Name: in vivo imaging method to distinguish acute and chronic inflammation
Uploaded: 2013-08-16T12:00:00
Description: Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

Dette arbejde blev støttet af Nancy Lurie Marks Foundation. Vi takker Dr. Nancy E. Kohl for kritisk læsning af manuskriptet. Vi takker Kin K. Wong for hans assistance i udarbejdelsen af ​​denne video rapport.

Bemærk: Alle dyreforsøg blev udført under godkendte institutionelle protokoller og dyrepleje retningslinjer. 1.. Reagenser og løsninger <ol><li> PMA løsning til sc podning: Forbered en stamopløsning af phorbol 12-myristat-13-acetat (PMA) ved 5 mg / ml i DMSO. Opbevar stamopløsningen ved -20 ° C. Før podning, tø stamopløsningen og fortyndes til 1 mg / ml PMA i PBS. </li><li> LPS løsning til sc podning: Opløs lipopolysaccharid (LPS fra Salmonella enterica</e...

<ol>
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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=Peroxisome+proliferator-activated+receptor+gamma+ligands+regulate+myeloperoxidase+expression+in+macrophages+by+an+estrogen-dependent+mechanism+involving+the+-463GA+promoter+polymorphism.">Peroxisome proliferator-activated receptor gamma ligands regulate myeloperoxidase expression in macrophages by an estrogen-dependent mechanism involving the -463GA promoter polymorphism.</a> J. Biol. Chem. 279, 8300-8315 (2004).</li><li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+inflammatory+phagocytes.">In vivo imaging of inflammatory phagocytes.</a> Chem Biol. 19, 1199-1209 (2012).</li><li>Gross, S., Gammon, S. 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=Bioluminescence+imaging+of+myeloperoxidase+activity+in+vivo.">Bioluminescence imaging of myeloperoxidase activity in vivo.</a> Nat. Med. 15, 455-461 (2009).</li><li>Kielland, A., Blom, 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=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radic. Biol. Med. 47, 760-766 (2009).</li><li>Zhou, J., Tsai, Y. 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=Noninvasive+assessment+of+localized+inflammatory+responses.">Noninvasive assessment of localized inflammatory responses.</a> Free Radic. Biol. Med. 52, 218-226 (2012).</li><li>Karlsson, A., Nixon, J. 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=Phorbol+myristate+acetate+induces+neutrophil+NADPH-oxidase+activity+by+two+separate+signal+transduction+pathways:+dependent+or+independent+of+phosphatidylinositol+3-kinase.">Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase.</a> J. Leukoc. Biol. 67, 396-404 (2000).</li><li>Taylor, R. G., McCall, C. 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=Histopathologic+features+of+phorbol+myristate+acetate-induced+lung+injury.">Histopathologic features of phorbol myristate acetate-induced lung injury.</a> Lab Invest. 52, 61-70 (1985).</li><li>Fukaya, S., Matsui, 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=Overexpression+of+TNF-alpha-converting+enzyme+in+fibroblasts+augments+dermal+fibrosis+after+inflammation.">Overexpression of TNF-alpha-converting enzyme in fibroblasts augments dermal fibrosis after inflammation.</a> Lab Invest. 93, 72-80 (2013).</li><li>Lu, Y. C., Yeh, W. 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I denne rapport, viser vi en bioluminescence metode til ikke-invasiv billeddannelse af betændelse i levende dyr. At drage fordel af to selvlysende substrater, luminol og lucigenin kan fremgangsmåden skelne mellem forskellige faser af inflammation. Luminol bioluminescens er forbundet med neutrofiler i den akutte fase af inflammation, hvorimod lucigenin bioluminescens medieres af makrofager i den kroniske fase. Relativt lille (MW = 177,16 g / mol) og elektrisk ladet, kan luminol hurtigt ind både plasma og phagosomal me...

Inflammation er en stærkt reguleret biologisk respons involveret i en række humane sygdomme, herunder mikrobiel infektion 1, sårheling 2, diabetes 3, kræft 4, hjerte 5 neurodegenerativ 6, og autoimmune sygdomme 7. Vævsinflammation kræver ordentlig koordinering af de forskellige immunceller for at opnå patogen clearance, væv reparation, og sygdom opløsning. Neutrofiler og makrofager er vigtige immune mediatorer af vævsinflammation. I den akutte fase af inflammation, er neutrofile de første respondenter til forskellige skadelige stimuli og vævsskader 8.. De neutrofile hurtigt ekstravasere fra cirkulation til stedet for skaden, hvor cellerne inaktivere invaderende mikrober ved at frigive antimikrobielle granulater og fagocytose. Under fagocytose opsluge neutrofiler invaderer mikrober i phagosomes, inden for hvilke celler producerer høje niveauer af Superoxide (O 2 · -). Phagosomal superoxid er den primære kilde til mange downstream reaktive ilt arter (ROS). For eksempel kan superoxid kan dismutated til hydrogenperoxid (H 2 O 2) ved spontan spaltning eller ved superoxiddismutase (SOD) 9, 10. Neutrofiler, yderligere myeloperoxidase (MPO) konverterer hydrogenperoxid til antimikrobiel chlorundersyrling (HOCI) 11. Da de inflammatoriske responser fortsætter, cirkulerende monocytter gradvist migrerer ind i læsionsstedet og differentiere til modne makrofager 2, hvis fagocytotisk funktion hjælpe med at fjerne inaktiverede patogener og cellerester. Hertil kommer, som en central regulator i den senere fase af inflammation makrofag fremmer væv reparation ved at producere anti-inflammatoriske cytokiner 12 og ved at skabe ekstracellulære ROS på et lavere niveau 9.. Den ROS genereret på dette senere tidspunkt regulerer vævsremodellering, nye fartøj dannelse, og reepitheliasering 13.. Fagocyt NADPH oxidase (phOx) er den primære kilde til superoxid produktion i både neutrofile og makrofager 9.. Phox er en multi-subunit kompleks hvis samling er stramt reguleret 9. Holoenzymet indeholder flere cytosoliske regulatoriske underenheder (P67 phox, P47 phox, P40 phox, og RAC) samt en membranbundet heterodimer cytochrom b 558 (bestå af underenhed CYBA og CYBB). Cytochrom bs 558 er reaktionen kerne, inden for hvilken CYBB underenhed (også kendt som P91 phox og NOX2) foretager den primære redox kædereaktion 9.. Interessant, dens montage sites er forskellige mellem neutrofile og makrofager. I hvilende neutrofiler, er cytochrom bs 558 meste til stede i membranen af intracellulære opbevaring granulat 14. Under fagocytose samle neutrofiler holoenzymes på phagosomes 9, hvor høje niveauer af MPO-aktivitet er også til stede. Neutrofil phOx hurtigt forbruger ilt og udøver sin mikrobicid magt ved ROS produktion, et fænomen kaldet respiratorisk burst 11.. I modsætning hertil har makrofager et lavere niveau af MPO udtryk og cytochrom b 558 er for det meste findes i plasmamembranen 15, 16. Således neutrofiler producerer høje niveauer af superoxid for anti-mikrobielle aktivitet, mens makrofager genererer mindre superoxid for regulerende funktioner 15. Da inflammation er en indviklet in vivo-processen, vil non-invasive billeddiagnostiske metoder er specifikke for de forskellige faser af inflammation tillade kvantitative og langsgående vurdering af sygdomsmodeller. Brug mekanistiske undersøgelser har vi tidligere vist anvendelsen af ​​to kemiluminescerende midler, luminol (5-amino-2 ,3-dihydro-1 ,4-phthalazinedione) og lucigenin (bis-N-methylacridinium nitrat)For ikke-invasiv billeddannelse af akutte og sene (kroniske) stadier af inflammation, henholdsvis 17. Luminol muliggør visualisering af neutrofil MPO aktivitet i den akutte fase af inflammation 18-20, hvorimod lucigenin bioluminescens kan anvendes til at vurdere makrofag aktivitet i forbindelse med den sene fase eller kronisk betændelse 17.. I dette manuskript, brugte vi to eksperimentelle betændelse modeller (sc PMA og SC LPS) til at vise disse billeddannende teknikker.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>P8139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype enteritidis</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>L2012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>A4685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucigenin (bis-N-methylacridinium nitrate)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>M8010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum imaging system with Living Imaging 4.2 software package</td>
			<td>Caliper LS/Perkin Elmer, Hopkinton, MA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vivo imaging method to distinguish acute and chronic inflammation

Inflammation er et grundlæggende aspekt af mange menneskelige sygdomme. I denne video rapporter, viser vi non-invasive bioluminescens imaging teknikker, der skelner akut og kronisk inflammation i musemodeller. Med vævsskader eller patogen invasion, er neutrofile den første linje i forsvaret, spiller en stor rolle i formidlingen af ​​den akutte inflammatoriske respons. Som den inflammatoriske reaktion skrider frem, cirkulerende monocytter gradvist migrerer ind i læsionsstedet og differentiere til modne makrofager, som medierer kronisk inflammation og fremme vævsreparation ved at fjerne vævsrester og producere anti-inflammatoriske cytokiner. Intraperitoneal injektion af luminol (5-amino-2 ,3-dihydro-1 ,4-phthalazinedione, natriumsalt) muliggør påvisning af akut betændelse i vid udstrækning medieres af væv-infiltrerende neutrofiler. Luminol reagerer specifikt med superoxid dannes inden for de phagosomes af neutrofiler siden bioluminescens resultater fra en myeloperoxidase (MPO) medieret reaktion. Lucigenin (bis-N-methylacridinium nitrat) også reagerer med superoxid for at generere bioluminescens. Men lucigenin bioluminescens er uafhængig af MPO og det udelukkende er afhængig af fagocyt NADPH oxidase (PHOX) i makrofager under kronisk inflammation. Sammen luminol og lucigenin tillade ikke-invasiv visualisering og langsgående vurdering af forskellige fagocyt populationer på tværs af både akutte og kroniske inflammatoriske faser. Grund af den vigtige rolle af inflammation i en række humane sygdomme, mener vi denne ikke-invasiv billeddannelse metode kan hjælpe undersøge differentielle roller neutrofiler og makrofager i en række patologiske tilstande.

Vi udførte langsgående bioluminescence imaging at vurdere akut og kronisk inflammation i eksperimentelle inflammation modeller. Phorbol 12-myristat-13-acetat (PMA) er et potent protein kinase C (PKC)-agonist, der aktiverer PHOX til superoxidanion produktion og udløser intense akutte inflammatoriske reaktioner 21.. SC injektion af 50 mg af PMA i NCR nøgne mus forårsagede hurtig hudirritation og akut inflammation ved injektionsstedet 18, 22, 23.. Vi udførte daglige billeddannelse i 4 dage med d...

Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Vi beskriver en ikke-invasiv billeddannelse fremgangsmåde til at skelne inflammatoriske stadier. Systemisk levering af luminol afbilder områder med akut betændelse afhængige MPO aktivitet i neutrofiler. I modsætning hertil muliggør injektion af lucigenin til visualisering af kronisk inflammation afhængig PHOX aktivitet i makrofager.

In vivo Imaging metode til at skelne akut og kronisk inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

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Immunology and Infection

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

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En optimeret fremgangsmåde til isolering og udvidelse Invariant Natural Killer T-celler fra musemilt

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

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

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 ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Ikke-invasiv<em&gt; In vivo</em&gt; Fluorescens optisk billeddannelse af inflammatorisk MMP Aktivitet Brug et aktiverbart Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Muse lymfocyt Mærkning af<sup&gt; 64</sup&gt; Cu-antistofreceptor Målretning for<em&gt; In vivo</em&gt; Cell Trafficking af PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

En effektiv og enkel metode til at etablere NK og T-cellelinier fra patienter med kronisk aktiv Epstein - Barr virusinfektion

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...