effective+mRNA+pulmonary+delivery+by+dry+powder+formulation+of+PEGylated+synthetic+KL4+peptide.">Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide.</a> Journal of Controlled Release. 314, 102-115 (2019).</li><li>Pfeifer, C., 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=Dry+powder+aerosols+of+polyethylenimine+(PEI)-based+gene+vectors+mediate+efficient+gene+delivery+to+the+lung.">Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.</a> Journal of Controlled Release. 154 (1), 69-76 (2011).</li><li>Kim, I., 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=Doxorubicin-loaded+highly+porous+large+PLGA+microparticles+as+a+sustained-+release+inhalation+system+for+the+treatment+of+metastatic+lung+cancer.">Doxorubicin-loaded highly porous large PLGA microparticles as a sustained- release inhalation system for the treatment of metastatic lung cancer.</a> Biomaterials. 33 (22), 5574-5583 (2012).</li><li>Tonnis, W. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+aerosol+generator+for+homogenous+distribution+of+powder+over+the+lungs+after+pulmonary+administration+to+small+laboratory+animals.">A novel aerosol generator for homogenous distribution of powder over the lungs after pulmonary administration to small laboratory animals.</a> European Journal of Pharmaceutics and Biopharmaceutics. 88 (3), 1056-1063 (2014).</li><li>Hoppentocht, M., Hoste, C., Hagedoorn, P., Frijlink, H. W., de Boer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+DP-4M+PennCentury+insufflator.">In vitro evaluation of the DP-4M PennCentury insufflator.</a> European Journal of Pharmaceutics and Biopharmaceutics. 88 (1), 153-159 (2014).</li><li>Liao, Q., 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=Porous+and+highly+dispersible+voriconazole+dry+powders+produced+by+spray+freeze+drying+for+pulmonary+delivery+with+efficient+lung+deposition.">Porous and highly dispersible voriconazole dry powders produced by spray freeze drying for pulmonary delivery with efficient lung deposition.</a> International Journal of Pharmaceutics. 560, 144-154 (2019).</li><li>Ito, T., Okuda, T., Takashima, Y., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+pDNA+Inhalation+Powder+Composed+of+Hyaluronic+Acid+Exhibits+High+Gene+Expression+in+the+Lungs.">Naked pDNA Inhalation Powder Composed of Hyaluronic Acid Exhibits High Gene Expression in the Lungs.</a> Molecular Pharmaceutics. 16 (2), 489-497 (2019).</li><li>Chaurasiya, B., Zhou, M., Tu, J., Sun, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+simple+device+for+insufflation+of+dry+powders+in+a+mice+model.">Design and validation of a simple device for insufflation of dry powders in a mice model.</a> European Journal of Pharmaceutical Sciences. 123, 495-501 (2018).</li></ol>

In this paper, custom-made devices for dry powder insufflation and intratracheal intubation are presented. In the powder loading step, dry powder are loaded into a 200 &#181;L gel-loading pipette tip. It is important to gently tap the tip to allow the loose packing of powder at the narrow end of the tip. However, if the powder are packed too tightly, they will get stuck in the tip and cannot be properly dispersed. It is recommended to neutralize the static charges of the powder and the pipette tip in order to facilitate ...

