Name: intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation
Uploaded: 2016-03-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

Alle Experimente wurden in Übereinstimmung mit den Richtlinien der Institutional Animal Care und Use Committee (IACUC) von Baylor Research Institute durchgeführt. 1. Anzucht nicht typisierbaren Haemophilus influenzae (NTHi) und Vorbereiten des Inokulums <ol><li> Platte NTHi auf einem Schokolade - Agar - Platte und halten die Platte den Kopf in einem befeuchteten CO 2 Inkubator über Nacht bei 37 ° C und 5% CO 2. Am folgenden Tag Kultur der Bakterien in Gehirn-Herz-Infusionsbrühe. Dann fügen Sie 1 ml der Brühe in eine sterile 1,5 ml-Tube. </li><li> Zentrifuge bei 4.000 xg für 5 min bei Raumtemperatur. Dann wird der Überstand verworfen und erneut zu suspendieren, das Pellet in 1 ml sterile 1x PBS. Wiederholen Sie einmal diesen Schritt. </li><li> Transfer von 100 &amp; mgr; l der resuspendierten Lösung in 900 ul 1x PBS und Messen der Extinktion der verdünnten Kultur bei 600 nm Wellenlänge in einem Spektrophotometer. </li><li> Bestimmen Sie die Lebensfähigkeit und die Kolonie bildende Einheits (CFUs) des Inokulums durch Züchten 01.10 Reihenverdünnungen auf Schokolade - Agarplatten und über Nacht bei 37 ° C und 5% CO 2 inkubiert. </li><li> Bezogen auf die Lebensfähigkeit und die CFUs aus dem vorherigen Schritt bestimmt wird , das Inokulum auf eine Endkonzentration von 20 x 10 6 CFU / ml einzustellen. </li></ol> 2. intratracheale (it) Einträufeln <ol><li> Injizieren Sie die Maus intraperitoneal (ip) mit 0,1 ml / 10 g Körpergewicht der Maus mit einer 150 mg / ml Ketamin + 20 mg / ml Xylazin Lösung. Das Verfahren sollte mit einer Lichtquelle in der Haube getan werden, um das Tier warm zu halten. </li><li> Drücken Sie den Zehenzwischenraum der Maus, um zu überprüfen, dass die Maus ausreichend betäubt. </li><li> Platzieren Sie die Maus auf dem Rücken. Shave den ventralen Bereich des Halses und desinfizieren mit Chlorhexidin und 70% Ethylalkohol. </li><li> Heben Sie die Haut des oberen ventralen Teil des Halses unter Verwendung von Rattenzahnzange. Machen Sie einen kleinen Schnitt (0,5-1,0 cm) über dem Thymus mit einem Skalpellund sorgfältig den Muskel sezieren, um die Luftröhre aus. <ol><li> Unter Verwendung einer 1-ml-Spritze mit einer 27 G-Nadel, die Injektion sollte langsam 0,05 ml Luft, gefolgt von 0,05 ml NTHi erhalten von Abschnitt 1 oder Salzlösung als Kontrolle, gefolgt von einem weiteren 0,05 ml Luft. Halten Sie Tier vertikal für ca. 1 min. </li></ol></li><li> Schließen Sie den Schnitt mit chirurgischen Klebstoff. Halten Sie die Maus seitlich liegend und warm für ca. 10 min. </li></ol> 3. BALF Sammlung <ol><li> Opfere die Maus durch Genickbruch 24 Stunden nach der Infektion. </li><li> Öffnen Sie die Bauchhöhle der Maus mit anatomischen Schere und schneiden