Name: the utilization of oropharyngeal intratracheal pamp administration and bronchoalveolar lavage to evaluate the host immune response in mice
Uploaded: 2014-04-02T17:00:00
Description: Watch this Scientific Journal Video about The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice at JoVE.com

Forfatterne takker VA-MD Regional College of Veterinary Medicine for at levere kerne og teknisk støtte til dette projekt. Dette arbejde understøttes af en NIH Career Development Award (K01DK092355).

Alle undersøgelser blev gennemført i henhold til godkendelse af Institutional Care og brug Udvalg (IACUC) for Virginia Tech og i overensstemmelse med National Institutes of Health Guide til Pleje og anvendelse af forsøgsdyr. 1.. Intratracheale (it) Podning af LPS Brug Oropharyngeal Administration <ol><li> Sørg for, at hvert dyr entydigt identificeres ved hjælp af enten en øre punch, øremærke eller andet institutionelt godkendt metode. </li><li> Optag baseline kropsvægt og te...

<ol>
	<li>Matute-Bello, 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=An+official+American+Thoracic+Society+workshop+report:+features+and+measurements+of+experimental+acute+lung+injury+in+animals.">An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals.</a> Am. J. respir. Cell Mol. Biol. 44, 725-738 (2011).</li><li>Egger, C., 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=Administration+of+bleomycin+via+the+oropharyngeal+aspiration+route+leads+to+sustained+lung+fibrosis+in+mice+and+rats+as+quantified+by+UTE-MRI+and+histology.">Administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by UTE-MRI and histology.</a> PloS one. 8, (2013).</li><li>Rayamajhi, 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=Nonsurgical+intratracheal+instillation+of+mice+with+analysis+of+lungs+and+lung+draining+lymph+nodes+by+flow+cytometry.">Nonsurgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry.</a> J. Vis. Exp. , (2011).</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>Allen, I. C., 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=Analysis+of+NLRP3+in+the+development+of+allergic+airway+disease+in+mice.">Analysis of NLRP3 in the development of allergic airway disease in mice.</a> J. Immunol. 188, 2884-2893 (2012).</li><li>Allen, I. C., 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=Characterization+of+NLRP12+during+the+in+vivo+host+immune+response+to+Klebsiella+pneumoniae+and+Mycobacterium+tuberculosis.">Characterization of NLRP12 during the in vivo host immune response to Klebsiella pneumoniae and Mycobacterium tuberculosis.</a> PloS one. , (2013).</li><li>Allen, I. C., 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=Expression+and+function+of+NPSR1/GPRA+in+the+lung+before+and+after+induction+of+asthma-like+disease.">Expression and function of NPSR1/GPRA in the lung before and after induction of asthma-like disease.</a> Am. J. Physiol. Lung Cell. Mol. Physiol. 291, 1005-1017 (2006).</li><li>Kebaier, C., 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=Staphylococcus+aureus+alpha-hemolysin+mediates+virulence+in+a+murine+model+of+severe+pneumonia+through+activation+of+the+NLRP3+inflammasome.">Staphylococcus aureus alpha-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome.</a> J. Infect. Dis. 205, 807-817 (2012).</li><li>Roberts, R. 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=Analysis+of+the+murine+immune+response+to+pulmonary+delivery+of+precisely+fabricated+nano-+and+microscale+particles.">Analysis of the murine immune response to pulmonary delivery of precisely fabricated nano- and microscale particles.</a> PloS one. 8, (2013).</li><li>Willingham, S. 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=NLRP3+(NALP3,+Cryopyrin)+facilitates+in+vivo+caspase-1+activation,+necrosis,+and+HMGB1+release+via+inflammasome-dependent+and+-independent+pathways.">NLRP3 (NALP3, Cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways.</a> J. Immunol. 183, 2008-2015 (2009).</li><li>Kline, J. 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=Variable+airway+responsiveness+to+inhaled+lipopolysaccharide.">Variable airway responsiveness to inhaled lipopolysaccharide.</a> Am. J. Respir. Crit. Med. 160, 297-303 (1999).</li><li>Cressman, V. L., Hicks, E. M., Funkhouser, W. K., Backlund, D. C., Koller, B. 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De mest kritiske trin for vellykket evaluering immunrespons værten i muselunger er som følger: 1) vælge den rette mus stamme og køn for modellen blive evalueret; 2) optimere PAMP levering til lungerne; 3) at indsamle og bearbejde BALF korrekt; og 4) fastsætte ordentligt og forberede af lungerne for histopatologiske vurderinger. Valget af musestamme er en vigtig faktor i evalueringen værtens immunrespons. C57BI / 6 mus typisk betragtes som den optimale muse baggrund for at studere medf?...

Lungeinfektioner forbundet med patogene bakteriearter er en almindelig årsag til den globale sygelighed og dødelighed. Bestemmelse af de mekanismer, der driver værtens immunrespons til disse patogener vil fremme udviklingen af ​​nye forebyggelsesstrategier og terapeutiske midler, der vil dæmpe virkningen af ​​disse infektioner. Det overordnede mål med protokollen beskrevet her er at give brugeren med en fleksibel metode til at evaluere respons værten medfødte immunforsvar til patogeninfektion hjælp af et patogen forbundet molekylær mønster (PAMP) som erstatning for levende bakterier. Hovedparten af ​​tidligere undersøgelser evaluerede svar værten medfødte immunforsvar bakterier har fokuseret på peritoneale modeller på grund af den relative lethed af udførelse. Mens disse modeller er meget nyttige, og har resulteret i betydelige fremskridt inden for host-patogen interaktioner og systemisk inflammation, de data, der genereres fra disse modeller er ikke altid hensigtsmæssig til undersøgelser involving åndedrætsorganerne. Her er en pulmonal model af akut lungebetændelse foreslået som en praktisk og klinisk relevant udvidelse af de klassiske intraperitoneal (ip) injektion modeller. Den foreslåede teknik gør det muligt for den lokale vurdering af medfødte immunrespons i en organspecifik modelsystem. De her beskrevne metoder er designet til at give en enkel og robust teknik til at tillade brugere at vurdere værtens immunrespons til LPS, som er en almindelig PAMP. Metoderne er baseret på intratracheal (it) instillation af LPS, som fremkalder en robust immunrespons medfødte i lungerne hos mus og efterligner mange af de patofysiologiske funktioner observerede i de humane patienter, der lider af luftvejsinfektioner og akut lungeskade 1. En primær fordel ved denne teknik er, at det giver brugeren mulighed for at vurdere værtens immunrespons uden forstyrrende faktorer og sikkerhedsmæssige problemer, der er forbundet med at gennemføre in vivo-undersøgelserbrug af levende bakterier. Ligeledes oropharyngeale det administration eksponeringsvej beskrevet i denne protokol har betydelige fordele frem for andre almindeligt anvendte teknikker, herunder intranasal (i) administration og kirurgisk det administration. For eksempel svælg det administration tillader relativt præcis dosaging og lungedeponering forhold til i administration, der typisk lider af øget variabilitet lungedeponering på grund af tabet af agenter i næsehulen og bihulerne 2-4. Det er det administrationsvej omgår disse hulrum og giver direkte adgang til luftrøret og luftveje. Ligeledes den kirurgiske det tilgang er et betydeligt mere morbid administration metode og kræver omfattende uddannelse til master. De her beskrevne protokoller omfatter også en beskrivelse af de almindelige teknikker og surrogat markører, der anvendes til at evaluere betændelse progression og ender med en protokol, der beskriver de korrekte teknikker til forberedelse af lungs til histopatologi vurderinger. Disse protokoller er fokuseret på at minimere antallet af mus, der kræves for hver undersøgelse ved at maksimere de genererede data fra hver enkelt dyr. De beskrevne protokoller er meget fleksible og kan let modificeres til at vurdere en bred vifte PAMPs og skader molekylære mønstre (dæmper). Desuden, med et par yderligere modifikationer, disse protokoller kan også anvendes til studier, der evaluerer allergisk luftvejs sygdomsprogression eller host-patogen interaktioner med levende bakterier, vira eller svampe 5-10.

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the utilization of oropharyngeal intratracheal pamp administration and bronchoalveolar lavage to evaluate the host immune response in mice

Svaret på patogener værtsimmunrespons er en kompleks biologisk proces. Hovedparten af undersøgelser in vivo klassisk anvendes til at karakterisere host-patogen interaktioner drage fordel af intraperitoneale injektioner af udvalgte bakterier eller patogener associeret molekylære mønstre (PAMPs) i mus. Selv om disse teknikker har givet enorme data i forbindelse med smitsomme sygdomme Patobiologi, intraperitoneal injektion modeller er ikke altid hensigtsmæssigt for host-patogen interaktionsundersøgelser i lungen. Ved hjælp af en akut lungebetændelse model i mus, er det muligt at gennemføre en høj opløsning analyse af værtens medfødte immunreaktion udnytte lipopolysaccharid (LPS). Her beskriver vi metoder til at administrere LPS ved hjælp af kirurgisk svælg intratracheal administration overvåge kliniske parametre, der er forbundet med sygdommen patogenese og anvende bronkoalveolærvaskevæsken at evaluere værtens immunrespons. De teknikker, der er beskreveter bredt anvendelig til at studere reaktion værten medfødte immun over for en bred vifte af PAMPs og patogener. Ligeledes, med mindre modifikationer, disse teknikker kan også anvendes i undersøgelser evaluerer allergisk luftvejsinflammation og farmakologiske applikationer.

Cellevæggene på gram-negative bakterier er sammensat af LPS, som er meget udbredte i miljøet. Indånding af LPS i følsomme befolkningsgrupper forværrer luftvejsreaktivitet og er i stand til at udløse en robust immunforsvar response11. LPS er også et fælles PAMP anvendes i musemodeller for at fremkalde en reaktion robust medfødte immunreaktion. I protokollen beskrevet her, fik musene en den dosis af LPS isoleret fra E. coli (serotype 0111: B4) under anvendelse af oropharyngeal det administration. I mode...

Watch this Scientific Journal Video about The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice at JoVE.com

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

Reaktionen på patogeninfektion værtsimmunrespons er en stramt reguleret proces. Ved hjælp af en lipopolysaccharid lunge eksponering model i mus, er det muligt at foretage høj opløsning evalueringer af de komplekse mekanismer, der er forbundet med sygdom patogenese.

Udnyttelsen af ​​Oropharyngeal Intratracheal PAMP Administration og Bronchoalveolære at evaluere Host immunrespons i mus

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

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A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Brug af RNA-interferens Undersøge Innate immunresponset i musemakrofager

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In vitro analyse af MyD88-medieret Cellular immunrespons til West Nile Virus mutantstamme Infektion

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Beskyttende Effekt og Pulmonal immunrespons efter subkutan og Intranasal BCG Administration i mus

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

pode<em&gt; Malariamyg</em&gt; Myg med perler til at fremkalde og måle melanin immunrespons

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolær Lavage af murine Lunger at analysere inflammatorisk celleinfiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Isolering af ekstracellulære vesikler fra Murine Bronchoalveolar Lavage væske ved hjælp af en ultrafiltrering centrifugering teknik

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Evaluering af vært-patogen svar og Vaccine effekt i mus

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

Udnyttelsen af ​​Oropharyngeal Intratracheal PAMP Administration og Bronchoalveolære at evaluere Host immunrespons i mus

Udnyttelsen af Oropharyngeal Intratracheal PAMP Administration og Bronchoalveolære at evaluere Host immunrespons i mus