The pulmonary route of administration offers various benefits in delivering therapeutics for both local and systemic actions. For the treatment of lung diseases, high local drug concentration can be achieved by pulmonary delivery, thereby reducing the required dose and lowering the incidence of systemic side effects. Moreover, the relatively low enzymatic activities in the lung can reduce premature drug metabolism. The lungs are also efficient for drug absorption for systemic action due to the large and well-perfused surface area, the extremely thin epithelial cell layer and the high blood volume in pulmonary capillaries1.
Inhaled dry powder formulations have been widely investigated for the prevention and treatment of various diseases such as asthma, chronic obstructive pulmonary disease, diabetes mellitus and pulmonary vaccination2,3,4. Drugs in the solid state are generally more stable than in the liquid form, and dry powder inhalers are more portable and user-friendly than nebulizers5,6. In the development of inhaled dry powder formulations, the safety, the pharmacokinetic profile and the therapeutic efficacy need to be evaluated in preclinical animal models following pulmonary administration7. Unlike humans who can inhale dry powder actively, pulmonary delivery of dry powder to small animals is challenging. It is necessary to establish an efficient protocol of delivering dry powder to the lungs of animals.
Mice are widely used as research animal models because they are economical and they breed well. They are also easy to handle and many disease models are well-established. There are two major approaches to administer dry powder to the lung&#160;of mouse: inhalation and intratracheal administration. For inhalation, the mouse is placed in a whole-body or nose-only chamber where dry powder is aerosolized and the animals breathe in the aerosol without sedation8,9. Expensive equipment is required and the drug delivery efficiency is low. While the whole-body chamber may be technically less challenging, the nose-only exposure chamber could minimize exposure of drugs to the body surface. Regardless, it is still difficult to precisely control and determine the dose delivered to the lungs. The dry powder is mainly deposited in the nasopharynx region where mucociliary clearance is prominent10. Moreover, mice inside the chamber are under significant stress during the administration process because they are constrained and deprived of food and water supply11. For intratracheal administration, it generally refers to the introduction of the substance directly into the trachea. There are two different techniques to achieve this: tracheotomy and orotracheal intubation. The former requires a surgical procedure that makes an incision in the trachea, which is invasive and seldom used for powder administration. Only the second technique is described here. Compared to the inhalation method, intratracheal administration is the more commonly used method for pulmonary delivery in the mouse because of its high delivery efficiency with minimal drug loss12,13. It is a simple and fast method to precisely deliver a small amount of powder within a few milligrams to the mouse. Although the mouse is anatomically and physiologically distinct to humans and anesthetization is required during the intubation process, intratracheal administration bypasses the upper respiratory tract and offers a more effective way to assess the biological activities of the dry powder formulation such as the pulmonary absorption, bioavailability and therapeutic effects14,15.
To administer dry powder intratracheally, the mouse has to be intubated, which could be challenging. In this paper, the fabrication of a custom-made dry powder insufflator and an intubation device is described. The procedures of intubation and insufflation of dry powder in the lung of the mouse are demonstrated.

<table>
	<tbody>
		<tr>
			<td>BALB/c mouse</td>
			<td></td>
			<td></td>
			<td>Female; 7-9 weeks old; Body weight 20-25 g</td>
		</tr>
		<tr>
			<td>CleanCap Firefly Luciferase mRNA</td>
			<td>TriLink Biotechnology</td>
			<td>L-7602</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Powder Insufflator</td>
			<td>PennCentury</td>
			<td>Model DP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 10%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Light emitting diode (LED) torch</td>
			<td>Unilite Internation</td>
			<td>PS-K1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol (Pearlitol 160C)</td>
			<td>Roquette</td>
			<td>450001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-filter round gel loading pipette tip (200 &#181;L)</td>
			<td>Labcon</td>
			<td>1034-800-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon floss</td>
			<td>Reach</td>
			<td>30017050</td>
			<td></td>
		</tr>
		<tr>
			<td>One milliliter syringe without needle</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fibre</td>
			<td>Fibre Data</td>
			<td>OMPF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG12KL4 peptide</td>
			<td>EZ Biolab</td>
			<td></td>
			<td>(PEG12)-KLLLLKLLLLKLLLLKLLLLK-NH2</td>
		</tr>
		<tr>
			<td>Plastic Pasteur fine tip pipette</td>
			<td>Alpha Labotatories</td>
			<td>LW4061</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>Braun</td>
			<td>D201</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zerostat 3 anti-static gun</td>
			<td>MILTY</td>
			<td>5036694022153</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

When a dry powder insufflator is used to deliver powder aerosol to the lung of an animal, the volume of air used is critical as it affects the safety as well as the powder dispersion efficiency. To optimize the method, different volumes of air (0.3 mL, 0.6 mL and 1.0 mL) were used to disperse the dry powder (1 mg of spray dried mannitol) and the weight of mice was monitored for 48 hours after administration (Figure 6). The use of 0.3 mL and 0.6 mL of air did not cause weight loss of the mice...

Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

Intratracheal Administration of Dry Powder Formulation in Mice

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 ...

Intratracheal administration of dry powder is essential to evaluate the performance and biological activities of an inhaled powder formulation, such as pulmonary absorption, bioavailability, and therapeutic effects in pre-clinical animal models. The intubation process is non-invasive and could deliver powder formulation to the mice safely and accurately. The custom-made dry powder insufflator is disposable, inexpensive, and efficient in disbursing powder formulations.The insufflators could be used in evaluating different formulations on multiple mice in the same experiments without a risk of cross-contamination from residual powder. The intubation process is challenging for non-experienced researchers. The ability to visualize and aim the finest tip of the cannula at the opening of the trachea is crucial for correct insertion of the guiding cannula.This technique requires practice to minimize improper insertion. My postdoc Carol and my PhD student Rachel are going to demonstrate the preparation and intubation procedure. To begin, neutralize the static charges of the dry powder and the 200 microliter non-filter round gel loading pipette tip with an anti-static gun or a balance with deionizing function.Prepare a weighing paper with an approximate size of four by four centimeters. Fold the paper in half diagonally and then unfold it. Then use it to weigh one to two milligrams of dry powder.Fill a gel loading pipette tip with powder through the wider opening and tap it gently to pack the powder until it forms loose agglomerates near the narrow end of the tip. Avoid packing the powder too tightly as it may hamper dispersion. Connect the powder loaded tip to a one milliliter syringe through a three-way stopcock holding the tip and syringe vertically during connection to prevent spillage of powder.The size of the syringe can be changed according to the volume of air used to disperse the powder. If administration is not performed immediately, use parafilm to seal the openings of the tip and store it temporarily under suitable conditions until administration. After anesthetizing the seven to nine-week-old BALB/c mouse with ketamine and xylazine, place it on a plexiglass platform mounted to a stand.Suspend the mouse by hooking its incisors on a nylon floss and secure its position with a piece of tape or a rubber band. Adjust the height and angle of the platform. Insert the optical fiber into the guiding cannula with the tip of the fiber level with the opening of the cannula.Then turn on the LED torch to illuminate. Gently protrude the tongue of the mouse with a pair of forceps to expose its trachea. Use the other hand to hold the guiding cannula with optical fiber and insert them through the oral cavity.With the illumination from the optical fiber, the opening of the trachea can be visualized as an orifice between the vocal cords. Align the bevel of the guiding cannula towards the midline of the opening and gently intubate it with the optical fiber into the trachea by aiming the finest tip of the cannula at the tracheal opening. Upon intubation, swiftly remove the optical fiber and leave the guiding cannula inside the trachea.Normal restoration should be observed. Hold the fine tip pipette at the opening of the guiding cannula and insufflate a small puff of air into the lung of the mouse. A slight inflation in the chest indicates proper intubation.Remove the fine tip pipette prior to powder administration. Hold the power loaded tip that is connected to the syringe, making sure that the air flow between the syringe and the tip is disconnected. Pull the syringe plunger backward to withdraw 0.6 milliliters of air.Turn the valve of the three-way stopcock to connect the air flow between the syringe and the power loaded tip. Then insert the power loaded tip into the guiding cannula which has already been placed in the trachea of the mouse. Hold the guiding cannula and push the syringe plunger forcefully in one continuous action to disperse the powder as aerosols into the lung minimizing any forward motion to avoid injury to the animal.Remove the tip and check if the powder has been emptied. Once the administration is complete, remove the guiding cannula from the trachea. Allow the mouse to recover by positioning it horizontally in a supine position with its tongue half protruded to avoid a blockage of the airways.To optimize the method, different volumes of air were used to disperse one milligram of the spray dried mannitol and the weight of mice was monitored. The use of 0.3 and 0.6 milliliters of air did not cause weight loss of the mice up to 48 hours post-administration. Dispersing the powder with one milliliter of air resulted in over 5%weight loss within 24 hours, which was not fully recovered after 48 hours.The mice were intratracheally administered with one milligram of SFD powder containing five micrograms of mRNA and the luciferase expression in the lungs was evaluated at 24 hours post-administration using an in vivo imaging system. The SFD powder was dispersed in the deep lung and luciferase expression was observed. As a comparison, the powder was reconstituted in water and administered with a micro sprayer using the same intubation procedure.The luciferase expression of the reconstituted formulation was significantly higher than the dry powder formulation, which could be due to the powder disillusion issue or different pharmacokinetic profile between powder and liquid form. The histological characteristics of the lungs treated with mRNA dry powder aerosol were compared with untreated control and LPS-treated groups. The lung without any treatment illustrated a healthy presentation while the lung treated with LPS showed irregular distribution of airspace and inflammatory cell infiltration into the interstitial and alveolar spaces.The lungs treated with SFD powder did not show any signs of inflammation. For successful powder dispersion, the powder in the loading tip should be tapped gently and not packed too tightly in the tip. This intubation setting can also be adapted to administer liquid formulations either with a pipette or a micro sprayer.

