Ringraziamo Daniel Bukala, Natalie Altmeyer e Funda Cay per un ottimo supporto tecnico. Ringraziamo Jonathan Cotton, Greg Bowden e Paul Soubiran per aver redatto il manoscritto. Questo lavoro è stato sostenuto dalla Fondazione Werner Siemens e dalla Facoltà di Medicina dell&#39;Università Eberhard Karls Tübingen (&#39;&#39; Promotionskolleg &#39;&#39;) e dal DFG attraverso la CRC 156 (progetto C3).

Tutte le procedure descritte in questo documento, hanno seguito le linee guida e standard internazionali di cura e l&#39;uso di animali da laboratorio e sono stati approvati dal Comitato locale il benessere degli animali ed etica del Paese Commissione Tuebingen, in Germania. 8 - topi 12 settimane BALB / ce C57BL / 6 sono stati tenuti al 12 h: 12 h di luce: buio ciclo e sono stati alloggiati in IVCS e condizioni ambientali standard a 22 ± 1 ° C in gruppi di 2 - 5 con acqua e alimento accesso ad libitum. 1. Preparazione Materiale <ol><li> Diluire il colorante OI per vicino infrarosso fluorescenza secondo il rispettivo foglio dati direttamente prima dell&#39;iniezione. Il colorante OI attivabile (sonda commerciale con eccitazione a 680 nm) per misurare MMP in vivo è pronto per l&#39;uso ad una concentrazione di 20 nmol in 1,5 ml di PBS 1x. Agitare delicatamente o vortice la soluzione prima dell&#39;uso. colorante OI può essere conservato a 2-8 ° C per un massimo di 6 mesi se protetto dalla luce. </li&gt; <li> Per via endovenosa (iv) iniezioni preparare un catetere venoso. Utilizzare un 20 U (0,5 mL) di insulina siringa riempita con una soluzione salina allo 0,9% (contenente 10 unità di iniezione di eparina in 50 mL), e un ago da 30 attaccato ad un catetere di polietilene. Iniettare la dose raccomandata di 2 nmol per il mouse del colorante OI. </li></ol> 2. Induzione di artrite reumatoide e cronica reazione di ipersensibilità di contatto <ol><li> Artrite reumatoide: <ol><li> Diluire 100 ml di siero (ottenuto da K / BXN topi 10) anticorpi contenenti (AB) contro glucosio-6-fosfato isomerasi (GPI) 1: 1 con PBS 1x (200 mL) per indurre RA. Conservare il siero diluito a -80 ° C. Procedure dettagliate per l&#39;induzione di RA sono descritti da Monach et al. 8. </li><li> Per indurre RA, sollevare ciascun topo delicatamente per la coda e iniettare 200 microlitri di siero diluito intraperitoneale (ip) al giorno 0 di tLui sperimenta. Dopo l&#39;iniezione, posizionare il mouse direttamente nella sua gabbia. </li><li> Facoltativamente, misurare i diametri della caviglia di ciascuna caviglia, prima delle iniezioni AB e definire un &quot;punteggio arthritico&quot; ( Figura 1A ). Continuare la misurazione del gonfiore alla caviglia quotidianamente fino al giorno 6 dopo il siero GPI utilizzando un dispositivo di misura meccanico (micrometro). </li><li> Iniettare il colorante OI attivo (punto 1.1 - 1.2), per misurare l&#39;attività MMP in vivo IV nella vena di coda di topi RA Il giorno 5 dopo iniezione di siero GPI e nella vena di coda dei topi di controllo. Effettuare esperimenti di imaging ottico 24 h dopo l&#39;iniezione della vena della coda. </li></ol></li><li> Reazione di ipersensibilità di contatto: <ol><li> Per la sensibilizzazione dei topi C57BL / 6, preparare una soluzione al 5% TNCB sciolta in una miscela di acetone / olio 4: 1. </li><li> Anestetizzare i topi C57BL / 6 utilizzando l&#39;1,5% di isoflurano vaporizzati in ossigeno al 100% (1,5 L / min). Una volta che i topi sono anestetizzati, metterlo in un automa-pazzo<html> Cono nasale (una siringa da 5 ml di polietilene, collegata al tubo isoflurano da 1,5 vol%) e mantenere l&#39;anestesia. Applicare unguento veterinario sugli animali anestetizzati per prevenire la secchezza degli occhi durante l&#39;anestesia. </li><li> Rasare l&#39;addome con cura (2 x 2 cm) usando un