Name: novel methods for intranasal administration under inhalation anesthesia to evaluate nose-to-brain drug delivery
Uploaded: 2018-11-14T14:45:00
Description: Watch this Scientific Journal Video about Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery at JoVE.com

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
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	<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 m...

<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 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 (Opneable 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>[14C]-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 ...

This method can help answer key questions in the pharmaceutical sciences field about intranasal administration and pharmacokinetics and pharmacology of drugs of interest. The main advantage of this technique is that it can be used for the quantitative evaluation of nose-to-brain delivery drug candidates and inhalation anesthesia with minimal stress to the animals. The implications of this technique extends towards the therapy of central nervous system diseases as it contributes to the development of drug delivery technologies via the nose-to-brain route.Demonstrating the procedure will be Mitsuyoshi Fukuda, a PhD student from my laboratory. For intranasal delivery via micropipette in an isolated radio isotope facility. Tape and anesthetize mouse to a cork board in the supine position, and administer 25 microliters of carbon-14 inulin solution in one to two microliter doses, alternatively, into the right and left nostrils of the animal.For reverse cannulation delivery from the airway side through the esophagus, place the anesthetized mouse under a dissecting microscope, and make a 1.5 centimeter skin incision over the throat. Use forceps to expand the incision until the trachea is exposed, and make a one millimeter incision in the exposed trachea. Insert a cannula 1.2 centimeters to the pre-marked position into the incision, and attach the opposite end of the cannula to the inside of an inhalation mask.After this, use forceps to expose the esophagus from under the trachea. Use scissors to make a one millimeter incision in the esophagus and insert a second cannula 1.4 centimeters to the pre-marked position toward the posterior end of the nasal cavity. It is important to insert cannula to an appropriate length according to the weight of the experimental animal and to adjust the length of the cannula in other ones.Ligate the esophageal cannula and attach a 27-gauge needle to a one milliliter syringe filled with an administration solution connected to a programmable microsyringe pump. Then deliver 25 microliters of carbon-14 inulin at a constant five microliter per minute rate until the entire volume of solution has been administered. A slow but constant diffusion rate is important for the retention of as much drug solution as possible within the nasal cavity.To quantify the amount of carbon-14 inulin that crossed the blood-brain barrier 30 minutes after the inulin administration, use scissors to carefully open the cranium of each experimentally-treated animal from the medulla oblongata side, and use a microspatula to carefully scoop out the brains. Place each brain on a piece of saline-moistened filter paper in a Petri dish on ice as it is harvested. And use a saline-soaked cotton swab to clean any blood from the surface of each brain.Next, quickly but carefully, dissect the brains into olfactory bulb, cerebrum, and medulla oblongata, plus pons sections. And place the brain samples in tissue solubilizer for one hour at 50 degrees Celsius, followed by the addition of 10 microliters of liquid scintillation cocktail. To determine the radioactivity of the applied solution, transfer a 25 microliter aliquot of the administration solution, dissolved in the scintillation cocktail, to a scintillation vial, and measure the disintegrations per minute of the carbon-14 radioactivity within the brain sample and the applied solution in a liquid scintillation counter.Under inhalation anesthesia, no experimental inter-individual variation is observed among intranasally-drug delivered animals. When the esophageal reverse cannula nasal cavity administration method is used, significantly higher levels of carbon-14 inulin are observed in the olfactory bulb, cerebrum, and medulla oblongata, than via micropipette delivery. Moreover, higher carbon-14 inulin is detected in the olfactory bulb and the medulla oblongata, both of which are prominently-evolved in the nose-to-brain pathway, than in the cerebrum.After its development, this technique paved the way for researchers in the field of nose-to-brain delivery to explore the bioactivity of large molecule therapeutic agents in the central nervous system.

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A Novel Rescue Technique for Difficult Intubation and Difficult Ventilation