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JoVE Journal

Medicine

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 ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

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 these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct 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. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

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

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

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 ...

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 ...

Customizable Disposable Dosators to Deliver Dry Powder Aerosol Drugs to Mice

Disposable Dosators Intended for Dry Powder Delivery to Mice

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models&#39; utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 &#181;g. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Author Spotlight: Developing a Disposable Dosator for Preclinical Testing of Dry Powder Inhalers in Small Animal Models 

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus ...

Assembling and Actuating a Disposable Dosator for Dry Powder Drug Delivery

To prepare the disposable dosator and its filling components, take a 2.5 centimeter or 1 inch blunt stainless steel needle of 21 to 25 gauge. Utilizing a precision sectioning saw or a belt sander, trim the plastic lure part of the needle, leaving about 2 to 3 millimeters of the plastic lure attached to the needle. Next, cut off approximately 1 to 1.5 centimeters from the tip of a 0.6 milliliter conical centrifuge tube.After that, fill this cut tip with 30 to 35 milligrams of the powder of interest. For storing or transporting the powder, place the cutoff tube cap onto the cut tip containing the powder. Use paraffin film to wrap the cover, minimizing the powder's exposure to ambient moisture.To load the dosator, tamp the trimmed stainless steel needle into the powder bed in the cut tip as many times as required to achieve the desired dose. Using a low lint wiper, gently wipe the sides of the needle to remove any excess powder. Then take a 3.81 centimeter or 1.5 inch polypropylene needle of 16 to 20 gauge and cautiously insert the loaded stainless steel needle into it, making sure not to dislodge any powder.Alternatively, use a 5.08 centimeter or 2 inch PTFE needle for the same. To actuate the dosator, first draw back a disposable syringe to the desired volume based on the application. Then attach the syringe to the lure lock on the polypropylene or PTFE needle before inserting the needle end of the dosator into the desired target.To analyze powder content and reproducibility, insert the dosator needle through a perforated rubber septum or paraffin film into a vial with a small quantity of liquid. Forcefully depress the syringe plunger, expelling the powder from the device into the collection vial. Finally, analyze the powder solution in the vial using UV's visible spectra photometry to monitor the dose of the powder released from the dosator.Four different spray dried powders visualized using scanning electron microscopy were considered for delivery using the demonstrated dosator. The delivered dose as a function of the number of Tamps in the powder bed, was obtained from UV visible spectra photometry of the powder solutions. Notably, all spray dried powders demonstrated a linear dose response from 1 to 4 Tamps.The first Tamp always incurred a larger dose of powder than subsequent Tamps. When compared to the demonstrated 21 gauge stainless steel with a 16 gauge polypropylene dosator, a slight increase in achievable dose was observed using a 21 gauge stainless steel with a 20 gauge PTFE dosator for powder SD 1. As expected, the achievable dose of SD 1 decreased for a 22 gauge stainless steel with an 18 gauge polypropylene dosator, having a smaller inner needle diameter.A 25 gauge stainless steel with a 20 gauge polypropylene dosator further decreased the initial and subsequent Tamp doses. This is a comparison of the four dosator systems that were evaluated, highlighting that the dosators can be customized to meet different dosage needs.