The authors have no acknowledgements.&#160;

All procedures were approved by the ethics committee of the medical association of the State Rhineland-Palatinate and performed in accordance with the Declaration of Helsinki.
1. Atomizer4
<ol>
	<li>Remove the set from package. Inspect the product to ensure there is no damage. 
	NOTE: The device is intended for one-time use. Please refer to the instructions for use for further information about storage and supply (see Materials and Equipment Table).</li>
</ol>
2. General Preparation of the Patient
<ol>
	<li>Administer premedication, e.g. Lorazepam 1 - 2.5 mg the evening before and/or 2 - 4 mg 1 - 2 h before the intervention (with attention to contraindications).</li>
	<li>Establish monitoring parameters such as pulse oximetry (SpO2), electrocardiography (ECG) and non-invasive blood pressure (NIBP). 
	NOTE: Monitor patient sedation using standards such as Ramsay Sedation Scale and Bispectral Index monitoring. Close monitoring of the level of sedation can help prevent potential complications such as respiratory depression.</li>
	<li>Secure a peripheral intravenous access.</li>
</ol>
3. Topical Anesthesia of the Upper Airway
<ol>
	<li>Apply 1 mL mixture of lidocaine 2% with phenylephrine 0.25% to each nostril.</li>
	<li>Apply lidocaine 10% spray twice directly onto the mucosa of the oropharynx, spraying from the tip to the back of the tongue. Ask the patient to gargle the lidocaine in the mouth for as long as possible. 
	NOTE: Anesthesia of the supraglottic airway can be performed alternatively using cotton-tipped swabs or nasopharyngeal airways, drops, gel, swish and gargling, aspirating and spraying local anesthetics.</li>
</ol>
4. Analgo-sedation
<ol>
	<li>Start analgo-sedation using i.v. medication, e.g. initial sufentanil bolus (&#60; 60 kg: 5 &#181;g; &#62; 60 kg: 10 &#181;g; &#62; 100 kg: 15 &#181;g). 
	NOTE: Dose drug-induced sedation at the lowest level possible in order to prevent apnea or airway constriction.</li>
	<li>Give an extra sufentanil i.v. bolus, e.g. 5 &#181;g, as &#39;rescue&#39; treatment until a Ramsay Sedation Score of 2 (cooperative, oriented, tranquil) is achieved.</li>
</ol>
5. Equipment Preparation
<ol>
	<li>Assemble and check the flexible intubation endoscope according to institutional standards.</li>
	<li>Connect the oxygen tube of the atomizer to the oxygen flow meter directly. 
	Caution! Oxygen flow meters with humidifier bottles must not be used.</li>
	<li>Attach the connecting tube to the Luer on the working channel of the endoscope. Set the oxygen flow rate to 10 L/min, as recommended in the instruction manual.</li>
	<li>Connect a 1 mL syringe containing the lidocaine 2% solution to the three-way sidearm fitting. 
	NOTE: Lidocaine 2% with its favorable efficacy profile is a suitable anesthetic.</li>
</ol>
6. Atomizer Technique
<ol>
	<li>Close the flow-control opening using a finger. Rapidly inject the medication. Immediately release the flow-control opening. 
	NOTE: A syringe must be connected to the three-way sidearm fitting to obtain a continuous low oxygen flow through the working channel.</li>
	<li>While advancing the flexible endoscope in the airway, inject the local anesthetic using the atomizer at the following sites: inside nostril, on posterior nares; epiglottis; glottis; and vocal cords.</li>
	<li>Repeat these steps as often as required while advancing the endoscope (&#34;spray-as-you-go&#34;). Before passing through the vocal cords, wait for 2 min to allow sufficient drug effect at the site. 
	NOTE: Monitor sedation level of patient and top up sedation using i.v. medication (e.g. sufentanil 5 &#181;g), as needed.</li>
</ol>
7. Flexible Endoscopic Intubation
<ol>
	<li>Advance the endoscope carefully through the glottis into the trachea and position the tip of the endoscope just above the carina. Railroad a suitable sized, cuffed and lubricated flexible endotracheal tube over the endoscope into the trachea under rotation. 
	NOTE: Avoid accidental advancement of the endoscope tip into a main stem bronchus.</li>
	<li>Confirm placement of the endotracheal tube in the trachea just above the carina by visual control via the endoscope. Remove the endoscope, leaving the endotracheal tube in place.</li>
	<li>Confirm the endotracheal position of the tube by measuring end tidal CO2 using capnography in the spontaneously breathing patient.</li>
	<li>Induce general anesthesia, e.g. intravenous Propofol 2 mg/kg/body weight. Start ventilating the patients according to current standards.</li>
</ol>
NOTE: The Enk Atomizer set is currently not available from Cook Medical.

<ol>
	<li>Simmons, S. T., Schleich, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+regional+anaesthesia+for+awake+fibreoptic+intubation.">Airway regional anaesthesia for awake fibreoptic intubation.</a> Reg Anesth Pain Med. 27, 180-192 (2002).</li><li>Benumof, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+the+difficult+adult+airway.+With+special+emphasis+on+awake+tracheal+intubation.">Management of the difficult adult airway. With special emphasis on awake tracheal intubation.</a> Anesthesiology. 75, 1087-1110 (1991).</li><li>Piepho, T., Thierbach, A. R., G&#246;bler, S. M., Maybaur, M. O., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+different+techniques+of+fiberoptic+intubation.">Comparison of two different techniques of fiberoptic intubation.</a> Eur J Anaesthesiol. 26, 328-332 (2009).</li><li>Pirlich, N., Lohse, J. A., Schmidtmann, I., Didion, N., Piepho, T., Noppens, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+atomizer+with+boluses+of+topical+anaesthesia+for+awake+fibreoptic+intubation.">A comparison of the atomizer with boluses of topical anaesthesia for awake fibreoptic intubation.</a> Anaesthesia. 71, 814-822 (2016).</li><li>Malcharek, M. J., 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=Comparison+of+the+Enk+Fibreoptic+Atomizer+with+translaryngeal+injection+for+topical+anaesthesia+for+awake+fibreoptic+intubation+in+patients+at+risk+of+secondary+cervical+injury:+a+randomised+controlled+trial.">Comparison of the Enk Fibreoptic Atomizer with translaryngeal injection for topical anaesthesia for awake fibreoptic intubation in patients at risk of secondary cervical injury: a randomised controlled trial.</a> Eur J Anaesthesiol. 32, 615-623 (2015).</li><li>Williams, K. A., Barker, G. L., Harwood, R. J., Woodall, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+nebulization+and+spray-as-you-go+topical+anaesthesia+of+the+airway.">Combined nebulization and spray-as-you-go topical anaesthesia of the airway.</a> Br J Anaesth. 95, 549-553 (2005).</li><li>O'Hare, D. O., Harwood, R., Woodall, N., Barker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+administering+topical+anaesthesia+for+flexible+airway+endoscopy.">A method of administering topical anaesthesia for flexible airway endoscopy.</a> Anaesthesia. 55, 616 (2000).</li><li>Long, T. R., Wass, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+to+transtracheal+injection+for+fiberoptic+intubation+in+awake+patients:+A+novel+noninvasive+technique+using+a+standard+multiorifice+epidural+catheter+through+the+bronchoscope+suction+port.">An alternative to transtracheal injection for fiberoptic intubation in awake patients: A novel noninvasive technique using a standard multiorifice epidural catheter through the bronchoscope suction port.</a> Anaesthesiology. 101, 1253 (2004).</li><li>Xue, F. S., 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=Spray-as-you-go+airway+topical+anesthesia+in+patients+with+a+difficult+airway:+a+randomized,+double-blind+comparison+of+2%+and+4%+lidocaine.">Spray-as-you-go airway topical anesthesia in patients with a difficult airway: a randomized, double-blind comparison of 2% and 4% lidocaine.</a> Anesth Analg. 108, 536-543 (2009).</li><li>Hershey, M. D., Hannenberg, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+distension+and+rupture+from+oxygen+insufflation+during+fiberoptic+intubation.">Gastric distension and rupture from oxygen insufflation during fiberoptic intubation.</a> Anesthesiology. 85, 1479-1480 (1996).</li><li>Ho, C. M., Yin, I. W., Tsou, K. F., Chow, L. H., Tsai, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+rupture+after+awake+fiberoptic+intubation+in+a+patient+with+laryngeal+carcinoma.">Gastric rupture after awake fiberoptic intubation in a patient with laryngeal carcinoma.</a> Br J Anaesth. 94, 856-858 (2005).</li><li>Chapman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+rupture+and+pneumoperitoneum+caused+by+oxygen+insufflation+via+a+fibreoptic+bronchoscope.">Gastric rupture and pneumoperitoneum caused by oxygen insufflation via a fibreoptic bronchoscope.</a> Anesth Analg. 106, 1592 (2008).</li><li>Ovassapian, A., Mesnick, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+insufflation+through+the+fiberscope+to+assist+intubation+is+not+recommended.">Oxygen insufflation through the fiberscope to assist intubation is not recommended.</a> Anesthesiology. 87, 183 (1997).</li></ol>

The authors have nothing to disclose.&#160;The Enk-Atomizer set is currently not available from Cook Medical. According to the inventor (personal communication) a comparable device will be available in the future.

Here we demonstrate topical anesthesia using an atomizer with high oxygen flow for awake-endoscopic intubation. The first study to evaluate the atomizer technique compared it with classical boluses of topical anesthesia via the working channel of the flexible intubating endoscope5. Patients in the atomizer group evaluated their level of comfort as better than those in the control group and experienced fewer coughs and distinct coughing episodes. In addition, the atomizer technique was faster and less local ane...

Topical anesthesia of the airway for awake intubation is generally recommended to improve patient comfort, which will help make the procedure a success. The spray-as-you-go technique with high oxygen flow is a simple and safe concept that is finding a high level of acceptance among patients. The atomization of the local anesthetic by oxygen at a flow rate of 10 L/min results in less stimulation of the cough and gag reflex.
Faced with negative feedback from several patients, we decided to refine the standard institutional spray-as-you-go technique. This classical bolus application of local anesthetic consisted of two injections, each of 5 mL lidocaine 2% plus 5 mL air in a 10 mL syringe, first into the vocal cords and then, after a 2-min waiting time, into the trachea. Patients complained about the feeling of being drowned by the splash of the bolus striking their airway mucosa.
The spray-as-you-go technique is commonly used worldwide1. This technique involves local anesthetic application through the working channel of a flexible intubating endoscope while advancing the tip through the upper and lower airway with the goal of intubating the patient&#180;s trachea. There are multiple approaches in practice and several descriptions of methods in the literature. Initial experiences with the spray-as-you-go-technique in combination with application of a constant oxygen flow through the working channel were published in the 1990s2. The focus here was on oxygen flow effects like cleaning the lens by dispersing mucus secretions and blood away from the lens and elevating the inspiratory oxygen delivery. The advantage of the oxygen flow in aiding atomization of local anesthetic was shown previously by Piepho and colleagues3. They described a vaporization technique, where the supply of 3 L/min oxygen resulted in less coughing by the patients. On the assumption that a flow of 10 L/min would atomize the local agent into finer particles that would evoke even less coughing, we successfully tested the atomizer at this higher flower rate. The atomizer consists of a small volume, kink-resistant, flexible, pressure-certified oxygen tube and a connecting tube. These are connected to a three-way sidearm fitting with a small flow-control opening.
The atomizer technique can be used for all airway situations. However, the device should be used cautiously distal from higher-grade airway stenosis caused by conditions such as tumor masses. The lack of backflow of oxygen could cause increased airway pressure and increases the risk of barotrauma. Also, the atomizer is a useful tool in an emergency setting. The preparation of the set needs less than a minute and the user needs no special training. In the following we present detailed operating instructions for the spray-as-you-go technique with high oxygen flow, illustrating the separate working steps.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lidocaine 10% pump spray&#160;</td>
			<td>e.g. AstraZeneca</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Opiod for i.v. administration</td>
			<td></td>
			<td>Any opiod can be used. We prefer either Sufentanil or Fentanyl</td>
		</tr>
		<tr>
			<td>Enk Fiberoptic Atomizer Set&#160;</td>
			<td>Cook Medical</td>
			<td>Catalogue number: C-EFNS-100&#160;</td>
		</tr>
		<tr>
			<td>Lidocaine 2%&#160;</td>
			<td>e.g. AstraZeneca</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intubation endoscope</td>
			<td>e.g. Karl Storz Endoscope</td>
			<td>We prefer a fibrescope with an outer diamter of 5 mm and a working channel with a diamter of 2 mm&#160;</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>e.g. R&#252;sch</td>
			<td>Size of the endotracheal tube needs to be adjusted to the patient. We prefer a ID 6.5mm for nasal approach and ID 7.0 mm for oral approach</td>
		</tr>
		<tr>
			<td>Anesthetic drug, e.g. Propofol 1%</td>
			<td>e.g. AstraZeneca</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

topical airway anesthesia for awake-endoscopic intubation using the spray-as-you-go technique with high oxygen flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

We performed awake flexible endoscopic intubation using the presented topical anesthesia technique in 48 patients. We succeeded in intubating all patients except one, who suffered from a stenosing tumor of the pharynx, which made it impossible to advance the tube.
The quality of awake intubation comfort was evaluated by patients, anesthetists and nurses using an 11-point visual analog scale (VAS) from 0 = not unpleasant to 10 = ...

Watch this Scientific Journal Video about Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow at JoVE.com

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

topical-airway-anesthesia-for-awake-endoscopic-intubation-using-spray

Research

JoVE Journal

Medicine

16.5K Views.  University Medical Center of the Johannes Gutenberg-University. The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

Video: Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

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 ...

Guidelines for Elective Pediatric Fiberoptic Intubation

Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small babies will desaturate very quickly if ventilation is interrupted mainly to high metabolic rate. We describe guidelines to perform a safe fiberoptic intubation while maintaining spontaneous breathing throughout the procedure. Steps requiring the use of propofol pump, fentanyl, glycopyrrolate, red rubber catheter, metal insuflation hook, afrin, lubricant and lidocaine spray are shown.

We describe guidelines to perform a safe and efficient elective fiberoptic intubation in pediatric patients while maintaining spontaneous ventilation.

Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Repeated Orotracheal Intubation in Mice

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

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

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

Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation

Magnetic resonance imaging (MRI) is an important tool for the clinical assessment of cardiovascular morphology and heart function. It is also the recognized standard-of-care for blood flow quantification based on phase contrast MRI. While such measurement of blood flow has been possible in adults for decades, methods to extend this capability to fetal blood flow have only recently been developed.
Fetal blood flow quantification in major vessels is important for monitoring fetal pathologies such as congenital heart disease (CHD) and fetal growth restriction (FGR). CHD causes alterations in the cardiac structure and vasculature that change the course of blood in the fetus. In FGR, the path of blood flow is altered through the dilation of shunts such that the oxygenated blood supply to the brain is increased. Blood flow quantification enables assessment of the severity of the fetal pathology, which in turn allows for suitable&#160;in utero&#160;patient management and planning for postnatal care.
The primary challenges of applying phase contrast MRI to the human fetus include small blood vessel size, high fetal heart rate, potential MRI data corruption due to maternal respiration, unpredictable fetal movements, and lack of conventional cardiac gating methods to synchronize data acquisition. Here, we describe recent technical developments from our lab that have enabled the quantification of fetal blood flow using phase contrast MRI, including advances in accelerated imaging, motion compensation, and cardiac gating.

Here we present a protocol for measuring fetal blood flow rapidly with MRI and retrospectively performing motion correction and cardiac gating.

Magnetic resonance imaging (MRI) is an important tool for the clinical assessment of cardiovascular morphology and heart function. It is also the ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...