Name: bioluminescence imaging of nadph oxidase activity in different animal models
Uploaded: 2012-10-22T12:00:00
Description: Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Dette arbejde blev finansieret af NIH RO1 AI079253 og Department of Veterans Affairs.

1. Dyremodeller <ol><li> Mus: Brug P47 phOx-/ - mus og alder-og køn-matchede C57BL6/DBA mus. Opnå godkendelse af forsøg fra Institutional Animal Care og Use Udvalg. </li><li> Anæstesi: Brug et kontinuerligt isofluran administration system til at fremkalde anæstesi. Fordamperen system (VetEquip) er fyldt med isofluran (2-3%). Bekræfter, at mus er fuldt bedøvet ved iagttagelse af respiration, bevægelse, og corneal refleks som reaktion på ydre stimuli. </li><li> Kirurgiske procedurer: Skrub d...

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&quot;Real-time&quot; måling af reaktive oxygenarter (ROS) i levende dyr kan opnås ved anvendelse af fluorescerende og kemiluminescerende prober. Mens fluorescerende prober lider med svage signal-til-støj-forhold 12, den billeddannende beskrevne teknik er mere følsom til detektering af lysemission efter en kemisk reaktion af ROS med luminol-baseret substrat L-012. Som alle bioluminescerende billeddannelsesteknikker, er denne metode begrænses af bølgelængde-afhængig lys absorption og spredning af organ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> L-012 </td><td> Wako Chemicals USA, Inc. </td><td> 120-04.891 </td><td></td></tr><tr><td> Zymosan </td><td> Sigma, St. Louis, MO </td><td> Z4250 </td><td></td></tr><tr><td> tetrachlormethan </td><td> Sigma, St. Louis, MO </td><td> 289.116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH-oxidase er et kritisk enzym, som medierer antibakteriel og antifungal værtsforsvar. Ud over dets rolle i antimikrobielt værtsforsvar har NADPH-oxidase kritiske signaleringsfunktioner som modulerer det inflammatoriske respons 1. Således er udviklingen af ​​en metode til måling i &quot;real-time&quot; kinetikken af ​​NADPH oxidase-afledt ROS generation forventes at være en værdifuld forskning værktøj til at forstå mekanismerne er relevante at være vært forsvar, inflammation og skade. Kronisk granulomatøs sygdom (CGD), er en arvelig sygdom af NADPH-oxidase er kendetegnet ved alvorlige infektioner og overdreven inflammation. Aktivering af fagocyt-NADPH-oxidase kræver translokation af de cytosoliske underenheder (P47 phOx, P67 phOx og p40 phOx) og Rac til et membranbundet flavocytochrome (bestående af en gp91 phOx og P22 phOx heterodimer). Tabaf funktion mutationer i nogen af ​​disse NADPH oxidase komponenter resulterer i CGD. Svarende til patienter med CGD, gp91 phOx-deficiente mus og P47 phOx-deficiente mus har defekt fagocyt NADPH-oxidaseaktivitet og nedsat værtsforsvar 13, 14. Ud over fagocytter, som indeholder NADPH-oxidase ovenfor beskrevne komponenter, udtrykke en række andre celletyper forskellige isoformer af NADPH-oxidase. Her beskriver vi en fremgangsmåde til kvantificering ROS produktion i levende mus, og for at afgrænse bidrag NADPH-oxidase til ROS-generering i modeller for inflammation og skader. Denne metode er baseret på ROS omsætning med L-012 (en analog af luminol) til at udsende luminescens, der registreres af et charge-coupled device (CCD). I den oprindelige beskrivelse af L-012-proben, blev L-012-afhængig kemiluminescens fuldstændigt ophævet af superoxiddismutase, hvilket indikerer, at den største ROS påvist i denne reaktion var superoxid anion 15. Efterfølgende undersøgelser har vist, at L-012 kan detektere andre frie radikaler, herunder reaktive nitrogenforbindelser 16, 17. Kielland et al. 17 viste, at topisk anvendelse af phorbolmyristatacetat, en potent aktivator af NADPH-oxidase, førte til NADPH-oxidase-afhængig ROS-generering, der kunne påvises i mus under anvendelse af luminescerende probe L-012. I denne model, viste de, at L-012-afhængig luminescens blev afskaffet i P47 phOx-deficiente mus. Vi sammenlignede ROS-generering i vildtype-mus og NADPH-oxidase-deficient P47 phOx-/ - mus 2 i de følgende tre modeller: 1) intratracheal administration af zymosan, en pro-inflammatorisk svampens cellevæg-afledt produkt, der kan aktivere NADPH-oxidase, 2) cecal ligering og punktering (CLP), en model af intra-abdominal sepsis med sekundær akut lunge-betændelse og skade, og 3) oral carbontetrachlorid(CCI4), en model af ROS-afhængig leverskade. Disse modeller blev specifikt udvalgt til at vurdere NADPH-oxidase-afhængig ROS-generering i forbindelse med ikke-infektiøs inflammation, polymikrobiale sepsis og toksin-induceret organ skade hhv. Sammenligning af bioluminescens i vildtype-mus til P47 phOx-/ - mus gør det muligt at afgrænse de specifikke bidrag ROS genereret af P47 phOx-holdig NADPH-oxidase til det bioluminescerende signal i disse modeller. Bioluminescens billeddannelse resultater, der viste forøget ROS-niveauer i vildtype-mus sammenlignet med P47 phOx-/ - mus viste, at NADPH-oxidase er den vigtigste kilde til ROS-generering som respons på inflammatoriske stimuli. Denne fremgangsmåde tilvejebringer en minimalt invasiv fremgangsmåde til &quot;real-time&quot; overvågning af ROS-generering under inflammation in vivo.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH-oxidase er den vigtigste kilde af reaktive oxygenarter (ROS) i fagocytter. På grund af den flygtige karakter af ROS, er det vanskeligt at måle og overvåge ROS niveauer i levende dyr. Et minimalt invasiv metode til seriel kvantificering af ROS i levende mus er beskrevet.

Bioluminescens Imaging af NADPH oxidase aktivitet i forskellige dyremodeller

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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