The authors have no acknowledgments. Alexandra Pop and Ioan Florin Marchis are featured in the video production that complements this article.

We designed a prospective, observational, single-center study evaluating the feasibility of tracheal intubation using the RTL with the approval of the University Ethics Committee no 432/24.11.2016 and registered with ClinicalTrials.gov NCT03341507. The study involved adult patients with ASA physical status 1&#8211;3, requiring general anesthesia for ear, parotid, and rhino-sinus surgery and having a presumed anatomically difficult airway as calculated with The Simplified Airway Risk Index (SARI) score8, with a score &#8805; 5, and no airway pathology.
NOTE: The pre-anesthetic exam focused on airway evaluation aiming to select patients with a modified Cormack-Lehane7 grade of glottic visualization &#8805;2b. The Cormack-Lehane classification defines the grade of glottis view during laryngoscopy, ranging from unrestricted glottic visualization-grade 1 to inability to see any laryngeal structure-grade 4. The SARI score8 is a multivariate risk score for predicting difficult tracheal intubation. A SARI score of 4 or above, 12 being maximum, enhances the chances of difficult intubation. Seven parameters contribute to the SARI score: mouth opening, thyromental distance, movement of the neck, Mallampati score, the ability to create an underbite, body weight, and previous intubation history.
1. Patient and equipment preparation
<ol>
	<li>Premedicate with 1 or 2 mg of midazolam intravenously for 10 min upon patient transportation in the operating theatre.</li>
	<li>Have the patient lie on the operating table with their head placed at a tilt in a sniffing position.</li>
	<li>Apply standard hemodynamic and respiratory monitoring: ECG, noninvasive arterial blood pressure, oxygen saturation, respiratory frequency, end-tidal CO2. Monitor patient anesthesia depth using bispectral index monitoring.</li>
	<li>Make sure that an experienced anesthetist or ENT physician is available in case of an emergency.</li>
	<li>Prepare a suction device with a suction probe in place and a difficult airway kit at hand. Prepare and check the connective piece with the light source attached with a set of rigid tubes of various sizes: three tubes that are 15-20 cm long with a 1.2-1.8 cm diameter for women and three tubes that are 20-25 cm long with a 1.2-2 cm diameter for men.</li>
	<li>Identify the thyroid cartilage (Adam&#39;s apple) by palpation of the anterior neck. Ask the patient to swallow and gently palpate the anterior neck with the thumb, the index, and the middle finger. The thyroid cartilage is the structure that moves while the patient swallows.
	<ol>
		<li>Ask the patient to extend the head and measure the distance between the labial commissure and the thyroid cartilage in a straight line using the thumb and the middle finger. Hold a rigid tube in the other hand while performing the measurement. The mandible impedes the use of a ruler to measure this distance.</li>
		<li>Note the length obtained on the rigid tube prepared, starting from its tip. Do this by directly translating the two fingers used to measure the distance mentioned in the previous step on the rigid tube.</li>
		<li>Expect to find the glottis when using the RTL at a depth equal with the distance between the labial commissure and thyroid cartilage. For a better grip and a useful lever effect, use a tube with a length of about double the above-measured distance.</li>
	</ol>
	</li>
</ol>
2. Induction of general anesthesia and conventional laryngoscopy
<ol>
	<li>Preoxygenate the patients for 5 min and start anesthesia induction with 2-3 &#181;g/kg fentanyl, 2-3 mg/kg propofol, and 1-1.5 mg/kg succinylcholine. Monitor the depth of anesthesia by checking the bispectral index level.</li>
	<li>After 1 min of mask ventilation with a pause during generalized muscular fasciculation provoked by succinylcholine, perform standard laryngoscopy with a curved blade laryngoscope, and register the modified Cormack-Lehane glottis visualization grade.
	<ol>
		<li>If the noticed degree is &#60;2b, resume the tracheal intubation conventionally with the curved blade laryngoscope.</li>
		<li>If the glottis view grade is &#8805;2b, meaning that, at the best, part of the arytenoids and the epiglottis are visible, withdraw the Macintosh laryngoscope, and maneuver further with the RTL. 
		CAUTION: If the mask ventilation is impossible or not efficient, the airway should be secured promptly following the institutional guidelines.</li>
	</ol>
	</li>
</ol>
3. Tracheal intubation with the rigid tube
<ol>
	<li>Cover the upper molars with a rubber teeth protector or a cotton swab on the side of the approach.</li>
	<li>Introduce the RTL in the oral cavity at the level of the right or left labial commissure with the bevel facing the superior arcade, the prismatic light deflector, and a functioning light source attached, held with the dominant hand with the light deflector inside the palm and the index following the tube.
	<ol>
		<li>Start with a 1.5-1.8 cm diameter tube to maximize the visual field.</li>
		<li>Displace the tongue gently to the contralateral site.</li>
		<li>If the pathology does not require otherwise, a righthanded practitioner should use the right retromolar space for approach.</li>
	</ol>
	</li>
	<li>Use the nondominant hand to open the patient&#8217;s mouth, hyperextend the patient&#39;s head, and advance the RTL towards the pharynx.</li>
	<li>Apply gentle pressure on the proximal end of the RTL against the protected superior molars as the tip of the tube is reaching the hypopharynx. That way, the tip of the RTL orientates anteriorly on the direction of the glottis. 
	NOTE: The thyroid cartilage is a landmark when moving the tip of the RTL from the retromolar space towards the midline.
	<ol>
		<li>If the epiglottis, the glottis or part of it is not getting into sight, withdraw the tube. At the second attempt, insert the tube in the oral cavity at the level of the incisors as that may make the tongue base and epiglottis easier to access. Once the epiglottis is in sight, slide the tube towards the labial commissure, keeping its tip in place, and advance further.</li>
		<li>Aspirate the secretions as needed with a flexible suction probe inserted through the RTL.</li>
		<li>Adjust the position of the larynx with the nondominant hand by gently pushing the thyroid cartilage and follow an imaginary line towards the thyroid cartilage as it improves the time needed for proper glottis visualization. 
		NOTE: When moving the cartilage while looking through the tube, the motion translates to the inner structures around the glottis and helps the practitioner orientate to reach his target.</li>
	</ol>
	</li>
	<li>Advance the RTL slowly until it reaches the epiglottis, and then lift the epiglottis with the tip of the device. Make sure that the bevel is oriented posteriorly. At that point, the vocal cords are visible at the distal end of the tube.
	<ol>
		<li>If, after lifting the epiglottis, the glottis is not in the visual field, consider using a smaller diameter RTL.</li>
	</ol>
	</li>
	<li>Once the glottis is in sight, place the intubating tube introducer through the RTL into the trachea. Do not insert the introducer too vigorously because the bronchial injury may occur.
	<ol>
		<li>Do not advance any more as soon as the glottis is visible after lifting the epiglottis because of the risk of vocal cord injury.</li>
	</ol>
	</li>
	<li>Extract the rigid tube.</li>
	<li>Place a standard cuffed lubricated intubating tube over the introducer into the trachea. Do not use force and gently rotate the intubating tube while advancing.
	<ol>
		<li>Make sure there is only a small gap between the inner diameter of the intubating tube and the introducer to reduce the chance of impingement.</li>
	</ol>
	</li>
	<li>Once the intubating tube is at the proper depth, 20-24 cm from the labial commissure, remove the intubating tube introducer leaving the intubating tube in place.</li>
	<li>Inflate the cuff and confirm tracheal intubation through lung auscultation and capnography.</li>
	<li>Inspect the upper lip and the teeth for damage. Be aware that the intubation over an intubating tube introducer carries the risk of vocal cords injury. Check the patient for sore throat and voice disturbance after surgery. 
	CAUTION: Stop the maneuver if it exceeds 100 s or if the patient desaturates to 80% and resume mask ventilation or an alternative method of securing the airway.</li>
</ol>

<ol>
	<li>Cook, T. M., Woodall, N., Frerk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+complications+of+airway+management+in+the+UK:+results+of+the+4th+National+Audit+Project+of+the+Royal+College+of+Anaesthetists+and+the+Difficult+Airway+Society.+Part+1+Anaesthesia.">Major complications of airway management in the UK: results of the 4th National Audit Project of the Royal College of Anaesthetists and the Difficult Airway Society. Part 1 Anaesthesia.</a> British Journal of Anaesthesia. 106 (5), 617-631 (2011).</li><li>Halligan, M., Charters, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+curve+for+the+Bonfils+intubation+fibrescope.">Learning curve for the Bonfils intubation fibrescope.</a> British Journal of Anaesthesia. 89, 671-672 (2002).</li><li>Smith, J. E., Jackson, A. P., Hurdley, J., Clifton, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+curves+for+fibreoptic+nasotracheal+intubation+when+using+the+endoscopic+video+camera.">Learning curves for fibreoptic nasotracheal intubation when using the endoscopic video camera.</a> Anaesthesia. 52 (2), 101-106 (1997).</li><li>N&#248;rskov, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+accuracy+of+anaesthesiologists'+prediction+of+difficult+airway+management+in+daily+clinical+practice:+a+cohort+study+of+188+064+patients+registered+in+the+Danish+anaesthesia+database.">Diagnostic accuracy of anaesthesiologists' prediction of difficult airway management in daily clinical practice: a cohort study of 188 064 patients registered in the Danish anaesthesia database.</a> Anaesthesia. 70 (3), 272-281 (2015).</li><li>Fagan, J., De Groot, M., za, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rigid+Laryngoscopy,+Oesophagoscopy+&amp;+Bronchoscopy+in+Adults.">Rigid Laryngoscopy, Oesophagoscopy &amp; Bronchoscopy in Adults.</a> Open Access Atlas of Otolaryngology, Head &amp; Neck Operative Surgery. , (2019).</li><li>Marchis, F., Radeanu, D., Pop, S., Chirila, M., Cosgarea, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rigid+Tube+of+Laryngoscopy+-+a+New+Intubation+Technique.">The Rigid Tube of Laryngoscopy - a New Intubation Technique.</a> Proceedings: National ENT, Head and Neck Surgery Conference. , 143-146 (2017).</li><li>Krage, R., 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=Cormack-Lehane+classification+revisited.">Cormack-Lehane classification revisited.</a> British Journal of Anaesthesia. 105 (2), 220-227 (2010).</li><li>N&#248;rskov, A. K., Rosenstock, C. V., Wetterslev, J., Lundstr&#248;m, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+unanticipated+difficult+airway+using+an+objective+airway+score+versus+a+standard+clinical+airway+assessment:+the+DIFFICAIR+trial+-+trial+protocol+for+a+cluster+randomized+clinical+trial.">Incidence of unanticipated difficult airway using an objective airway score versus a standard clinical airway assessment: the DIFFICAIR trial - trial protocol for a cluster randomized clinical trial.</a> Trials. 14, 347 (2013).</li><li>Cook, T. M., MacDougall-Davis, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+and+failure+of+airway+management.">Complications and failure of airway management.</a> British Journal of Anaesthesia. 109 (1), 68-85 (2012).</li><li>Abdullah, H. R., Li-Ming, T., Marriott, A., Wong, T. 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+comparison+between+the+Bonfils+Intubation+Fiberscope+and+McCoy+laryngoscope+for+tracheal+intubation+in+patients+with+a+simulated+difficult+airway.">A comparison between the Bonfils Intubation Fiberscope and McCoy laryngoscope for tracheal intubation in patients with a simulated difficult airway.</a> Anesthesia &amp; Analgesia. 117 (5), 1217-1220 (2013).</li><li>Henderson, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+paraglossal+straight+blade+laryngoscopy+in+difficult+tracheal+intubation.">The use of paraglossal straight blade laryngoscopy in difficult tracheal intubation.</a> Anaesthesia. 52 (6), 552-560 (1997).</li><li>Bein, B., 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=Tracheal+intubation+using+the+Bonfils+intubation+fibrescope+after+failed+direct+laryngoscopy.">Tracheal intubation using the Bonfils intubation fibrescope after failed direct laryngoscopy.</a> Anaesthesia. 59, 1207-1209 (2004).</li><li>Webb, A., 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+Bonfils+and+Levitan+optical+stylets+for+tracheal+intubation:+a+clinical+study.">Comparison of the Bonfils and Levitan optical stylets for tracheal intubation: a clinical study.</a> Anaesthesia and Intensive. 39 (6), 1093-1097 (2011).</li><li>Paul, A., Gibson, A. A., Robinson, O. D., Koch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+traffic+light+bougie:+a+study+of+a+novel+safety+modification.">The traffic light bougie: a study of a novel safety modification.</a> Anaesthesia. 69 (3), 214-218 (2014).</li></ol>

The authors have nothing to disclose. We did not find a current commercial offer for the rigid tube and the connective piece we used in this study.

The most prevalent mistake when dealing with an unexpected problematic airway is to insist on the same tool and the same technique only to realize that it is not working. This makes the situation only worse by promoting edema or bleeding9.
We had the idea of testing this tool when we anesthetized a patient with a grade 4 glottic visualization at conventional laryngoscopy, according to the Cormack-Lehane classification. Since the rigid tube was prepared to investigate th...

Airway management incidents represent a large proportion of anesthesia-related mortality and severe morbidity despite the development of new tools for airway control and extensive educational resources1. The video laryngoscope and flexible or rigid endoscopes are big steps forward, but they require a supplementary investment in addition to training2,3. Difficult intubation is often unanticipated, so it is the practitioner&#39;s responsibility to have a backup plan and use the appropriate tool from those available4. This study aims to show that a cheap and straightforward tool, the rigid tube for laryngoscopy (RTL), could be efficiently used for tracheal intubation in patients with poor glottis visualization during conventional laryngoscopy.
The rigid tube for laryngoscopy (Figure 1) is a 15-25 cm long, straight, round hollow tube with a 5-20 mm diameter and a bevel end distally. It resembles a rigid bronchoscope or esophagoscope, but shorter5. A connective piece that has a prismatic light deflector with connection to a light source is attached to the proximal end of the rigid tube when in use, and the image is obtained directly, looking through this assembly. It is an instrument used by ear, nose, and throat specialists to visualize the larynx and its vicinity. In a pilot study, 20 patients with supraglottic pathology and anatomically challenging airways were intubated with the rigid tube, and the results were encouraging6.
This research included patients with anatomical criteria for difficult intubation and a modified Cormack-Lehane7 glottis view grade &#8805;2b when using a curved blade laryngoscope. The purpose was to demonstrate that laryngoscopy using the rigid tube with a retromolar approach could offer a quick glottic view in cases with poor glottic visualization at conventional laryngoscopy and the right conditions to intubate the trachea over the intubating tube introducer.

<table>
	<tbody>
		<tr>
			<td>Airway management set</td>
			<td>Karl Storz</td>
			<td>11300 B3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Draeger</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic drug: Fentanyl</td>
			<td>Chiesi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic drug: propofol</td>
			<td>Fresenius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic drug: succinylcholine</td>
			<td>Takeda</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic drugs: midazolam</td>
			<td>Aguettant</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intubation Tube</td>
			<td>Touren</td>
			<td></td>
			<td>A 7-7.5 mm diameter is suitable for tracheal intubation over the bougie</td>
		</tr>
		<tr>
			<td>Intubation Tube Introducer (Bougie)</td>
			<td>Ontex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricating gel</td>
			<td>Dynarex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mcintosh laryngoscope</td>
			<td>Heine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rigid tube with a removable connective piece that has a prismatic light deflector attached</td>
			<td>Explorent GMBH</td>
			<td></td>
			<td>The connective piece serial number is: 650021 
			We did not find a current commercial offer for the rigid tube and the connective piece we used in this study.</td>
		</tr>
		<tr>
			<td>Table top light source with light cable</td>
			<td>Karl Storz</td>
			<td>20134001, 61594GW</td>
			<td></td>
		</tr>
	</tbody>
</table>

the rigid tube as an alternative in controlling the problematic airway

The unexpected problematic airway represents a large proportion of anesthesia-related morbidity and mortality. The retromolar or paraglossal approach is an alternative to the majority of the rigid instruments used for tracheal intubation, which follow the midline to access the glottis. This single-center, prospective case-series study offers an option to conventional laryngoscopy in case of a poor glottic view, introducing an instrument (the rigid tube for laryngoscopy) that uses the retromolar approach to accomplish tracheal intubation. If after anesthesia induction, the modified Cormack-Lehane glottis view grade &#62;2b, the intubation is carried further with the rigid tube. The tube follows the direction of the thyroid cartilage while advancing from the labial commissure, displacing the tongue to the contralateral side. Adjusting the position of the larynx with the nondominant hand by gently pushing the thyroid cartilage and following an imaginary line towards it while advancing it improves the time needed for proper glottis visualization. Once the epiglottis is in sight, the practitioner progresses slowly, lifting the epiglottis and aiming the tip of the tube more anteriorly. When the glottis appears in the visual field, the intubating tube introducer is placed in the trachea, and a lubricated cuffed intubating tube is advanced over the introducer after the rigid tube is extracted. This tool was tested on 30 patients with an unsatisfactory glottic view when using the Macintosh laryngoscope and obtained excellent results with respect to intubation time and complications. The reduced visual field is the main limitation of this method, which requires a training period for reasonable expertise. This simple, robust, and cheap instrument could be a rescue option in case of a difficult airway.

Over 24 months, we included 64 patients with a SARI &#8805;5, predictive for difficult intubation (Supplemental File 1). Thirty of them presented a modified Cormack-Lehane glottis view grade &#8805;2b during the laryngoscopy performed with the curved blade laryngoscope, so they were attempted to be tracheal intubated with the RTL (Table 1). In all cases, with one exception, the procedure with the rigid tube was successful without any incidents occurring. The patient we did not intubate, ...

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The Rigid Tube as an Alternative in Controlling the Problematic Airway

Presented here is a protocol of tracheal intubation over an intubating tube introducer using a rigid tube for laryngoscopy with an attached light source. The main characteristics of this maneuver are the retromolar approach and the use of the thyroid cartilage as a landmark while advancing the rigid tube.

The unexpected problematic airway represents a large proportion of anesthesia-related morbidity and mortality. The retromolar or paraglossal approach is ...

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6.3K Views.  Iuliu Hatieganu University of Medicine and Pharmacy. Presented here is a protocol of tracheal intubation over an intubating tube introducer using a rigid tube for laryngoscopy with an attached light source. The main characteristics of this maneuver are the retromolar approach and the use of the thyroid cartilage as a landmark while advancing the rigid tube.

Video: The Rigid Tube as an Alternative in Controlling the Problematic Airway

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

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 ...

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.

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 ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of epididymal OA since 2000. There are two challenges in classic microscopic vasoepididymostomy. First, anastomosis of the vas deferens and epididymis is performed with double-needle sutures. However, there is a lack of good-quality and cost-effective double-needle sutures in China, which leads to increased difficulty and poor success rates of anastomosis. Second, the separation of the vas deferens does not retain vasculature, although the vas deferens vasculature plays an important role in the blood supply to the vas deferens, epididymis, and testis. This affects the blood supply to the anastomotic area and epididymis.
Therefore, this team has made innovative improvements to address these problems. Good-quality, cost-effective, single-needle sutures, which are easy to purchase in China and other countries, were used in microsurgical longitudinal intussusception vasoepididymostomy. This can optimize the operation procedure and shorten the operation time while ensuring the success rate of the anastomosis. The surgical method of preserving the vas deferens vessels was innovatively proposed because the etiology of epididymal OA is mostly inflammatory in China. The protection of the blood supply to the vas deferens and epididymis is maximized using microsurgical forceps to separate and protect the vasculature. Patency reached 81.7% in the postoperative follow-up, indicating a better surgical treatment effect.

The present protocol describes a vessel-sparing, longitudinal intussusception vasoepididymostomy using readily available single-needle sutures in China, a safe and effective procedure, which may improve patients' patency and natural pregnancy rates.

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...