Name: non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent
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Description: Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

Vi takker Daniel Bukala, Natalie Altmeyer og Funda Cay for fremragende teknisk support. Vi takker Jonathan Cotton, Greg Bowden og Paul Soubiran for at redigere manuskriptet. Dette arbejde blev støttet af Werner Siemens-fonden og det medicinske fakultet for Eberhard Karls Universitet Tübingen (&#39;&#39; Promotionskolleg &#39;&#39;) og af DFG gennem CRC 156 (projekt C3).

Alle procedurer beskrevet i dette papir fulgte retningslinjer og internationale standarder for pleje og brug af forsøgsdyr og blev godkendt af den lokale dyrevelfærd og etiske udvalg i landkommissionen Tuebingen, Tyskland. 8-12 uger gamle BALB / c og C57BL / 6 mus blev opbevaret i en 12 h: 12 h lys: mørk cyklus og blev anbragt i IVC&#39;er og standardiserede miljøforhold ved 22 ± 1 ° C i grupper på 2-5 med vand og Adgang til mad ad libitum . 1. Materialepræparation <ol><li...

<ol>
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Applications in small animal research.</a> Nuklearmedizin. 55 (2), 63-70 (2016).</li><li>Mahling, 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=A+Comparative+pO2+Probe+and+[18F]-Fluoro-Azomycinarabino-Furanoside+([18F]FAZA)+PET+Study+Reveals+Anesthesia-Induced+Impairment+of+Oxygenation+and+Perfusion+in+Tumor+and+Muscle.">A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle.</a> PLoS One. 10 (4), 0124665 (2015).</li><li>Fuchs, 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=Oxygen+breathing+affects+3'-deoxy-3'-18F-fluorothymidine+uptake+in+mouse+models+of+arthritis+and+cancer.">Oxygen breathing affects 3'-deoxy-3'-18F-fluorothymidine uptake in mouse models of arthritis and cancer.</a> J Nucl Med. 53 (5), 823-830 (2012).</li><li>Fuchs, 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=Impact+of+anesthetics+on+3'-[18F]fluoro-3'-deoxythymidine+([18F]FLT)+uptake+in+animal+models+of+cancer+and+inflammation.">Impact of anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in animal models of cancer and inflammation.</a> Mol Imaging. 12 (5), 277-287 (2013).</li><li>Liu, N., Shang, J., Tian, F., Nishi, H., Abe, K. <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+optical+imaging+for+evaluating+the+efficacy+of+edaravone+after+transient+cerebral+ischemia+in+mice.">In vivo optical imaging for evaluating the efficacy of edaravone after transient cerebral ischemia in mice.</a> Brain Res. 1397, 66-75 (2011).</li><li>Sheth, R. A., Maricevich, M., Mahmood, U. <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+optical+molecular+imaging+of+matrix+metalloproteinase+activity+in+abdominal+aortic+aneurysms+correlates+with+treatment+effects+on+growth+rate.">In vivo optical molecular imaging of matrix metalloproteinase activity in abdominal aortic aneurysms correlates with treatment effects on growth rate.</a> Atherosclerosis. 212 (1), 181-187 (2010).</li><li>Chen, 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=Near-infrared+fluorescent+imaging+of+matrix+metalloproteinase+activity+after+myocardial+infarction.">Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction.</a> Circulation. 111 (14), 1800-1805 (2005).</li><li>Wallis de Vries, B. 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H., Mahmood, U., Bogdanov, A. <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+tumors+with+protease-activated+near-infrared+fluorescent+probes.">In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.</a> Nat Biotechnol. 17 (4), 375-378 (1999).</li><li>Cortez-Retamozo, V., 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=Real-time+assessment+of+inflammation+and+treatment+response+in+a+mouse+model+of+allergic+airway+inflammation.">Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.</a> J Clin Invest. 118 (12), 4058-4066 (2008).</li><li>McIntyre, J. O., 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=Development+of+a+novel+fluorogenic+proteolytic+beacon+for+in+vivo+detection+and+imaging+of+tumour-associated+matrix+metalloproteinase-7+activity.">Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.</a> Biochem J. 377, 617-628 (2004).</li><li>Scherer, R. L., VanSaun, M. N., McIntyre, J. O., Matrisian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+matrix+metalloproteinase-7+activity+in+vivo+using+a+proteolytic+nanobeacon).">Optical imaging of matrix metalloproteinase-7 activity in vivo using a proteolytic nanobeacon).</a> Mol Imaging. 7 (3), 118-131 (2008).</li><li>Olson, E. 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S., Nicolay, K., Grull, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+targeting+of+MMP-2/9+activatable+cell-penetrating+imaging+probes+is+caused+by+tumor-independent+activation).">Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation).</a> J Nucl Med. 52 (2), 279-286 (2011).</li><li>Schafers, M., Schober, O., Hermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-metalloproteinases+as+imaging+targets+for+inflammatory+activity+in+atherosclerotic+plaques.">Matrix-metalloproteinases as imaging targets for inflammatory activity in atherosclerotic plaques.</a> J Nucl Med. 51 (5), 663-666 (2010).</li><li>Wagner, 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=A+new+18F-labelled+derivative+of+the+MMP+inhibitor+CGS+27023A+for+PET:+radiosynthesis+and+initial+small-animal+PET+studies.">A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal PET studies.</a> Appl Radiat Isot. 67 (4), 606-610 (2009).</li><li>Waschkau, B., Faust, A., Schafers, M., Bremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+new+fluorescence-labeled+MMP+inhibitor+to+image+tumor+MMP+activity+in+vivo+in+comparison+to+an+MMP-activatable+probe.">Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.</a> Contrast Media Mol Imaging. 8 (1), 1-11 (2013).</li><li>Vogl, 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=Alarmin+S100A8/S100A9+as+a+biomarker+for+molecular+imaging+of+local+inflammatory+activity.">Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.</a> Nat Commun. 5, 4593 (2014).</li></ol>

OI er et meget nyttigt, hurtigt og billigt værktøj til ikke-invasiv in vivo molekylær billeddannelse i præklinisk forskning. En særlig styrke af OI er evnen til at overvåge meget dynamiske processer som inflammatoriske reaktioner. Desuden giver OI mulighed for at følge sygdomsforløbet i længere tid, alt fra dage til uger. OI har flere fordele i forhold til andre in vivo billeddannelsesmodaliteter som positronemissionstomografi (PET) eller magnetisk resonansbilledda...

Autoimmune sygdomme som reumatoid arthritis (RA) eller psoriasis vulgaris er klassificeret som hypersensitivitetsreaktioner med forsinket type (DTHR). 1 RA er en almindelig autoimmun sygdom præget af erosiv synovitis og ledd ødelæggelse. 2 inflamtrerede leddgiktskanaler viser infiltration og proliferation af inflammatoriske celler, et øget udtryk for proinflammatoriske celler, der fører til dannelse af pannus, brusk og knogleredbrydning. 3 , 4 Spaltningen af ​​ekstracellulære matrixmolekyler, såsom kollagen ved matrixmetalloproteinaser (MMP&#39;er), er essentiel for vævsomdannelse og angiogenese og forårsager vævsdestruktioner. 5 , 6 Kontakt overfølsomhedsreaktioner (CHR) er kendetegnet ved aggregering af neutrofiler, der fører til en oxidativ udbrud. 7 Ligesom RA er MMP&#39;er i CHR involveretub i væv konvertering, cellevandring og angiogenese for at etablere kronisk inflammation. At undersøge RA, blev glucose-6-phosphat-isomerase (GPI) -serum injektion musemodel anvendes. 8 Serum fra transgene K / BxN-mus, der indeholder antistoffer mod GPI, blev injiceret i naive BALB / c-mus, hvorefter reumatisk inflammation begyndte at udvikle sig inden for 24 timer med et maksimum på ankel hævelse på dag 6 efter GPI-serum injektion (se 1.1). At analysere kronisk CHR, blev C57BL / 6-mus sensibiliseret med trinitrochlorbenzen (TNCB) på maven. Det højre øre blev udfordret op til 5 gange startende 1 uge efter sensibilisering (se også 1.1 og 1.2). Noninvasive lille dyr OI er en teknik baseret på in vivo undersøgelse af fluorescent-, chemiluminescent- og bioluminescerende-signaler, der hovedsagelig anvendes i præklinisk forskning. Den erhvervede semi-kvantitative data giver indsigt i, Molecular mekanismer i organer og væv fra raske såvel som syge eksperimentelle dyremodeller, og muliggør langsgående opfølgning målinger (f.eks at vurdere terapeutiske responsprofiler in vivo). En stor fordel af langsgående undersøgelser er reduktionen af ​​antallet af dyr, som de samme dyr kan måles i opfølgende undersøgelser på flere tidspunkter i stedet for at bruge forskellige mus pr tidspunkt. Løsningen af ​​OI tillader detaljeret funktionel billeddannelse af organer og endnu mindre vævsstrukturer i forsøgsdyr. Anvendelsen af ​​specifikke excitations- og emissionsfiltre med en smal transmissionsspektrum, en beskyttelse mod spredt lys ved en lystæt &quot;mørk box&quot; og en følsom Charge Coupled indretning (CCD) kamera, som afkøles i mange enheder ned til -70 ° C tillader meget specifikke og følsomme målinger af fluorescenssignaler. Ved anvendelse af fluorescerende midler med excitations- ogEmissionsspektre i det nær-infrarøde fluorescensvindue (650-950 nm), kan signal-til-støjforholdene forbedres signifikant. Det nær-infrarøde fluorescensvindue er karakteriseret ved en relativt lav absorption af signalet med hæmoglobin og vand såvel som en lav baggrunds-auto-fluorescens. 9 Dette giver mulighed for en indtrængningsdybde på op til 2 cm i små dyrs væv. OI-prober kan adressere et mål direkte ( fx ved et fluorescensmærket antistof) eller kan aktiveres i målvævet ( f.eks. Ved proteaser). Aktiverbare OI-prober er optisk tavse i deres inaktiverede form på grund af Förster-resonansenergioverførslen (FRET) til en slukningsdel, som overfører excitationsenergien inden for molekylet til et andet domæne. Hvis farvestoffet spaltes (f.eks. Ved en protease) overføres energi ikke længere inden i molekylet, og et fluorescerende signal kan detekteres af OI. Dette gør det muligt at designe OI-prober med høj specifikationY for forskellige biologiske processer og fremragende signal-til-støj-forhold. Den følgende protokol forklarer detaljeret fremstillingen af ​​dyrene, OI-målingerne ved anvendelse af en aktiverbar OI-probe til billede MMP-2, -3, -9 og -13 aktivitet in vivo og to eksperimentelle modeller af inflammation (RA, CHR).

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		<tr>
			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
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		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
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			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
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			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
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			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
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		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
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		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
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		<tr>
			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
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			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
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		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
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			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
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		<tr>
			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
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			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
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			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
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			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
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			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
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		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
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		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
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		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
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non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

Dette papir beskriver en ikke-invasiv metode til billeddannelse af matrix-metalloproteinaser (MMP) -aktivitet ved hjælp af en aktiverbar fluorescerende probe via in vivo fluorescensoptisk billeddannelse (OI) i to forskellige musemodeller af inflammation: en reumatoid arthritis (RA) og en kontakt Overfølsomhedsreaktion (CHR) model. Lys med en bølgelængde i det nærliggende infrarøde (NIR) vindue (650-950 nm) muliggør en dybere vævspennetrækning og minimal signalabsorption sammenlignet med bølgelængder under 650 nm. De største fordele ved hjælp af fluorescens OI er, at det er billigt, hurtigt og nemt at implementere i forskellige dyremodeller. Aktiverbare fluorescerende prober er optisk tavse i deres inaktiverede tilstande, men bliver stærkt fluorescerende, når de aktiveres af en protease. Aktiverede MMP&#39;er fører til ødelæggelse af væv og spiller en vigtig rolle for sygdomsprogression i hypersensitivitetsreaktioner med forsinket type (DTHR&#39;er) såsom RA og CHR. Endvidere er MMP&#39;erde centrale proteaser til brusk- og knoglenedbrydning og fremkaldes af makrofager, fibroblaster og chondrocytter i afhængighed proinflammatoriske cytokiner. Her bruger vi en sonde, der aktiveres af de centrale MMP&#39;er som MMP-2, -3, -9 og -13 og beskrive et billeddannende protokol for nær infrarød fluorescens OI af MMP-aktivitet i RA og kontrolmus 6 dage efter sygdomsinduktion samt som i mus med akut (1x udfordring) og kronisk (5x udfordring) CHR på højre øre i forhold til raske ører.

For at inducere rheumatoid arthritis (RA) hos naive BALB / c-mus, blev dyr injiceret ip med auto-antistoffer (1: 1 fortynding med 1x PBS) mod GPI på dag 0. Den maksimale betændelse (ankel hævelse) i denne GPI-serum induceret RA model er på dag 6 efter injektionen 11. Derfor, 2 nmol den aktiverbare OI farvestof blev fremstillet og injiceret iv i halevenen på mus med arthritis og raske kontroldyr på dag 5. 24 timer efter injektion (dag 6) ble...

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

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

Dette papir forklarer anvendelsen af fluorescerende billeddannelse under anvendelse af et aktiverbart optisk billeddannelse probe at visualisere in vivo-aktiviteten af centrale matrixmetalloproteinaser i to forskellige forsøgsmodeller for inflammation.

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 ...

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Immunology and Infection

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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