This study was supported in part by the Private University Research Branding Project from MEXT; a Grant-in-Aid for Scientific Research (C) (17K08249 [to T.K. and T.S.]) from the Japan Society for the Promotion of Science (JSPS); a grant for cooperative research from the Hamaguchi Foundation for the Advancement of Biochemistry [to T.S.], and the Takeda Science Foundation [to T.K.]. We thank Mr. Yuya Nito and Ms. Akiko Asami for their valuable technical assistance in conducting the experiments.

This animal study (#AP17P004) was performed in accordance with the guidelines approved by the Nihon University Animal Care and Use Committee (Tokyo, Japan). This study (#17-0001) was approved by the Radioisotope Center of the School of Pharmacy, Nihon University.
1. Animals Used for Intranasal Administration Under Inhalation Anesthesia
<ol>
	<li>House the experimental mice in stainless-steel cages under a 12-h light/dark cycle (light on 8:00 AM&#8211;8:00 PM), with a controlled temperature maintained at 23 &#177; 1&#176;C, humidity of 50% &#177; 10%, and ad libitum access to food and water.</li>
	<li>Prior to experimentation, anesthetize the mice via inhalation of 2% isoflurane, following initiation at a concentration of 4%. Confirm the requisite level of anesthetization by checking for the disappearance of surface righting.&#160;&#160;</li>
</ol>
2. Preparation of the Administration Solution
<ol>
	<li>Prepare an administration solution of [14C]-inulin (50 &#956;M, 0.5 &#956;Ci/mL per mouse) by diluting in phosphate-buffered saline, and store at 4 &#176;C until use.</li>
</ol>
3. Intranasal Administrations for Mice
<ol>
	<li>Micropipette method using a temporarily openable inhalation mask (Figure 1) 
	Note: This technique is a modification of the intranasal administration protocol using a micropipette established by Frey et al.3
	<ol>
		<li>Fix mice in the supine position on a corkboard by taping their limbs under inhalation anesthesia with 2% isoflurane (Figure 1A).</li>
		<li>Administer a total volume of 25-&#956;L administration solution to each mouse at 30-s intervals, via 1- to 2-&#956;L doses alternatively administered into the left and right nostrils while the mice are fixed under inhalation anesthesia (Figure 1B and C).</li>
		<li>Note:&#160;Waste anesthetic gas is&#160;scavenged by active means&#160;(fume hood, hard-ducted biosafety cabinet, vacuum, etc.) when the&#160; mask is opened and by passive&#160;means(waste anesthetic gas cannister)&#160; when closed&#160;</li>
	</ol>
	</li>
	<li>Reverse cannulation method from the airway side through the esophagus (Figure 2) 
	Note: This technique is a modification of the intranasal absorption protocol for rats established by Hirai et al.7
	<ol>
		<li>Fix mice in the supine position on a corkboard by taping their limbs under inhalation anesthesia with 2% isoflurane.</li>
		<li>Hair in the neck&#160;is shaved and&#160;prepped via betadine or chlorhexidine application followed by an alcohol rinse.</li>
		<li>Expose the trachea and esophagus by expanding the skin below the throat with forceps after making a small incision (1.5 cm) with scissors.</li>
		<li>Make an incision (1 mm) in the trachea using scissors.</li>
		<li>Insert a cannula (internal diameter: 0.58 mm, exterior diameter: 0.965 mm) to a length of 1.2 cm and attach the opposite end of the cannula to the inside of the inhalation mask.</li>
		<li>Make an incision (1 mm) in the esophagus using scissors, insert a cannula (internal diameter: 0.28 mm, exterior diameter: 0.61 mm) to a length of 1.4 cm toward the posterior part of the nasal cavity, and ligate it (Figure 2A and B). 
		Note: Procedures 3.2.2 to 3.2.4 were carried out under a stereoscopic microscope at &#215;10 magnification.</li>
		<li>Attach a needle (27G &#215; 1/2) to a 1-mL syringe filled with an administration solution and connect to a programmable micro-syringe pump.</li>
		<li>Connect above needle to the cannula that had been inserted into the esophagus at 3.2.5 (Figure 2C).</li>
		<li>Administer a total volume of 25 &#956;L [14C]-inulin solution at a constant rate (5 &#956;L/min) (Figure 2C and 2D).</li>
	</ol>
	</li>
</ol>
4. Quantitative Experiment Using Radio-Labeled Water-Soluble Macromolecules ([14C]-inulin)
<ol>
	<li>Decapitate the experimental mice under anesthesia&#160;and open up their craniums, using scissors and from the side of the medulla oblongata, while taking care not to damage the brains.</li>
	<li>Extract carefully the whole brain by scooping using a micro-spatula from cranium.</li>
	<li>Place a filter paper moistened with saline solution on a Petri dish that is stored on ice.</li>
	<li>Place the extracted brains on the moistened filter paper.</li>
	<li>Wipe off blood adhering to the surface of the brain with a cotton swab moistened with saline solution to eliminate at least the influence of [14C]-inulin in the blood on the surface of the brain.</li>
	<li>Dissect brains rapidly, and divide them into three parts: the olfactory bulb, cerebrum, and medulla oblongata (including the pons).</li>
	<li>Place brain samples in tissue solubilizer at 50 &#176;C for 1 h.</li>
	<li>Add 10 &#956;L of the liquid scintillation cocktail to the brain samples.</li>
	<li>Transfer a 25 &#956;L aliquot of the administration solution dissolved in scintillation cocktail to a scintillation vial to determine the radioactivity of the applied solution.</li>
	<li>Measure the disintegrations per minute of [14C] radioactivity in the brain sample ([14C] Xbrain) and the applied solution ([14C] XIN dose) in a liquid scintillation counter equipped with an appropriate crossover correction for 3H and 14C.</li>
</ol>
5. Data Analysis
<ol>
	<li>Calculate the drug distribution levels (%) for the injected dose (ID%) using the following equation: 
	ID%&#8260;g brain = ([14C] Xbrain/[14C] XIN dose) &#215; 100, 
	where Xbrain (dpm/g brain) is the amount of [14C]-inulin measured in the brain tissue and XIN dose (dpm/25 &#956;L solution) is the concentration of the [14C]-inulin in the solution used for intranasal administration.</li>
</ol>

