Die Autoren danken Brian Johnson von der Histologie und Imaging-Core an der Universität von Washington für die Hilfe bei der Trichrom-Färbung und Analyse. Diese Arbeit wurde vom NIH Zuschüsse HL098067 und HL089455 unterstützt.

Die Institutional Animal Care und Verwenden Komitees (IACUC) an der University of Washington und Cedars-Sinai Medical Center haben das Tier Arbeit notwendig für diese Studien zugelassen. 1. Vorbereitung <ol><li> Sterilisieren sowohl dem stumpfen Ende einer Pinzette und den Niederhalter über Autoklaven. </li><li> Mit einem biologischen Sicherheitsschrank, bereiten eine Arbeits Lager von BLM in PBS aus dem gefriergetrocknetes Pulver. Beschallen die Lösung für 10 min bei 35 kHz auch sicherzustellen Mischen. Hinweis: Ein Gesamtvolumen von 30 bis 45 &amp; mgr; l wird empfohlen Pipettieren Variation am unteren Ende zu verhindern, und Erstickung mit größeren Volumina. </li><li> Bereiten einer sauberen Arbeitsbereich, der sich etwa 1 m 2 für das Verfahren umfasst, sowie vorgesehenen Stellen für Käfige vor und nach dem Eingriff. </li><li> Befestigen Sie die Basis des Verfahrens Bord der Bank unmittelbar vor dem Forscher durch Verlegung 2 oder 3 Streifen von Labor Band über der Basis und underlying Bank. Siehe Abbildung 1 für weitere Angaben auf ein Brett zu schaffen. </li><li> Binden Sie eine Einzellänge von Größe 4,0 Faden zwischen den beiden Stellschrauben des Verfahrens Bord. </li><li> Generieren Sie einen provisorischen Spirometer durch Entfernen und Verwerfen des Kolbens von drei 1 ml Spritzen und Ablegen 60 ul PBS in der Spitze jedes Fass eine luftdichte Abdichtung zu bilden. Sichern Sie die Nabe des Katheters lose an einer der Spritzen und legen Sie es auf der einen Seite der Platine. </li><li> Saugen Sie 300 ul von Luft in einer 1-ml-Spritze und legen Sie es auf der einen Seite der Platine. </li><li> Schneiden Sie ein zusätzliches Stück Band ca. 6 cm in der Länge und zur Seite legen. Diese werden verwendet, um das Tier zu dem Brett in Schritt 2.4 zu sichern. </li><li> Stellen Sie eine Isofluran Kammer auf. Bringen O 2, Isofluran und Vakuum an die entsprechenden Anschlüsse sowohl auf der Belichtungskammer und der Frei Vakuum. Alternativ verwalten Betäubung in eine Isofluran-kompatibel biologischenSicherheitswerkbank. </li></ol> 2. Intubation <ol><li> Betäuben die Maus mit Isofluran in der Kammer, bis er das Bewusstsein verliert und die Atmung verlangsamt zu einer angemessenen Geschwindigkeit. Eine typische Belichtung enthält 4% Isofluran und 2% O 2 für 3 bis 4 min, und das ideale Ergebnis ist 2 bis 2,5 min der Sedierung. Dies entspricht einer Atemfrequenz von 1 Luft alle 2 sec. </li><li> Während des Wartens auf Sedierung in zu setzen, aspirieren zwischen 30 und 45 &amp; mgr; l der BLM in eine Pipette und zur Seite legen. </li><li> Wenn Sie bereit sind, unterbrechen die sediert Maus mit seinem oberen Schneidezähne aus der zu den Stellschrauben des Verfahrens