We describe a novel non surgical technique to maintain oxygenation and ventilation in a case of difficult intubation and difficult ventilation, which works especially well with poor mask fit.
Can not intubate, can not ventilate" (CICV) is a potentially life threatening situation. In this video we present a simulation of the technique we used in a case of CICV where oxygenation and ventilation were maintained by inserting an endotracheal tube (ETT) nasally down to the level of the naso-pharynx while sealing the mouth and nares for successful positive pressure ventilation.
A 13 year old patient was taken to the operating room for incision and drainage of a neck abcess and direct laryngobronchoscopy. After preoxygenation, anesthesia was induced intravenously. Mask ventilation was found to be extremely difficult because of the swelling of the soft tissue. The face mask could not fit properly on the face due to significant facial swelling as well. A direct laryngoscopy was attempted with no visualization of the larynx. Oxygen saturation was difficult to maintain, with saturations falling to 80%. In order to oxygenate and ventilate the patient, an endotracheal tube was then inserted nasally after nasal spray with nasal decongestant and lubricant. The tube was pushed gently and blindly into the hypopharynx. The mouth and nose of the patient were sealed by hand and positive pressure ventilation was possible with 100% O2 with good oxygen saturation during that period of time. Once the patient was stable and well sedated, a rigid bronchoscope was introduced by the otolaryngologist showing extensive subglottic and epiglottic edema, and a mass effect from the abscess, contributing to the airway compromise. The airway was secured with an ETT tube by the otolaryngologist.This video will show a simulation of the technique on a patient undergoing general anesthesia for dental restorations.

We describe a technique to maintain oxygenation and ventilation using an endotracheal tube inserted nasally to the level of the naso-pharynx while sealing the mouth and nares for successful positive pressure ventilation.

We describe a novel non surgical technique to maintain oxygenation and ventilation in a case of difficult intubation and difficult ventilation, which ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

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 ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

CO2-Lasertonsillotomy Under Local Anesthesia in Adults

Tonsil-related complaints are very common among the adult population. Tonsillectomy under general anesthesia is currently the most performed surgical treatment in adults for such complaints. Unfortunately, tonsillectomy is an invasive treatment associated with a high complication rate and a long recovery time. Complications and a long recovery time are mostly related to removing the vascular and densely innervated capsule of the tonsils. Recently, CO2-lasertonsillotomy under local anesthesia has been demonstrated to be a viable alternative treatment for tonsil-related disease with a significantly shorter and less painful recovery period. The milder side-effect profile of CO2-lasertonsillotomy is likely related to leaving the tonsil capsule intact. The aim of the current report is to present a concise protocol detailing the execution of CO2-lasertonsillotomy under local anesthesia. This intervention has been performed successfully in our hospital in more than 1,000 patients and has been found to be safe and to be associated with a steep learning curve.

CO2-Lasertonsillotomy Under Local Anesthesia in Adults

CO2-lasertonsillotomy under local anesthesia is an interesting alternative treatment method for tonsillectomy under general anesthesia for tonsil-related complaints in adults. This report presents a step-by-step protocol detailing the execution of CO2-lasertonsillotomy under local anesthesia.

Tonsil-related complaints are very common among the adult population. Tonsillectomy under general anesthesia is currently the most performed surgical ...

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 ...

Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards

Electroretinogram (ERG) is the only clinical objective test available to assess retinal function. Full-field ERG (ffERG) measures the panretinal rod and cone photoreceptor function as well as inner retinal function and is an important measure in the diagnosis and management of inherited retinal diseases as well as inflammatory, toxic, and nutritional retinopathies. Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid and reliable dark-adapted (scotopic) and light-adapted (photopic) ffERG responses. Performing ffERG in infants and children is challenging and often requires general anesthesia in the operating room. However, maintaining retinal dark adaptation in the operating room is becoming increasingly difficult given the numerous light sources from anesthesiology monitoring systems and other equipment. A practical and widely applicable method for ffERG testing is described in the operating room that optimizes retinal dark adaptation. The method reduces operating room time by dark-adapting the patient before general anesthesiology is instituted. The operating room is modified for dark adaptation and any remaining light source in the darkened operating room is minimized with the use of a modified portable foldable darkroom that encloses the patient&#8217;s head and the ERG examiner during ffERG scotopic recordings. The simple method adheres to ffERG international standards and provides valid reliable scotopic and photopic ffERG recordings that are critical to assess objective retinal function in this young age group where subjective assessment of visual function such as visual acuity and visual fields are not possible. Furthermore, the ffERG is the gold standard clinical test in detecting early onset inherited retinal diseases including Leber congenital amaurosis where approved gene therapy has become available. In sedated conditions, very low amplitude ffERG signals can be detected due to minimal orbicularis muscle activity interference, which is particularly relevant in patients after gene therapy to detect improved amplitude responses.

Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field&#160;electroretinogram for infants and children under sedation or general anesthesia in the operating room setting.

Electroretinogram (ERG) is the only clinical objective test available to assess retinal function. Full-field ERG (ffERG) measures the panretinal rod and ...