piccolo trimmer di animali. Evitare di danneggiare la pelle, in quanto ciò può portare a segnali fluorescenti non specificati all&#39;interno dell&#39;animale e può influenzare i risultati dello studio. </li><li> Applicare lentamente 80 μL della soluzione di TNCB del 5% all&#39;area addominale rasata usando una pipetta da 100 μL per sensibilizzare l&#39;animale. Riposiziona delicatamente il mouse nella sua gabbia, accertandosi che gli occhi del mouse non vengano a contatto con la biancheria per evitare lesioni corneali e evitare temperature corporee durante la fase di recupero. </li><li> Sei giorni dopo la sensibilizzazione, preparare una soluzione TNCB da 1% (disciolta in una miscela di acetone / olio 9: 1) e applicare 20 μL all&#39;orecchio destro usando una pipetta per provocare CHSR acuta e cronica.&quot;&gt; 1 <ol><li> Iniziare con la prima sfida su entrambi i lati dell&#39;orecchio destro con 20 μL di 1% soluzione TNCB utilizzando una pipetta da 100 μL e ripetere la sfida ogni secondo giorno fino a cinque volte (giorno 15 dopo la sensibilizzazione) per ottenere la CHR ( figura 1B ) . Misurare lo spessore dell&#39;orecchio ogni giorno, utilizzando un dispositivo di misurazione del micrometro. </li></ol></li><li> Iniettare il colorante OI attivo (una sonda commerciale con eccitazione a 680 nm) (fase 1.1-1.2) per misurare l&#39;attività MMP in vivo in topi CHR 12 h dopo la sfida. Eseguire esperimenti di imaging ottico 24 h iniezione venosa della coda della coda ( iv ). </li></ol></li></ol> 3. Preparazione degli animali per l&#39;imaging ottico <ol><li> Spostare il topo del mouse (almeno 3 giorni prima dell&#39;immagine) a bassa oa fluorescenza ( ad esempio , senza manganese) per evitare l&#39;interferenza di auto-fluorescenza (circa 700 nm) con il segnale di fluorescenza degli agenti OI usati. </li><li> Mettere l&#39;animale inad una scatola di anestesia e anestetizzare con 1,5% vol isoflurano vaporizzato con ossigeno (1,5 L / min) (ossigeno può essere sostituita da aria, ma deve essere standardizzato entro un esperimento). </li><li> Quando il mouse è visibilmente anestetizzato, collocarlo in un&#39;ogiva fatto da sé (costruito da un taglio 5 mL polietilene siringa, collegato al tubo% isoflurano 1,5 vol) e mantenere l&#39;anestesia. </li><li> Radere l&#39;animale con cura sul sito di destinazione (2 centimetri x 2 cm) con un piccolo trimmer peli. Evitare ferire la pelle, poiché ciò può portare a segnali fluorescenti aspecifici all&#39;interno dell&#39;animale e può influenzare i risultati dello studio. NOTA: peli può assorbire il segnale di fluorescenza durante OI (dipendente dal ceppo di topi, più o meno si osserva assorbimento) a seconda della regione di interesse (ROI). </li><li> Per l&#39;iniezione endovenosa, posizionare la coda del topo in acqua riscaldata, per indurre vasodilatazione, delicatamente detergere la pelle nel sito di iniezione con alcool, e inizia posizionando il catetereIl sito distale della coda. Posizionare il bordo &quot;tagliato&quot; dell&#39;ago in un angolo di 20 ° nella vena della coda e verificare la corretta collocazione del catetere riattivando la siringa. </li><li> Se il catetere è posizionato correttamente, sostituire la siringa e iniettare la sonda OI (2 nmol). Dopo l&#39;iniezione, sostituire la siringa e iniettare 25 μL di soluzione salina di 0,9% per liberare completamente il volume morto del tubo in polietilene. NOTA: Il tempo di dimezzamento del tessuto del colorante OI usato (una sonda commerciale con eccitazione a 680 nm) per misurare l&#39;attività MMP in vivo è di 72 ore. Per garantire una completa libertà, non è raccomandata una reinserzione del colorante prima di 7 giorni dopo un&#39;iniezione preventiva. </li></ol> 4. Imaging ottico <ol><li> Posizionare un foglio