Sie die Bauchaorta zu bluten. Setzen Sie das ventrale Teil der Lunge. </li><li> Setzen Sie die Luftröhre und intubieren es mit einem 20-Gauge-Katheter. Instill 0,8 ml BALF Puffer (1x PBS, 0,1% Dextrose, 10 U / ml Heparin) und die Spülung durch leichtes Ansaugen sammeln. Wiederholen Sie diesen Vorgang dreimal. </li><li> Zählen der Gesamtzahl der Zellen in 100 &amp; mgr; l BALF Suspension unter Verwendung eines hemocytoMeter unter 10-facher Vergrößerung mit Trypan-Blau-Färbung. </li><li> Bereiten Sie Folien für modifizierte Giemsa. Aliquot 100 ul BALF in die entsprechenden Vertiefungen der Cytospin und Zentrifuge bei 500 xg für 5 min. Trocknen der Objektträger für 10 min und färben die Zellen modifiziert Giemsa-Färbung unter Verwendung von nach Protokollen des Herstellers. </li><li> Verwenden , um die verbleibenden Zellen für eine nachfolgende Analyse, wie beispielsweise Fluoreszenz - aktivierte Zellsortierung (FACS), die konfokale Mikroskopie, die Genexpression und die Western - Blot - 11-13. </li></ol> 4. Histopathologie Vorbereitung <ol><li> Für histopathologische Analyse, infizieren Mäuse mit NTHi wie in Schritt 2.4 beschrieben. Nach 24 Stunden der Infektion opfern, um die Tiere durch Genickbruch. Schneiden Sie die Brusthöhle mit anatomischen Schere öffnen die Lunge und die Luftröhre freizulegen, wie bereits erwähnt. Dann legen Sie die 20-Gauge-Katheter in die Trachea. </li><li> Füllen Sie die Lunge mit 2% warme Agarose-Lösung in 4% Formalin präpariert. Sobald derLungen sind voll aufgeblasen, statt Eis auf der Oberseite der Lunge die Agarose-Lösung zu verfestigen. </li><li> Dann entfernen Sie vorsichtig die Brust Stecker und steckte es in eine 50 ml-Lösung von 4% Formalin in 1x PBS, bis sie für Hämatoxylin und Eosin (H &amp; E) Abschnitte verarbeitet wird. </li></ol> 5. FACS Zellen in der BALF Anfärben <ol><li> Stellen 1 x 10 5 Zellen von der BALF Fluid in einer 96-Well - V-Boden Mikrotiterplatte. </li><li> Wasche die Zellen mit 300 ul kaltem PBS 1x und Schleudern bei 4.000 × g für 3 min bei 4 ° C. </li><li> Stain die Zellen mit Zellfarblösung in 100 ul 1x PBS (1: 1000 Verdünnung) und halten bei Raumtemperatur für 15 min. </li><li> Wasche die Zellen mit 300 ul kaltem PBS 1x bei 4.000 × g für 3 min bei 4 ° C. </li><li> Halten Zellen in 100 &amp; mgr; l FACS-Waschpuffer [Waschpuffer (1x PBS, 2% FBS, 1 mM EDTA, 0,1% Natriumazid], das 1: 100 verdünnt Fc-Block-für 15 min bei 4 ° C. </li><li> Centrifuge Zellen bei 4.000 × g für 3 min und entsorgen supernatant. </li><li> Stain Zellen mit 100 &amp; mgr; l Oberflächenfärbung Cocktail (1: 100 Verdünnung) in FACS-Waschpuffer für 30 min bei 4 ° C. </li><li> Waschen Zellen dreimal mit 300 &amp; mgr; l FACS-Waschpuffer bei 4.000 × g für 3 min bei 4 ° C. </li><li> Fix-Zellen in 100 &amp; mgr; l von 4% Paraformaldehyd für 15 min bei Raumtemperatur. </li><li> Wasche die Zellen zweimal mit 300 ul 1x PBS bei 400 × g für 3 min bei 4 ° C und resuspendieren in 300 &amp; mgr; l FACS-Waschpuffer. </li><li> Machen Sie einfarbige Färbung Kontrollen unter Verwendung von Durchflusszytometrie Perlen. <ol><li> Geben Sie einen Tropfen Perlen in den 96-Well-V-Boden-Mikrotiterplatte und waschen mit 200 ul 1x PBS. </li><li> 1 &amp; mgr; l-Färbung Antikörper auf 100 ul 1x PBS und Perlen und Inkubation für 5 min bei Raumtemperatur. </li><li> Waschen Sie Perlen zweimal mit 300 ul 1x PBS. </li></ol></li><li> Analysieren Sie einzelne gebeiztem Kontrollen und Zellen mittels Durchflusszytometrie 14. </li></ol> 6. Homogenisieren von Lungengewebe RNA zu isolieren <ol><li> Nach der Infektion über Nacht ein kleines Stück Lunge (20-40 mg) und halten Sie sie in RNA-stabilisierende Reagenz bei 4 ° C zu sammeln. Dann speichern Sie es bei -80 ° C bis zum nächsten Schritt. </li><li> Waschen Sie die Lungengewebe in kaltem 1x PBS loswerden der RNA-Stabilisierung Reagenz zu erhalten. </li><li> Platzieren das Gewebe in einem 1,5 ml Zentrifugenröhrchen, das Lyse-Puffer (1 ml Lyse-Puffer + 10 &amp; mgr; l β-Mercaptoethanol). </li><li> Hacken Sie das Gewebe in kleine Stücke spitzen Schere und homogenisieren für 20-40 sec ein schnurloses Motor Pellet zerstoßen. </li><li> Halten Sie das Lysat auf Eis für 5 min und fahren Sie mit Schritt 7.1. </li></ol> 7. RNA-Isolierung aus der Lunge <ol><li> Zentrifugieren Sie das Lysat bei 18.000 × g für 3 min. Entfernen Sie den Überstand durch Pipettieren und vorsichtig Transfer in ein neues 1,5-ml-Röhrchen. </li><li> 1 Volumen von 70% Ethanol zu dem Lysat. Mischen Sie sofort durch Pipettieren und warten 2-4 min. </li><li> Übertragen Sie bis zu 700 &amp; mgr; l Lysat auf eine Spin-Säule platziertin einem 2 ml Sammelröhrchen. </li><li> Zentrifuge bei 9.600 × g für 15 Sekunden und den Durchlauf verwerfen. </li><li> In 350 ul strengen Waschpuffer und Zentrifuge bei 9.600 × g für 15 Sekunden. Verwerfen Sie den Durchfluss durch. </li><li> In 80 ul DNase I direkt auf die Spinsäule Membran und halten Sie sie für 15 Minuten bei Raumtemperatur. </li><li> In 350 ul strengen Waschpuffer auf die Spin-Säule und Zentrifuge bei 9.600 × g für 15 Sekunden. Verwerfen Sie den Durchfluss durch. </li><li> In 500 ul RNA Waschpuffer und Zentrifuge bei 9.600 × g für 15 Sekunden. Verwerfen Sie den Durchfluss durch. </li><li> In 500 ul RNA Waschpuffer und Schleudern bei 9.600 × g für 2 min. Verwerfen Sie den Durchfluss durch. </li><li> Legen Sie die Spin-Säule in ein neues 2 ml Sammelröhrchen und Zentrifuge bei 9.600 × g für 1 min. Verwerfen Sie den Durchfluss durch. </li><li> Hinzufügen 30-50 &amp; mgr; l RNase-freiem Wasser direkt auf die spin-Säule zentrifugieren bei 9.600 × g für 1 min. Store RNA bei -80 ° C. </li><li> Führen RNA-Seq - Analyse 14. </li></ol>

<ol>
	<li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+physiological+roles+of+inflammation.">Origin and physiological roles of inflammation.</a> Nature. 454, 428-435 (2008).</li><li>Grivennikov, S. I., Greten, F. R., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity,+inflammation,+and+cancer.">Immunity, inflammation, and cancer.</a> Cell. 140, 883-899 (2010).</li><li>Barnes, 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=Immunology+of+asthma+and+chronic+obstructive+pulmonary+disease.">Immunology of asthma and chronic obstructive pulmonary disease.</a> Nat Rev Immunol. 8, 183-192 (2008).</li><li>Sethi, S., Murphy, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+in+the+pathogenesis+and+course+of+chronic+obstructive+pulmonary+disease.">Infection in the pathogenesis and course of chronic obstructive pulmonary disease.</a> N Engl J Med. 359, 2355-2365 (2008).</li><li>King, P. T., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lung+Immune+Response+to+Nontypeable+Haemophilus+influenzae+(Lung+Immunity+to+NTHi).">The Lung Immune Response to Nontypeable Haemophilus influenzae (Lung Immunity to NTHi).</a> J Immunol Res. 2015, 706376 (2015).</li><li>Kapur, N., Grimwood, K., Masters, I. B., Morris, P. S., Chang, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+airway+microbiology+and+cellularity+in+children+with+newly+diagnosed+non-CF+bronchiectasis.">Lower airway microbiology and cellularity in children with newly diagnosed non-CF bronchiectasis.</a> Pediatr Pulmonol. 47, 300-307 (2012).</li><li>King, P. T., Holdsworth, S. R., Freezer, N. J., Villanueva, E., Holmes, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiologic+follow-up+study+in+adult+bronchiectasis.">Microbiologic follow-up study in adult bronchiectasis.</a> Respir Med. 101, 1633-1638 (2007).</li><li>Shuto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+NF-kappa+B+by+nontypeable+Hemophilus+influenzae+is+mediated+by+toll-like+receptor+2-TAK1-dependent+NIK-IKK+alpha+/beta-I+kappa+B+alpha+and+MKK3/6-p38+MAP+kinase+signaling+pathways+in+epithelial+cells.">Activation of NF-kappa B by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha /beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells.</a> Proc Natl Acad Sci U S A. 98, 8774-8779 (2001).</li><li>Moghaddam, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemophilus+influenzae+lysate+induces+aspects+of+the+chronic+obstructive+pulmonary+disease+phenotype.">Haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype.</a> Am J Respir Cell Mol Biol. 38, 629-638 (2008).</li><li>Rajagopalan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+exposure+to+bacterial+superantigens+induces+airway+inflammation+in+HLA+class+II+transgenic+mice.">Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice.</a> Infect Immun. 74, 1284-1296 (2006).</li><li>Ganesan, 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=Elastase/LPS-exposed+mice+exhibit+impaired+innate+immune+responses+to+bacterial+challenge:+role+of+scavenger+receptor+A.">Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge: role of scavenger receptor A.</a> Am J Pathol. 180, 61-72 (2012).</li><li>Hogner, 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=Macrophage-expressed+IFN-beta+contributes+to+apoptotic+alveolar+epithelial+cell+injury+in+severe+influenza+virus+pneumonia.">Macrophage-expressed IFN-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.</a> PLoS Pathog. 9, e1003188 (2013).</li><li>Karisola, P., 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=Invariant+Natural+Killer+T+Cells+Play+a+Role+in+Chemotaxis,+Complement+Activation+and+Mucus+Production+in+a+Mouse+Model+of+Airway+Hyperreactivity+and+Inflammation.">Invariant Natural Killer T Cells Play a Role in Chemotaxis, Complement Activation and Mucus Production in a Mouse Model of Airway Hyperreactivity and Inflammation.</a> PloS one. 10, e0129446 (2015).</li><li>Theivanthiran, 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=The+E3+ubiquitin+ligase+Itch+inhibits+p38alpha+signaling+and+skin+inflammation+through+the+ubiquitylation+of+Tab1.">The E3 ubiquitin ligase Itch inhibits p38alpha signaling and skin inflammation through the ubiquitylation of Tab1.</a> Sci Signal. 8, 22 (2015).</li><li>Venuprasad, K., Zeng, M., Baughan, S. L., Massoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+role+of+the+ubiquitin+ligase+Itch+in+immune+regulation.">Multifaceted role of the ubiquitin ligase Itch in immune regulation.</a> Immunol Cell Biol. , (2015).</li><li>Decramer, M., Janssens, W., Miravitlles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+obstructive+pulmonary+disease.">Chronic obstructive pulmonary disease.</a> Lancet. 379, 1341-1351 (2012).</li></ol>

The authors have nothing to disclose.

Hier beschreiben wir eine einzigartige und minimal-invasive Verfahren, um die Lungen von Mäusen, die mit einer bakteriellen Lungen Pathogen zu beimpfen. Wir zeigen, dass dieses Verfahren verwendet werden kann, um die Funktion der verschiedenen Gene zu untersuchen, die in Mäusen unter Verwendung von Genen von Entzündungssignalwege defizient sind. Dieses Verfahren kann auch die Entzündungsreaktionen gegen virale und fungale Infektionen der Lunge zu untersuchen verwendet werden. Die Vorteile dieses Verfahrens gegenübe...

Die Entzündung ist eine grundlegende Immunabwehrmechanismus gegen infektiöse Erreger. Es fördert die Erreger Beseitigung und Reparatur von beschädigtem Gewebe. Es erleichtert auch die Erholung zu einem normalen , gesunden Zustand 1. Jedoch führt dysregulated Entzündung oft zu chronischen entzündlichen Erkrankungen 2. Entzündung der Atemwege ist ein erster Auslöser für verschiedene Lungenerkrankungen wie chronische obstruktive Lungenerkrankung (COPD), Asthma und Lungenfibrose 3. Die nicht-typisierbaren (nicht eingekapselten) Haemophilus influenzae (NTHi) mit chronischen oberen und unteren Lungenentzündungserkrankungen 4,5 verbunden. Es ist die dominante Spezies aus den unteren Atemwege von Kindern und Erwachsenen mit chronisch - obstruktiver Lungenerkrankung 4,6,7 isoliert. Die Entzündungsantwort nach der NTHi-Infektion wird durch die Hochregulation von proinflammatorischen Zytokinen (wie TNF und IL-1β) gekennzeichnet, und es wird vermittelt by Mitogen - aktivierte Proteinkinase (MAPK) und nuclear factor-kappaB (NF-kappaB) über Toll-like Rezeptoren (TLR) 8. Mausmodelle sind sehr nützliche Werkzeuge zur Analyse der zugrundeliegenden Pathologie der Lungenentzündungskrankheit aufgrund der Verfügbarkeit der verschiedenen Gen-defizienten Linien. Live / attenuierten Bakterien und bakterielle Produkte zu inokulieren, einschließlich intranasale Instillation und aerosolized Inhalations 9,10 mehrere Verfahren verwendet worden. Hier zeigen wir, intratracheale Instillation. Obwohl weniger häufig verwendet wird, ist dieser Ansatz effizienter und hoch reproduzierbare wegen der direkten Abgabe des Inokulums auf den unteren Atemwege.

<table><tbody><tr><td>Chocolate agar plate</td><td>Fisher Scientific</td><td>CAS50-99-7</td><td></td></tr><tr><td>Dextrose Anhydrous</td><td>Themo Scientific</td><td>R01300</td><td></td></tr><tr><td>Heparin</td><td>Hospira,Inc</td><td>RL-3010</td><td></td></tr><tr><td>Deft quick solution</td><td>Sigma</td><td>GS500-500ML</td><td></td></tr><tr><td>Syringe needle 20/26G&amp;nbsp;</td><td>BD</td><td>(REF305115/175)</td><td></td></tr><tr><td>Iml syringe</td><td>BD</td><td>REF 309602</td><td></td></tr><tr><td>Catheter 20GA</td><td>BD</td><td>REF 381433</td><td></td></tr><tr><td>Dissecting Scissors, straight, 10 cm long</td><td>kentscientific</td><td>INS600393</td><td></td></tr><tr><td>Iris Forceps, serrated, 10cm long</td><td>kentscientific</td><td>INS650915</td><td></td></tr><tr><td>Tweezer #5 Stainless steel, 11cm long</td><td>kentscientific</td><td>INS600095</td><td></td></tr><tr><td>10% Formalin</td><td>Fisher Scientific</td><td>CAS 67-56-1</td><td></td></tr><tr><td>Agarose</td><td>peqlab</td><td>35-1020</td><td></td></tr><tr><td>5ml polystyrene round-bottom tubes</td><td>BD</td><td>REF 352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Light Labs</td><td>A-7001-R</td><td></td></tr><tr><td>Reasy Mini&amp;nbsp; kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Pellet pestile motor (Tissue homoginizer)</td><td>Sigma</td><td>Z359971-1EA</td><td></td></tr><tr><td>96 well microtiter plates V bottom</td><td>Thermo</td><td>2605</td><td></td></tr><tr><td>1X PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>CD45.2-APC</td><td>eBioscience</td><td>17-0454-81</td><td>Working dilution 1:100</td></tr><tr><td>Ly-6G-eFlor 450</td><td>eBioscience</td><td>48-5931-82</td><td>Working dilution 1:100</td></tr><tr><td>BSA</td><td>HyClone</td><td>SH30574.03</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>Live/Dead fixable aqua dead cell stain kit</td><td>Invitrogen</td><td>L-34957</td><td></td></tr><tr><td>EDTA (0.5M)</td><td>lifetechnologies</td><td>15575-020</td><td></td></tr><tr><td>CD16/CD32 FcBlock</td><td>BD</td><td>553142</td><td></td></tr><tr><td>Facs tubes polystyrene round bottom tube</td><td>BD</td><td>352052</td><td></td></tr><tr><td>Formaldehyde</td><td>Polyscience</td><td>4018</td><td></td></tr></tbody></table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

Hier beschreiben wir ein detailliertes Verfahren , um effizient und direkt Haemophilus influenzae liefern in die unteren Atemwege von Mäusen. Wir zeigen das Verfahren zur Herstellung von H. influenzae Inokulum, intratracheale Instillation von H. influenzae in die Lunge, Sammlung von broncho-alveolären Lavage (BALF), Analyse von Immunzellen in der BALF und RNA - Isolation für Differentialgenexpressionsanalyse. Dieses Verfahren kann verwendet werden, um die Lungenentzündungsreaktion auf alle Bakterien, Viren oder Pilze zu untersuchen. Direkt tracheale Instillation wird meist über intranasale oder Aerosolinhalationsverfahren bevorzugt, da es effizienter das bakterielle Inokulum in die unteren Atemwege mit geringer Mehrdeutigkeit liefert.

Intratracheale Instillation führte zu einer deutlich erhöhten Anzahl von Leukozyten in der BALF (1A, linkes Feld) als Anlage mit Kochsalzlösung. Die Differentialzählung Analyse der Leukozyten zeigte deutlich erhöhte neutrophile Infiltration (Abbildung 1, rechts). Die FACS - Analyse der Zellen in der BALF bestätigt weiter die erhöhte Anzahl von Neutrophilen (1B). Histologische Analyse von H &amp; E-gefärbten Schnitten des Lungenge...

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intratracheale Verabreichung von<em&gt; Haemophilus influenzae</em&gt; In Mausmodelle zur Untersuchung Atemwegsentzündung

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

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Immunology and Infection

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

10.1K Aufrufe.  Baylor Research Institute. We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models. 

Serie: Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Nicht-chirurgische Intratrachealbiopersistenztest von Mäusen mit Analyse der Lungen-und Lungenforschung Lymphknoten mittels Durchflusszytometrie

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Forschung

Immunologie und Infektion

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 ...

Medizin

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

Die Nutzung der Oropharyngeale Intratracheale PAMP Verwaltung und bronchoalveoläre Lavage, um die Immunantwort in Mäusen Bewerten

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Die intranasale Verabreichung von rekombinantem Influenza Vaccines in chimären Maus-Modelle zu Mucosale Immunität Studieren

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studieren Mikrobielle Gemeinschaften<em&gt; In-vivo-</em&gt;: Ein Modell von Host-vermittelte Interaktion zwischen<em&gt; Candida albicans</em&gt; Und<em&gt; Pseudomonas aeruginosa</em&gt; In den Atemwegen

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Intubation vermittelte Intratracheale (IMIT) Instillation: eine nichtinvasive, lungenspezifischen Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Ein Nicht-invasive und in technischer Hinsicht nicht intensive Verfahren zur Induktion und Phänotypisierung für experimentelle bakterielle Pneumonie bei Mäusen

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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.

Ein verbessertes Verfahren zur schnellen Intubation der Luftröhre in Mice

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

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Bildgebung Zelle Interaktion in Trachealen Schleimhaut während der Influenza-Virus-Infektion mit Intravitalen zwei-Photonen-Mikroskopie

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Bewertung der Wirt-Pathogen-Antworten und Wirksamkeit des Impfstoffes bei Mäusen

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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