<ol>
	<li>Sakane, T., Yamashita, S., Yata, N., Sezaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transnasal+delivery+of+5-fluorouracil+to+the+brain+in+the+rat.">Transnasal delivery of 5-fluorouracil to the brain in the rat.</a> Journal of Drug Targeting. 7 (3), 233-240 (1999).</li><li>Illum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+drugs+from+the+nasal+cavity+to+the+central+nervous+system.">Transport of drugs from the nasal cavity to the central nervous system.</a> European Journal of Pharmaceutical Science. 11 (1), 1-18 (2000).</li><li>Hanson, L. R., Frey, W. H. <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+bypasses+the+blood-brain+barrier+to+target+therapeutic+agents+to+the+central+nervous+system+and+treat+neurodegenerative+disease.">Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease.</a> BMC Neuroscience. 9 (Suppl 3), S5 (2008).</li><li>Chapman, C. 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=Intranasal+treatment+of+central+nervous+system+dysfunction+in+humans.">Intranasal treatment of central nervous system dysfunction in humans.</a> Pharmaceutical Research. 30 (10), 2475-2484 (2012).</li><li>Kanazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+non-invasive+drug+delivery+system+to+the+brain+for+brain+diseases+therapy.">Development of non-invasive drug delivery system to the brain for brain diseases therapy.</a> Yakugaku-Zasshi. 138 (4), 443-450 (2018).</li><li>Kozlovskaya, L., Abou-Kaoud, M., Stepensky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+drug+delivery+to+the+brain+via+nasal+route.">Quantitative analysis of drug delivery to the brain via nasal route.</a> Journal of Controlled Release. 189, 133-140 (2014).</li><li>Hirai, S., Yashiki, T., Matsuzawa, T., Mima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorption+of+drugs+from+the+nasal+mucosa+of+rat.">Absorption of drugs from the nasal mucosa of rat.</a> International Journal of Pharmaceutics. 7 (4), 317-325 (1981).</li><li>Lochhead, J. J., Thorne, R. G. <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+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Advances in Drug Delivery Reviews. 64 (7), 614-628 (2011).</li><li>Lalatsa, A., Schatzlein, A. G., Stepensky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+deliver+peptide+drugs+to+the+brain.">Strategies to deliver peptide drugs to the brain.</a> Molecular Pharmaceutics. 11 (4), 1081-1093 (2014).</li><li>Kanazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+delivery+of+small+interfering+ribonucleic+acid+and+drugs+through+intranasal+administration+with+nano-sized+polymer+micelles.">Brain delivery of small interfering ribonucleic acid and drugs through intranasal administration with nano-sized polymer micelles.</a> Medical Devices. 8, 57-64 (2015).</li><li>Kanazawa, T., 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=Enhancement+of+nose-to-brain+delivery+of+hydrophilic+macromolecules+with+stearate-+or+polyethylene+glycol-modified+arginine-rich+peptide.">Enhancement of nose-to-brain delivery of hydrophilic macromolecules with stearate- or polyethylene glycol-modified arginine-rich peptide.</a> International Journal of Pharmacology. 530 (1-2), 195-200 (2017).</li><li>Kamei, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+an+enhanced+nose-to-brain+delivery+of+insulin+on+mild+and+progressive+memory+loss+in+the+senescence-accelerated+mouse.">Effect of an enhanced nose-to-brain delivery of insulin on mild and progressive memory loss in the senescence-accelerated mouse.</a> Molecular Pharmaceutics. 14 (3), 916-927 (2017).</li><li>Suzuki, T., Oshimi, M., Tomono, K., Hanano, M., Watanabe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+transport+mechanism+of+pentazocine+across+the+blood-brain+barrier+using+the+in+situ+rat+brain+perfusion+technique.">Investigation of transport mechanism of pentazocine across the blood-brain barrier using the in situ rat brain perfusion technique.</a> Journal of Pharmaceutical Science. 91 (11), 2346-2353 (2002).</li></ol>

The authors have nothing to disclose.

The nose-to-brain delivery of drugs is expected to have a pronounced effect on central nervous system disorders because this pathway represents a direct transportation route that bypasses the BBB. Three different nose-to-brain pathways have been reported to date8. The first is the olfactory nerve pathway, which passes from the olfactory mucosa in the nasal mucosa to the forebrain via the olfactory nerve. The second is the trigeminal nerve pathway, which passes from the respiratory mucosa in the na...

Biomedicines such as peptides, oligonucleotides, and antibodies are considered to have potential application as novel therapeutic agents for refractory central nervous system disorders that currently have no curative therapy. However, because most biomedicines are water-soluble macromolecules, delivery from the blood into the brain via intravenous or oral administration is extremely difficult due to impedance of the blood-brain barrier (BBB).
In recent years, intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that avoids the BBB1,2,3,4,5. However, there have been relatively few reports regarding the quantitative analysis of nose-to-brain pathway delivery6. Moreover, there have been virtually no reports on established optimal administration conditions and dosing regimens, such as volume, times, time-periods, and speed, for investigations of nose-to-brain delivery. The aforementioned deficiencies can be ascribed to the following reasons: (i) an optimal method of intranasal administration for mice has yet to be established, and (ii) intranasal administration by pipetting, which is generally used, is typically characterized by interindividual variation among animals due to mucociliary clearance (MC), thereby often leading to underestimations of the actual nose-to-brain delivery potential of a particular drug.
Inhalation anesthesia using isoflurane (initiation: 4%, maintenance: 2%) with an inhalation mask for rodents has gained widespread use, with the aim of reducing or eliminating the pain associated with surgery performed on experimental animals. The use of masks makes it relatively straightforward to perform typical drug administration in experimental animals under inhalation anesthesia via the subcutaneous, intraperitoneal, and intravenous routes. However, in the case of intranasal administration, the mask needs to be temporarily removed from the animals for drug administration. With maintenance under 2% isoflurane, animals typically awaken rapidly from inhalation anesthesia. When the administration volume per dose is large, this could cause the drug solution to flow from the nasal cavity into the esophagus, and therefore a single large dose may need to be broken down into multiple smaller doses for intranasal administration to small animals. As intranasal administration necessitates mask removal for repeated administration and sufficient time for sustained nasal cavity delivery, there is a high probability that mice would awaken from anesthesia during the administration procedure. This makes it very difficult to perform intranasal administration under a stable anesthetic state, and probably contributes to the observed interindividual variation of nose-to-brain delivery among rodents.
In this study, we therefore developed two novel methods of stable intranasal administration under inhalation anesthesia, which impose minimal physical stress on the experimental animals. For the first method, we used a temporarily openable mask that enables intranasal administration during inhalation anesthesia. The openable part of the mask incorporates a silicone plug that can be used in accordance with administration timing to facilitate stable intranasal administration using a pipette. For the second method, a cannula was surgically inserted to pass from the esophagus into the nasal cavity, and a syringe pump was then attached to this so that the drug solution could be directly and reliably delivered into the nasal cavity under stable inhalation anesthesia. This method can enhance the delivery of drugs into the brain via the nose-to-brain route, because by substantially minimizing the effects of MC, drug retentively in the nasal cavity would be improved. In addition, we describe a method for quantitatively evaluating drug distribution levels (% for the injected dose/g brain) in the brain using radio-labeled [14C]-inulin [molecular weight (MW): 5,000] as a model substrate of water-soluble macromolecules.

<table><tbody><tr><td>ddY mouse</td><td>Japan SLC, Inc.</td><td></td><td>Male, 4-6 weeks, 20-30 g</td></tr><tr><td>Isoflurane</td><td>Pfizer</td><td>v002139</td><td></td></tr><tr><td>Isoflurane setup</td><td>SHINANO manufacturing CO. LTD.</td><td>SN-487-OTAir, SN-489-4</td><td></td></tr><tr><td>Isoflurane mask</td><td>SHINANO manufacturing CO. LTD.</td><td></td><td>For small rodents</td></tr><tr><td>Isoflurane mask (openable type)</td><td>SHINANO manufacturing CO. LTD.</td><td></td><td>Special orders</td></tr><tr><td>Anesthesia Box</td><td>SHINANO manufacturing CO. LTD.</td><td>SN-487-85-02</td><td></td></tr><tr><td>Animal experiments scissors-1</td><td>NATSUME SEISAKUSHO CO., LTD.</td><td>B-27H</td><td></td></tr><tr><td>Animal experiments scissors-2</td><td>NATSUME SEISAKUSHO CO., LTD.</td><td>B-13H</td><td></td></tr><tr><td>Tweezers-1</td><td>FINE SCIENCE TOOLS Inc.</td><td>11272-30</td><td>Dumont #7 Dumoxel</td></tr><tr><td>Tweezers-2</td><td>NATSUME SEISAKUSHO CO., LTD.</td><td>A-12-1</td><td></td></tr><tr><td>Cannula tube (PE-50)</td><td>Becton, Dickinson and Company.</td><td>5069773</td><td>I.D.: 0.58 mm, O.D.: 0.965 mm</td></tr><tr><td>Cannula tube (SP-10)</td><td>NATSUME SEISAKUSHO CO., LTD.</td><td>KN-392</td><td>I.D.: 0.28 mm, O.D.: 0.61 mm</td></tr><tr><td>Shaver</td><td>MARUKAN, LTD.</td><td>DC-381</td><td></td></tr><tr><td>Stereoscopic microscope</td><td>Olympus Corporation</td><td>SZ61</td><td></td></tr><tr><td>Needle 27G 1/2 in 13 mm</td><td>TERUMO CORPORATION</td><td>NN-2738R</td><td></td></tr><tr><td>1 mL syringe</td><td>TERUMO CORPORATION</td><td>SS-01T</td><td></td></tr><tr><td>Syringe pump</td><td>Neuro science</td><td>NE-1000</td><td></td></tr><tr><td>Cellulose membrane</td><td>Toyo Roshi Kaisya, Ltd.</td><td>00011090</td><td></td></tr><tr><td>Micro spatula</td><td>Shimizu Akira Inc.</td><td>91-0088</td><td></td></tr><tr><td>Micropipette (0.5-10 uL)</td><td>Eppendorf AG</td><td>Z368083</td><td></td></tr><tr><td>Pipette chip</td><td>Eppendorf AG</td><td>0030 000.811</td><td></td></tr><tr><td>Tape</td><td>TimeMed Labeling System, Inc.</td><td>T-534-R</td><td>For fixing mouse</td></tr><tr><td>[&lt;sup&gt;14&lt;/sup&gt;C]-Inulin</td><td>American Radiolabeled Chemicals Inc.</td><td>ARC0124A</td><td>0.1 mCi/mL</td></tr><tr><td>EtOH</td><td>Wako Pure Chemical Industries, Ltd.</td><td>054-00461</td><td></td></tr><tr><td>Liquid scintillation counter</td><td>Perkin Elmer Life and Analytical Sciences, Inc</td><td>Tri-Carb 4810TR</td><td></td></tr></tbody></table>