di plastica o carta nera nella scatola nera dello scanner OI al centro del campo visivo (FOV). </li><li> Impostare un protocollo di misurazione e scegliere la lunghezza d&#39;onda destra (eccitazione: 680 ± 10 nm e eMissione: 700 ± 10 nm) e parametri di imaging. NOTA: in alcuni sistemi OI, è predefinito l&#39;impostazione per diversi colori di imaging. </li><li> Per scegliere il protocollo corretto per l&#39;imaging di fluorescenza, aprire il software di imaging (fornito dal produttore) e inizializzare il sistema. La maggior parte delle telecamere CCD deve raffreddarsi alla loro temperatura di funzionamento, e questo può richiedere 10 minuti. Per risultati affidabili, attendere che il sistema sia pronto. </li><li> Osservate che il pannello di controllo di acquisizione del sistema di imaging in vivo si apre e ciascuna coppia di filtri selezionata rappresenterà un&#39;immagine nella sequenza. In questo caso, acquisire un&#39;immagine con le coppie di filtri per la tintura commerciale con una lunghezza d&#39;onda di eccitazione e emissione rispettivamente di 680 ± 10 nm e 700 ± 10 nm e avviare la misurazione (premere &quot;Acquisire sequenza&quot;). Per istruzioni più dettagliate, consultare il manuale del produttore. </li><li> Etichettare le immagini in modo appropriato e salvare le informazioni nella &quot;Modifica immagine&quot;Etichette &quot;, che verrà visualizzata dopo la&quot; sequenza Acquisizione &quot;. </li><li> Prendere una scansione di base di ciascun animale prima dell&#39;iniezione del colorante OI, o utilizzare animali di controllo ingenui per differenziare il segnale di sfondo. </li><li> H dopo l&#39;iniezione della vena della coda del colorante OI, mettere gli animali al centro del FOV, in grado di misurare il segnale più alto nel sistema OI e avviare le misurazioni. NOTA: Importante: l&#39;ipotermia può influenzare sensibilmente la distribuzione degli agenti di imaging. Assicurarsi che la fase sia riscaldata fino a 37 ° C per evitare l&#39;ipotermia dell&#39;animale. L&#39;imaging può essere eseguito simultaneamente con gli animali in gruppi da 1 a 5 topi. Scegliere la dimensione del FOV a seconda del numero di animali da misurare contemporaneamente. È possibile eseguire un&#39;immagine a campo luminoso per verificare se ciascun mouse è visibile. </li></ol> 5. Analisi dei dati NOTA: Esegui un&#39;analisi dei dati utilizzando il seguente software di immagineProtocollo del produttore. <ol><li> Per le misurazioni di RA, utilizzare l&#39;unità calibrata dell&#39;emissione di fotoni come mostrato in Figura 2 , mentre la CHSR cronica è rappresentata come intensità del segnale in percentuale (efficienza). </li><li> Per disegnare manualmente i ROI, utilizzare la piastra utensile. Per l&#39;analisi delle immagini RA usate un cerchio standardizzato, posizionato intorno al segnale più alto in tutte le caviglie e zampe di ogni animale. Per analizzare il CHSR, posizionare i ROI intorno all&#39;orecchio destro e sinistro in base all&#39;immagine del campo luminoso. </li><li> Per misurare le emissioni di fotoni oi valori di intensità del segnale nella specifica ROI disegnata, premere &quot;misura&quot;. Il sistema fornirà valori nel ROI disegnato per analisi statistiche descrittive. </li></ol>

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OI è uno strumento molto utile, veloce e poco costoso per l&#39;imaging molecolare non invasivo in vivo nella ricerca preclinica. Una particolare forza di OI è la capacità di monitorare processi altamente dinamici come risposte infiammatorie. Inoltre, l&#39;OI permette di seguire il corso di una malattia per un lungo periodo di tempo, che va dai giorni alla settimana. L&#39;OI ha diversi vantaggi rispetto ad altre modalità di imaging in vivo , come la tomografi...

Le malattie autoimmuni come l&#39;artrite reumatoide (RA) o la psoriasi vulgaris sono classificate come reazioni di ipersensibilità di tipo ritardato (DTHR). 1 RA è una malattia autoimmune comune caratterizzata da sinovite erosiva e distruzione congiunta. 2 Le articolazioni artrite infiammate dimostrano l&#39;infiltrazione e la proliferazione delle cellule infiammatorie, una maggiore espressione di cellule pro-infiammatorie che portano alla formazione di pannus, alla cartilagine e alle distruzioni dell&#39;osso. 3 , 4 La scissione di molecole di matrice extracellulare, come il collagene mediante matrici metalloproteinasi (MMPs), è essenziale per la conversione e l&#39;angiogenesi dei tessuti e provoca distruzioni del tessuto. 5 , 6 Le reazioni di ipersensibilità di contatto (CHR) sono caratterizzate dall&#39;aggregazione di neutrofili che portano ad uno scoppio ossidativo. 7 Simili a RA, i MMP in CHR sono involved nella conversione dei tessuti, la migrazione cellulare e l&#39;angiogenesi per stabilire infiammazione cronica. Per studiare RA, il glucosio-6-fosfato isomerasi (GPI) iniezione -serum modello di topo è stato utilizzato. 8 siero di topi K / BXN transgeniche contenenti anticorpi contro GPI, è stato iniettato in topi BALB / c naive dopodiché infiammazione reumatica cominciato a svilupparsi entro 24 h con un massimo di edemi declivi il giorno 6 dopo l&#39;iniezione GPI-siero (vedi 1.1). Per analizzare CHR cronica, topi C57BL / 6 sono stati sensibilizzati con trinitrochlorobenzene (TNCB) sull&#39;addome. L&#39;orecchio destro è stato sfidato fino a 5 volte a partire 1 settimana dopo la sensibilizzazione (vedi anche 1.1 e 1.2). Non invasiva piccoli animali OI è una tecnica basata sulle indagini in vivo del fluorescente-, chemiluminescent- e bioluminescenti-segnali, che vengono utilizzati principalmente nella ricerca preclinica. I dati semiquantitativa acquisita dà intuizioni nel MolecMeccanismi Ular negli organi e tessuti sani e modelli animali sperimentali malati, e consente longitudinale follow up misure (ad esempio per valutare profili di risposta terapeutica in vivo). Un grande vantaggio di studi longitudinali è la riduzione del numero di animali, come gli stessi animali possono essere misurati in studi di follow up in diversi punti temporali invece di utilizzare mouse diversi per punto temporale. La risoluzione di OI permette imaging funzionale dettagliata di organi e strutture tissutali più piccole in animali da esperimento. L&#39;uso di specifici filtri eccitazione e di emissione con uno spettro di trasmissione ristretta, una protezione contro luce diffusa da una tenuta di luce &quot;scatola scura&quot; ed un dispositivo charged-coupled sensibili (CCD), che viene raffreddata in molti dispositivi fino a -70 ° C , permette altamente specifico e misurazioni sensibili dei segnali di fluorescenza. Utilizzando agenti fluorescenti con excitation- eGli spettri di emissione nella finestra di fluorescenza ad infrarossi vicino (650 - 950 nm), i rapporti segnale-rumore possono essere migliorati significativamente. La finestra a fluorescenza a infrarossi vicino è caratterizzata da un assorbimento relativamente basso del segnale da parte dell&#39;emoglobina e dell&#39;acqua così come una bassa fluorescenza di fondo. 9 Questo permette una profondità di penetrazione fino a 2 cm nel tessuto dei piccoli animali. Le sonde OI possono indirizzare direttamente un bersaglio ( ad esempio mediante un anticorpo a fluorescenza) oppure possono essere attivate nel tessuto bersaglio (per es. Mediante proteasi). Le sonde OI attuabili sono otticamente silenziose nella loro forma inattivata a causa del trasferimento di energia di risonanza di Förster (FRET) ad una parte di quenching, che trasferisce l&#39;energia di eccitazione all&#39;interno della molecola ad un altro dominio. Se il colorante viene ceduto (ad esempio da una proteasi), l&#39;energia non è più trasferita all&#39;interno della molecola e un segnale fluorescente può essere rilevato da OI. Ciò consente la progettazione di sonde OI ad alta specificitày per processi biologici distinti ed eccellenti segnale-rumore-ratio. