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>

<ol>
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L., 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=The+mouse+as+a+model+for+human+biology:+a+resource+guide+for+complex+trait+analysis.">The mouse as a model for human biology: a resource guide for complex trait analysis.</a> Nat Rev Genet. 8, 58-69 (2007).</li><li>Baron, R. M., Choi, A. J., Owen, C. A., Choi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+manipulated+mouse+models+of+lung+disease:+potential+and+pitfalls.">Genetically manipulated mouse models of lung disease: potential and pitfalls.</a> Am J Physiol Lung Cell Mol Physiol. 302, 485-497 (2012).</li><li>Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome.">Acute respiratory distress syndrome.</a> BMJ Clin Evid. 2010, (2010).</li><li>Saguil, A., Fargo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome:+diagnosis+and+management.">Acute respiratory distress syndrome: diagnosis and management.</a> Am Fam Physician. 85, 352-358 (2012).</li><li>Wilson, M. S., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+fibrosis:+pathogenesis,+etiology+and+regulation.">Pulmonary fibrosis: pathogenesis, etiology and regulation.</a> Mucosal Immunol. 2, 103-121 (2009).</li><li>Wuyts, W. 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=The+pathogenesis+of+pulmonary+fibrosis:+a+moving+target.">The pathogenesis of pulmonary fibrosis: a moving target.</a> Eur Respir J. 41, 1207-1218 (2013).</li><li>Mouratis, M. A., Aidinis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+pulmonary+fibrosis+with+bleomycin.">Modeling pulmonary fibrosis with bleomycin.</a> Curr Opin Pulm Med. 17, 355-361 (2011).</li><li>Moeller, A., Ask, K., Warburton, D., Gauldie, J., Kolb, 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+bleomycin+animal+model:+a+useful+tool+to+investigate+treatment+options+for+idiopathic+pulmonary+fibrosis?.">The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?.</a> Int J Biochem Cell Biol. 40, 362-382 (2008).</li><li>Myll&#228;rniemi, M., Kaarteenaho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+treatment+of+idiopathic+pulmonary+fibrosis+-+preclinical+and+clinical+studies+of+pirfenidone,+nintedanib,+and+N-acetylcysteine.">Pharmacological treatment of idiopathic pulmonary fibrosis - preclinical and clinical studies of pirfenidone, nintedanib, and N-acetylcysteine.</a> European Clinical Respiratory Journal. 2, (2015).</li><li>Reinert, T., Baldotto, C. S. d. R., Nunes, F. A. P., Scheliga, A. A. d. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin-Induced+Lung+Injury.">Bleomycin-Induced Lung Injury.</a> Journal of Cancer Research. 2013, 1-9 (2013).</li><li>Lakatos, H. F., 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=Oropharyngeal+aspiration+of+a+silica+suspension+produces+a+superior+model+of+silicosis+in+the+mouse+when+compared+to+intratracheal+instillation.">Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation.</a> Exp Lung Res. 32, 181-199 (2006).</li><li>Helms, M. 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. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+endotracheal+intubation+of+mice.">A novel and simple method for endotracheal intubation of mice.</a> Lab Anim. 41, 128-135 (2007).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> J Vis Exp. , e50601 (2013).</li><li>Starcher, B., WIlliams, I. <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+for+intratracheal+instillation+of+endotoxin+into+the+lungs+of+mice.">A method for intratracheal instillation of endotoxin into the lungs of mice.</a> Lab Anim. 23, 234-240 (1989).</li><li>Daubeuf, F., Frossard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+bronchoalveolar+lavage+in+the+mouse.">Performing bronchoalveolar lavage in the mouse.</a> Curr Protoc Mouse Biol. 2, 167-175 (2012).</li><li>Li, Y., 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=Severe+lung+fibrosis+requires+an+invasive+fibroblast+phenotype+regulated+by+hyaluronan+and+CD44.">Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44.</a> J Exp Med. 208, 1459-1471 (2011).</li><li>Grazioli, 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=CYR61+(CCN1)+overexpression+induces+lung+injury+in+mice.">CYR61 (CCN1) overexpression induces lung injury in mice.</a> Am J Physiol Lung Cell Mol Physiol. 308, L759-L765 (2015).</li><li>Redente, 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 border="0" cellpadding="0" cellspacing="0" width="1528">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Bleomycin For Injection, 30 units/vial</td>
			<td style="width:304px;">APP Pharmaceuticals, LLC</td>
			<td style="width:139px;">103720</td>
			<td style="width:604px;">For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt End Forceps</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4&#34;)&#160;</td>
			<td style="width:304px;">Covidien</td>
			<td>E1551G</td>
			<td>Before use, create a 45 degree bend 1.5 cm&#160; from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exel Safelet Catheter 22G X 1&#34;</td>
			<td style="width:304px;">Exel International</td>
			<td>26746</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)</td>
			<td style="width:304px;">BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2ml Pipettor and Filter Tips</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator</td>
			<td style="width:304px;">Dolan Jenner Industries, Inc.</td>
			<td style="width:139px;">181-1</td>
			<td>Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Intubation Board</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>See Diagram 1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colored Label Tape: 0.5&#160;in. Wide</td>
			<td style="width:304px;">Fisherbrand</td>
			<td>15-901-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen&#160;</td>
			<td style="width:304px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 1X</td>
			<td style="width:304px;">Corning</td>
			<td>21-040-CV</td>
			<td>Product should be sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0</td>
			<td style="width:304px;">Harvard Apparatus</td>
			<td>517698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table Top Anesthesia Machine Isoflurane</td>
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			<td>http://www.highlandmedical.net/</td>
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			<td height="21" style="height:21px;">Slide Top Induction Mouse Isoflurane Chamber</td>
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			<td height="21" style="height:21px;">FORANE (isoflurane, USP) Liquid For Inhalation 100 mL&#160;</td>
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			<td height="21" style="height:21px;">&#160;Nanozoomer Digital Pathology system</td>
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			<td height="21" style="height:21px;">IgM ELISA Quantification Kit&#160;</td>
			<td style="width:304px;">Bethyl Laboratories</td>
			<td style="width:139px;">E90-101</td>
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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...

Watch this Scientific Journal Video about An Improved Method for Rapid Intubation of the Trachea in Mice at JoVE.com

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

25.0K 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

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.

Richtlinien für die Wahlfächer Pediatric Fiberoptische Intubation

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

Forschung

Medizin

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Orthotopen Aortic Transplantation in Mäuse für das Studium der Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

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  ...

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

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

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

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

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

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

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

Bewertung und Charakterisierung von Hyaloidgefäßen bei Mäusen

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Eine überarbeitete Methode zur Induktion von Sekundärlymphödemen im Hinterglied von Mäusen

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...

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 ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Semi-Minimal Invasive Methode zur Induzieren von Myokardinfarkt bei Ratten und der Beurteilung der Herzfunktion durch ein isoliertes funktionierendes Herzsystem

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Eine modifizierte einfache Methode zur Induktion von Myokardinfarkt bei Mäusen

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...