Plattform befestigt Faden. Stellen Sie sicher, dass das Tier die Rücken liegt flach auf der Plattformoberfläche. </li><li> Man aufpassen, nicht Lüftung zu beschränken, legen Sie ein Stück Klebeband lose über die untere (kaudale) Teil der Brusthöhle, direkt über der Membran. Eine Platzierung sollte fest genug sein, die richtige Ausrichtung während der Prozedur zu haltenEDURE, aber nicht so eng, daß sie die Atmung einschränkt. </li><li> Schalten Sie den Illuminator auf zwischen 80% und 100% Intensität und orientieren den Schwanenhals, so dass es 1 bis 2 cm von der Oberfläche der Haut ist in der Nähe des Sonnengeflecht. Überprüfen Sie regelmäßig die Spitze der Schwanenhals für Wärme, um sicherzustellen, dass es nicht die Maus nicht verletzen. </li><li> Hinter der Plattform mit Hilfe der sterilen, stumpfen Ende Zange die Zunge zu lokalisieren. Wobei darauf geachtet, die unteren Schneidezähne, sanft Griff und ziehen die Zunge in der Mundhöhle zu vermeiden. </li><li> Mit dem restlichen Hand, legen Sie die Niederhalter und es verwenden, um die Zunge gegen den Boden der Mundhöhle zu glätten. Lassen Sie die Zange, aber die Niederhalter an Ort und Stelle für die nächsten beiden Schritte lassen. </li><li> Richten Sie das Licht, so dass die Luftröhre durch die Führung des Schwanenhals proximal von der Höhe des Solarplexus sichtbar ist, bis sie das Niveau des Hauptbronchus Bronchien erreicht. Anmerkung: Die Luftröhre kann durch die Wirkung der Atmung leicht unterschieden werden, WHIch bewirkt, dass das emittierte Licht in Intensität schwankt. Wenn richtig positioniert, wird diese Struktur in der axialen Ebene als einer zentral angeordneten Stift von Licht mit minimaler Umgebungslicht in der Mundhöhle selbst erkennbar sein. </li><li> Winkeln Sie die Spritze so, dass sie den natürlichen Weg der Luftröhre folgt, und die 22-G Katheterspitze senken, mit dem angebrachten Spritze das Tröpfchen, gerade in das Lumen enthält. Die PBS-Blase beginnt mit jedem Atemzug bei erfolgreicher Platzierung zu steigen und fallen. Hinweis: Diese Aktion kann durch mehrere Sekunden infolge der tiefen Sedierung verzögert werden. </li><li> Führen Sie den Katheter in einem zusätzlichen 5 mm. Entfernen Sie den Spatel. </li><li> Schieben Sie die Spritze mit der anderen Hand, und Greifen der Nabe, sanft gegen die Spritze entfernen. </li><li> Ablagerung zwischen 30 und 45 &amp; mgr; l von BLM in der Mitte des Inneren des Katheteransatzes, der zweite Spritze befestigen und 300 &amp; mgr; l von Luft in der Nabe verzichtet werden. </li><li> Setzen Sie die zweiteSpritze mit dem zunächst die Blase PBS mit. Die Blase wird weiter steigen und fallen, wenn das Verfahren erfolgreich durchgeführt worden ist. </li></ol> 3. Post-Verfahrenspflege <ol><li> Entfernen Sie den Katheter und Band, und legen Sie das Tier in einem trockenen warmen Ort, bis er das Bewusstsein wiedererlangt - in der Regel innerhalb von ein paar Minuten. </li></ol>

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N., Torres-Gonzalez, E., Goodson, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tracheal+instillation+of+solutes+into+mouse+lung.">Direct tracheal instillation of solutes into mouse lung.</a> J Vis Exp. , (2010).</li><li>Osier, M., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+inhalation+vs+intratracheal+instillation:+differences+in+particle+effects.">Intratracheal inhalation vs intratracheal instillation: differences in particle effects.</a> Fundam Appl Toxicol. 40, 220-227 (1997).</li><li>Driscoll, K. E., 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=Intratracheal+instillation+as+an+exposure+technique+for+the+evaluation+of+respiratory+tract+toxicity:+Uses+and+Limitations.">Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: Uses and Limitations.</a> Toxicol Sci. 55, 24-35 (2000).</li><li>MacDonald, K. D., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+simple+method+of+mouse+lung+intubation.">An improved simple method of mouse lung intubation.</a> J Appl Physiol (1985). 106, 984-987 (1985).</li><li>Spoelstra, E. 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The authors have nothing to disclose.

In Fällen, in denen Aerosolisierung aufgrund der begrenzten Verfügbarkeit Reagenz unpraktisch ist, Sicherheit oder Kosten, ist direkte tracheale Verabreichung eine überlegene Methode zur Abgabe von exogenen Mitteln in die Lungen 16 Transtracheale Instillation diese weit verbreitet zu bewerkstelligen ist. Jedoch, wie bei allen chirurgischen Eingriff, es führt auch damit das Potential für durch das Verfahren verursachten Komplikationen selbst und nicht notwendigerweise das Mittel eingeflößt wurde. 1...

Trotz einiger anatomischen und physiologischen Unterschiede, ein Mausmodell weiterhin für die Modellierung menschlichen Biologie und Pathogenese der Erkrankung. 2 von einer Haltung Standpunkt, Mäuse sind leicht zu handhaben, haben eine geringe Brutzeit, eine beschleunigte Lebensdauer und sind relativ preiswert von unschätzbarem Wert sein unterzubringen. Mit der Entwicklung der verschiedenen genetischen Stämme und Strategien (z. B. bedingte Knock-outs, Reporter Mäusen Abstammung-Tracing-Ansätze, etc.), sowie die große Auswahl an erhältlichen Reagenzien (z. B. Antikörper, rekombinante Proteine, Inhibitoren, etc.), Mäuse sind zu einem wesentlichen Modell wirbel~~POS=TRUNC-Organismus aufzudecken menschliche Homöostase und Krankheitsprozesse geworden. 3 Mäuse haben besonders wertvoll gewesen Lungenstudienbedingungen, einschließlich akutem Lungenversagen (ALI) und Lungenfibrose. 4 ALI beim Menschen können durch ein Trauma, Verletzungen hervorgerufen werden, oder Sepsis und wird von epithelialen gekennzeichnet undendothelialen Leck (dh., Ödem), Entzündung und werdende Fibrose. Bei vielen Patienten schreitet ALI seiner schweren Form, akutem Atemnotsyndrom (ARDS), die oft in Fibrose und Tod durch Atemstillstand führt. 5,6 Lungenfibrose eine progressive, fatale Pathologie ist durch das überschüssige Ablagerung von extrazellulärer Matrix gekennzeichnet geben, vor allem I-Kollagen, was zu einer Beeinträchtigung der Lungenfunktion. 7,8 Verabreichung von Bleomycin (BLM) ist die am weitesten verbreitete und am besten charakterisierte Modell zur Induktion ALI und Fibrose bei Versuchstieren. 9 Obwohl Lungenfibrose bei Nagetieren-BLM induziert tut rekapitulieren nicht vollständig die menschlichen fibrotische Phänotypen, 10 Maus-Studien mit diesem Modell zur Entdeckung von vielen wichtigen Faktoren geführt haben das Auftreten und Fortschreiten der Erkrankung zu beeinflussen. 11 Während der genaue Mechanismus (n) hinter BLM-induzierte Fibrogenese unbekannt sind, das auslösende Verletzungvermutlich aus Kontakt-abhängigen DNA-Strangbrüche in den Epithelzellen entstehen die leitenden Atemwege und Lungenbläschen auskleiden, und insbesondere, Typ-1-Pneumozyten. 12 Die Notwendigkeit für den direkten Kontakt zwischen BLM und die Lungen Epithel unterstreicht die Bedeutung eines robusten Lieferroute und diese Bedenken sind auch für eine breite Palette an Anwendungen auf die Atemwege distal ausgerichtet, einschließlich rekombinanter Proteine, Antikörper, siRNA, Viren, Bakterien, Teilchen und mehr relevant. Oropharyngealen Aspiration (OPA) wurde zu diesem Zweck 13, aber ein Haupt Ein Nachteil OPA ist, dass ein Teil der Mittel kann geliefert werden Verschlucken in den Magen-Darm-Trakt, was zu Ungenauigkeiten in der verabreichten Dosis eingesetzt. Eine andere verbreitete Ansatz ist transtracheal Einträufeln, die die Luftröhre und die Applikation von einem Agenten aufzudecken direkt in die Atemwege Tracheotomie unter starker Betäubung beinhaltet. 14 Aber nicht nur kann soeine Prozedur aufgrund seiner Invasivität unerwünscht sein, aber es ist auch zeitaufwendig und erfordert ein gutes Stück der Ausbildung, und verursacht eine starke Schädigung der Atemwege. 15,16 verschiedene Protokolle, die die direkte Verabreichung von Wirkstoffen in die entwickelt wurden, umfassen Luftröhre ohne die Notwendigkeit für einen chirurgischen Eingriff, 16,17,18,19,20 aber diese Verfahren erweitert Erholungszeiten von mächtigen Anästhetika verursacht beinhalten, die Verwendung von teuren Geräten (dh., Otoskop / Laryngoskop, im Handel erhältliche Verfahren Platten, Glasfaser Drähte, etc.), ein Überschuß der Manipulation in der Mundhöhle, und der Unsicherheit in Bezug auf die Dosierung. Dieses Papier beschreibt eine relativ einfache Methode zur Verabreichung über Intubation, die ein Forscher, preiswert, schnell ermöglicht und zuverlässig ein Reagens in die Mäuselunge mit begrenztem Risiko von Rest Schäden an den umgebenden Gewebe vermitteln.

<table><tbody><tr><td>Bleomycin For Injection, 30 units/vial</td><td>APP Pharmaceuticals, LLC</td><td>103720</td><td>For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots</td></tr><tr><td>Blunt End Forceps</td><td></td><td></td><td></td></tr><tr><td>Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4&quot;)&amp;nbsp;</td><td>Covidien</td><td>E1551G</td><td>Before use, create a 45 degree bend 1.5 cm&amp;nbsp; from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.&amp;nbsp;</td></tr><tr><td>Exel Safelet Catheter 22G X 1&quot;</td><td>Exel International</td><td>26746</td><td></td></tr><tr><td>1 ml Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)</td><td>BD</td><td>309659</td><td></td></tr><tr><td>0.2 ml Pipettor and Filter Tips</td><td></td><td></td><td></td></tr><tr><td>Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator</td><td>Dolan Jenner Industries, Inc.</td><td>181-1</td><td>Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.</td></tr><tr><td>Intubation Board</td><td></td><td></td><td>See Diagram 1.</td></tr><tr><td>Colored Label Tape: 0.5&amp;nbsp;in. Wide</td><td>Fisherbrand</td><td>15-901-15A</td><td></td></tr><tr><td>Oxygen&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Phosphate-Buffered Saline, 1X</td><td>Corning</td><td>21-040-CV</td><td>Product should be sterile</td></tr><tr><td>Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0</td><td>Harvard Apparatus</td><td>517698</td><td></td></tr><tr><td>Table Top Anesthesia Machine Isoflurane</td><td>Highland Medical Equipment</td><td></td><td>http://www.highlandmedical.net/</td></tr><tr><td>Slide Top Induction Mouse Isoflurane Chamber</td><td>MIP / Anesthesia Technologies</td><td>AS-01-0530-SM</td><td></td></tr><tr><td>FORANE (isoflurane, USP) Liquid For Inhalation 100 mL&amp;nbsp;</td><td>Baxter</td><td>1001936040</td><td></td></tr><tr><td>&amp;nbsp;Nanozoomer Digital Pathology system</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>IgM ELISA Quantification Kit&amp;nbsp;</td><td>Bethyl Laboratories</td><td>E90-101</td><td></td></tr></tbody></table>

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.

Intubierten Mäusen wurden täglich auf Gewichtsverlust und Not überwacht und getötet, 4, 10 oder 17 Tage später durch eine intraperitoneale Injektion von 2,5% 2,2,2-Tribromethanol. Bronchoalveoläre Lavage (BAL) wurde in drei Waschungen mit PBS gesammelt, wie an anderer Stelle beschrieben 21 und die rechte Lunge wurde in 10% Formalin, Paraffin eingebettet und gefärbt mit Masson Trichrom der von der University of Washington Histology and Imaging Kern...

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An Improved Method for Rapid Intubation of the Trachea in Mice

Ein verbessertes Verfahren zur schnellen Intubation der Luftröhre in Mice

Dieser Artikel stellt eine schnelle und einfache Methode zur Bleomycin direkt in die Luftröhre Maus über Intubation verabreicht wird. Die wichtigsten Vorteile dieser Methode sind, dass sie in hohem Maße reproduzierbar, leicht zu beherrschen ist, und nicht spezialisierte Ausrüstung oder langwierige Wiederherstellungszeiten erfordern.

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

an-improved-method-for-rapid-intubation-of-the-trachea-in-mice

Research

JoVE Journal

Medicine

25.1K Aufrufe.  University of Washington.  Dieser Artikel stellt eine schnelle und einfache Methode zur Bleomycin direkt in die Luftröhre Maus über Intubation verabreicht wird. Die wichtigsten Vorteile dieser Methode sind, dass sie in hohem Maße reproduzierbar, leicht zu beherrschen ist, und nicht spezialisierte Ausrüstung oder langwierige Wiederherstellungszeiten erfordern. 

Serie: An Improved Method for Rapid Intubation of the Trachea in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Pressure Controlled Ventilation zu Acute Lung Injury in Mäusen zu induzieren

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Forschung

Medizin

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Intubation bei Mäusen<em&gt; Über</em&gt; Direkte Laryngoskopie Mit einem Otoskop

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Eine einfache Methode der Maus Lung Intubation

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Nichtinvasive Intratracheale Intubation, die Pathologie und Physiologie der Maus Lung Study

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Eine reversible, nicht-invasive Methode für Airway Resistance-Messungen und bronchoalveolären Lavage Sampling in Mäuse

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biologie

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Eine geschlossen Kasten Modell quer Aortenstenose Verengung bei Mäusen zu induzieren

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Ein Mausmodell der orotrachealen Intubation und der beatmeten Lungenischämie-Reperfusionschirurgie

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Immunologie und Infektion

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Maus Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Neurowissenschaften

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.

Wiederholte orotracheale Intubation bei Mäusen

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

<meta charset="utf8"></head><body>Tracheotomie-basiertes endotracheales Intubationsprotokoll für Rattenmodelle, die nicht überleben

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that require controlled or high ventilation pressures or access to the thoracic cavity and organs. Conventional endoorotracheal intubation using the anatomically existing route through the mouth is well suited for survival experiments. However, this procedure poses some challenges, including generally higher levels of the required experience and technical skill, more advanced equipment, and greater time effort with relevant intubation failure rates and complications such as tracheal perforation, temporary systemic hypooxygenation, and relevant aerial leakage.
This manuscript, therefore, presents a detailed step-by-step protocol for endotracheal intubation through tracheotomy in non-survival rat models when guaranteed intubation success, constant oxygenation levels, high ventilation pressures, or open thoracotomy are required.
The protocol emphasizes the importance of meticulous surgical technique to ensure consistent and reliable outcomes, especially for researchers who are inexperienced or lack routine in the technique of endoorotracheal intubation via direct laryngoscopy. This procedure is, therefore, expected to minimize animal suffering and unnecessary animal losses.

<meta charset="utf8"></head><body>Endotracheale Intubation mittels Tracheotomie und anschließender Thorakotomie bei Ratten für Anwendungen ohne Überleben

Here we introduce a standardized procedure for endotracheal intubation via tracheotomy followed by thoracotomy in rats, aimed at enhancing the precision and reproducibility of non-survival applications requiring invasive ventilation and exposure of thoracic organs in in vivo rat models.

Endotracheale Intubation mittels Tracheotomie und anschließender Thorakotomie bei Ratten für Anwendungen ohne Überleben

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that ...