novel methods for intranasal administration under inhalation anesthesia to evaluate nose-to-brain drug delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Figure 3 shows the [14C]-inulin levels (ID%&#8260;g brain) in the olfactory bulb (A), cerebrum (B), and medulla oblongata (C) obtained using the two types of intranasal administration assessed in the present study. Intranasal administration using the pipette method enabled delivery of [14C]-inulin into the brain using openable inhalation masks (Figure 1). Under inhalation anesthesia, the quantitative results...

Watch this Scientific Journal Video about Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery at JoVE.com

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

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19.9K Views.  Nihon University. Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Video: Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

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 ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the extremely low delivery efficiency (&#60; 1%) to the olfactory mucosa that directly connects the brain. It is crucial to develop novel techniques to deliver neurological medications more effectively to the olfactory region. The objective of this study is to develop a numerical platform to simulate and improve intranasal olfactory drug delivery. A coupled image-CFD method was presented that synthetized the image-based model development, quality meshing, fluid simulation, and magnetic particle tracking. With this method, performances of three intranasal delivery protocols were numerically assessed and compared. Influences of breathing maneuvers, magnet layout, magnetic field strength, drug release position, and particle size on the olfactory dosage were also numerically studied.
From the simulations, we found that clinically significant olfactory dosage (up to 45%) were feasible using the combination of magnet layout and selective drug release. A 64 -fold higher delivery of dosage was predicted in the case with magnetophoretic guidance compared to the case without it. However, precise guidance of nasally inhaled aerosols to the olfactory region remains challenging due to the unstable nature of magnetophoresis, as well as the high sensitivity of olfactory dosage to patient-, device-, and particle-related factors.

This manuscript reviews the modeling and simulations of different protocols to deliver medications to the olfactory region in image-based nasal airway models. Multiple software modules are used to develop the anatomically accurate nose model, generate computational mesh, simulate nasal airflows, and predict particle deposition at the olfactory region.

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the ...

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 ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Immunology and Infection

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Neuroscience

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 ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Biology

Compound Administration III

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound administration in laboratory mice and rats. However, certain protocols may require the use of less commonly used routes, including intradermal, intranasal, and intracranial injections. Specialized training is essential for these procedures to be performed successfully. Justification for these routes may need to be provided to gain Institutional Animal Care and Use Committee (IACUC) approval.

Compound Administration in Rodents- Intradermal, Intranasal &amp; Intracranial Routes

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound ...