Name: a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice
Uploaded: 2016-09-28T14:00:00
Description: Watch this Scientific Journal Video about A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice at JoVE.com

This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (Z01 ES102005).

Alle forsøg blev udført i overensstemmelse med dyreværnsloven US Public Health Service Policy på Humane Pleje og anvendelse af forsøgsdyr, og efter gennemgang af Animal Care og brug Udvalg NIEHS. 1. Fremstilling af K. pneumoniae Kultur Forsigtig: Udfør alle trin i et bio-niveau 2 (BSL2) hætte eller andet BSL2 udpegede område og kassere affald pr institut BSL2 retningslinjer. <ol><li> For ophængning vækst af K. pneumoniae, tø en glyc...

<ol>
	<li>Mizgerd, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+lower+respiratory+tract+infection.">Acute lower respiratory tract infection.</a> N Engl J Med. 358 (7), 716-727 (2008).</li><li>Waterer, G. W., Rello, J., Wunderink, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+community-acquired+pneumonia+in+adults.">Management of community-acquired pneumonia in adults.</a> Am J Respir Crit Care Med. 183 (2), 157-164 (2011).</li><li>Mizgerd, J. P., Skerrett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+pneumonia.">Animal models of human pneumonia.</a> Am J Physiol Lung Cell Mol Physiol. 294 (3), L387-L398 (2008).</li><li>Revelli, D. A., Boylan, J. A., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+intratracheal+inoculation+method+for+the+study+of+pulmonary+melioidosis.">A non-invasive intratracheal inoculation method for the study of pulmonary melioidosis.</a> Front Cell Infect Microbiol. 2, 164 (2012).</li><li>Morales-Nebreda, 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=Intratracheal+administration+of+influenza+virus+is+superior+to+intranasal+administration+as+a+model+of+acute+lung+injury.">Intratracheal administration of influenza virus is superior to intranasal administration as a model of acute lung injury.</a> J Virol Methods. 209, 116-120 (2014).</li><li>Aujla, 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=IL-22+mediates+mucosal+host+defense+against+Gram-negative+bacterial+pneumonia.">IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia.</a> Nat Med. 14 (3), 275-281 (2008).</li><li>Chen, 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=Th17+cells+mediate+clade-specific,+serotype-independent+mucosal+immunity.">Th17 cells mediate clade-specific, serotype-independent mucosal immunity.</a> Immunity. 35 (6), 997-1009 (2011).</li><li>Draper, D. W., 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=ATP-binding+cassette+transporter+G1+deficiency+dysregulates+host+defense+in+the+lung.">ATP-binding cassette transporter G1 deficiency dysregulates host defense in the lung.</a> Am J Respir Crit Care Med. 182 (3), 404-412 (2010).</li><li>Robinson, K. M., 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=Influenza+A+exacerbates+Staphylococcus+aureus+pneumonia+by+attenuating+IL-1beta+production+in+mice.">Influenza A exacerbates Staphylococcus aureus pneumonia by attenuating IL-1beta production in mice.</a> J Immunol. 191 (10), 5153-5159 (2013).</li><li>Mizgerd, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+neutrophil+recruitment+elicited+by+bacteria+in+the+lungs.">Molecular mechanisms of neutrophil recruitment elicited by bacteria in the lungs.</a> Semin Immunol. 14 (2), 123-132 (2002).</li><li>Balamayooran, G., Batra, S., Fessler, M. B., Happel, K. I., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neutrophil+accumulation+in+the+lungs+against+bacteria.">Mechanisms of neutrophil accumulation in the lungs against bacteria.</a> Am J Respir Cell Mol Biol. 43 (1), 5-16 (2010).</li><li>Quinton, L. 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=Hepatocyte-specific+mutation+of+both+NF-kappaB+RelA+and+STAT3+abrogates+the+acute+phase+response+in+mice.">Hepatocyte-specific mutation of both NF-kappaB RelA and STAT3 abrogates the acute phase response in mice.</a> J Clin Invest. 122 (5), 1758-1763 (2012).</li><li>Gowdy, K. M., 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=Key+role+for+scavenger+receptor+B-I+in+the+integrative+physiology+of+host+defense+during+bacterial+pneumonia.">Key role for scavenger receptor B-I in the integrative physiology of host defense during bacterial pneumonia.</a> Mucosal Immunol. 8 (3), 559-571 (2015).</li><li>Madenspacher, J. H., 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=p53+Integrates+host+defense+and+cell+fate+during+bacterial+pneumonia.">p53 Integrates host defense and cell fate during bacterial pneumonia.</a> J Exp Med. 210 (5), 891-904 (2013).</li><li>Brain, J. D., Knudson, D. E., Sorokin, S. P., Davis, M. 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+distribution+of+particles+given+by+intratracheal+instillation+or+by+aerosol+inhalation.">Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation.</a> Environ Res. 11 (1), 13-33 (1976).</li><li>Card, J. W., 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=Gender+differences+in+murine+airway+responsiveness+and+lipopolysaccharide-induced+inflammation.">Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation.</a> J Immunol. 177 (1), 621-630 (2006).</li><li>Ivanov, I. I., 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=Induction+of+intestinal+Th17+cells+by+segmented+filamentous+bacteria.">Induction of intestinal Th17 cells by segmented filamentous bacteria.</a> Cell. 139 (3), 485-498 (2009).</li><li>Hooper, L. V., Littman, D. R., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+the+microbiota+and+the+immune+system.">Interactions between the microbiota and the immune system.</a> Science. 336 (6086), 1268-1273 (2012).</li></ol>

Murine modeller af bakteriel lungebetændelse, indgået et samarbejde med gen-targeting og in vivo biologiske og farmakologiske interventioner, har givet kritiske indsigt i pulmonal vært forsvar reaktion. Store fremskridt er blevet gjort især i vores forståelse af de kemokiner og adhæsionsmolekyler, der styrer rekruttering af neutrofiler til den inficerede luftrum 10,11. In vivo modeller af lungebetændelse, i modsætning til cellebaserede eller alternative tilgange, også har givet vigt...

Pneumoni fortsat den hyppigste dødsårsag fra infektion i USA, med lidt samlede ændring i dødelighed i løbet af de sidste 40 år på trods af forbedringer i vaccination og antibiotika strategier 1,2. På trods af den manglende mærkbare fremskridt på det offentlige sundhed niveau, i de seneste år er blevet gjort dramatiske fremskridt i vores forståelse af den molekylære og cellulære patogenese lungebetændelse, med mange af disse fremskridt gjort mulig ved brug af musemodeller af lungeinfektion. Den genetiske sporbarhed af musen, har ligheden af ​​murine og humane immunforsvar, og det enorme opbud af murine målrettet immunologiske reagenser, er blevet kommercielt tilgængelige sammen lettet hurtige fremskridt af feltet. Musemodeller for bakteriel lungebetændelse beskrevet i litteraturen har generelt påberåbes en af ​​fire tekniske ruter for patogen podning: i) aerosolisering; ii) intranasal levering; iii) peroral levering; og iv) kirurgisk intratracheal injektion (dvs. tracheotomi) 3. Alle smitteveje har fordele og ulemper 3. Især relative eksponering af den øvre luftvej, potentiale for iblanding af oronasal flora, krav til generel anæstesi, variabilitet af inoculum leveret til den distale lunge, lobar fordeling af de leverede patogener, tekniske krav ekspertise, og proceduremæssige sygelighed varierer meget i disse tilgange. Almindeligt anvendte perorale infektion teknikker omfatter endotracheal (translaryngeal) kanylering via enten en &quot;blind&quot; (ikke-visualiseret) tilgang, eller i direkte larynx visualisering 3-5. Begge metoder, mens robust, kræve betydelige uddannelse og også bære risikoen for traumer til de øvre luftveje. I nærværende rapport beskriver vi en teknisk ikke-intensive, non-invasiv, billig og hurtig metode til peroral infektion, wret bakterier (Klebsiella pneumoniae i eksemplet billede) afpipetteret i oropharynx af en bedøvet mus leveres til lungerne via aspiration (dvs. inhalation). Vi og andre har brugt aspirationspneumoni teknik held 6-9. Denne alsidige og let lært lunge-levering metode kan udvides til levering af mange yderligere ikke-ætsende midler til lungerne, herunder cytokiner og andre proteiner, patogen-associerede molekyler (fx lipopolysaccharid), celler (dvs. adoptiv overførsel), og toksiner (fx bleomycin). Ud over at drøfte vigtige tekniske overvejelser, beskriver vi også en integreret, kvantitativ tilgang til vurdering af efterfølgende vært reaktion på lungebetændelse, herunder nedstrøms måling af bakteriel clearance (dvs. kolonidannende enhed [CFU] kvantificering i lunge- og perifere organer) og leukocyt ophobning i luftrummet.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Klebsiella pneumoniae, serotype 2</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:119px;">43816</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tryptic soy broth</td>
			<td style="width:169px;">Becton Dickenson</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Safelet IV Catheters, 18G x 1 1/4&#34;</td>
			<td>Claflin Medical Equipment</td>
			<td style="width:119px;">MEDC-031122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Solution 1</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Solution 2</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Fixative</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">27&#189; gauge tuberculin syringes</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">14-826-87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lithium heparin plasma collectors</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">2675187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">L-shaped disposable spreaders</td>
			<td style="width:169px;">Lab Scientific</td>
			<td style="width:119px;">DSC</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:243px;">1x PBS, pH 7.4</td>
			<td style="width:169px;">prepared in-house</td>
			<td style="width:119px;">n/a</td>
			<td>Distilled water (5 L), NaCl (40 g), KCl (1 g), Na2HPO4 (5.75 g), KH2PO4 (1 g)&#160;&#160;&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:243px;">ACK lysis buffer</td>
			<td style="width:169px;">prepared in-house</td>
			<td style="width:119px;">n/a</td>
			<td>NH4Cl (4.145 g), KHCO3 (0.5 g), EDTA (18.6 mg), bring up to 500 ml with distilled water and pH to 7.4</td>
		</tr>
	</tbody>
</table>

a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice

Selvom community-erhvervet pneumoni er fortsat et stort problem for folkesundheden, har murine modeller af bakteriel lungebetændelse nylig lettet betydelige prækliniske fremskridt i vores forståelse af den underliggende cellulære og molekylære patogenese. In vivo musemodeller fange integrerede fysiologi og modstandskraft værtens forsvar reaktion i en måde, som ikke afsløret af alternative, forenklede ex vivo metoder. Adskillige fremgangsmåder er blevet beskrevet i litteraturen til intrapulmonal inokulering af bakterier i mus, herunder aerosolisering, intranasal levering, peroral endotrachealt kanyle under &#39;blind&#39; og visualiseret betingelser, og transkutan endotracheal kanyle. Alle metoder har relative fordele og begrænsninger. Heri beskriver vi detaljeret en non-invasiv, teknisk ikke-intensive, billig og hurtig fremgangsmåde til intratrakeal levering af bakterier, der involverer aspiration (dvs. inhalation) af musenaf en infektiøs inokulum pipetteret i oropharynx, mens under generel anæstesi. Denne metode kan anvendes til pulmonal fremføring af en lang række ikke-ætsende biologiske og kemiske stoffer, og det er relativt let at lære, selv for laboratorier med minimal forudgående erfaring med pulmonale procedurer. Ud over at beskrive aspirationspneumoni metode, vi også give trin-for-trin procedurer til analyse af efterfølgende in vivo pulmonal medfødte immunreaktion af musen, navnlig fremgangsmåder til kvantificering bakterieclearance og det cellulære immunrespons af den inficerede luftvej. Denne integrerede og enkel tilgang til lungebetændelse vurdering tillader hurtig og robust evaluering af effekten af ​​genetiske og miljømæssige manipulationer ved pulmonal medfødte immunitet.

C57BL / 6-mus blev inficeret med 2000 CFU af K. pneumoniae 43816 (serotype 2) via svælg optagelse i lungerne. Ved denne dosis mus typisk begynder at vise kliniske symptomer 12-24 timer efter infektion, herunder sløvhed, pjusket pels, og vægttab på 5-10% (figur 2A). Inden 48-72 timer efter infektion, mange af de mus viser symptomer på sygdom og sygelighed, der typisk indledes med et gennemsnit på 20% vægttab og resulterer i hunched stillinger med nedsat ak...

Watch this Scientific Journal Video about A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice at JoVE.com

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

Adskillige fremgangsmåder er blevet beskrevet i litteraturen til modellering bakteriel lungebetændelse i mus. Heri beskriver vi en ikke-invasiv, billig, hurtig metode til induktion lungebetændelse via aspiration (dvs. inhalation) af et bakterielt podestof pipetteret ind i oropharynx. Downstream metoder til vurdering af pulmonal medfødte immunforsvar er også detaljeret.

En ikke-invasiv og Teknisk Ikke-intensive Metode til induktion og fænotype for Eksperimentel bakteriel lungebetændelse i mus

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

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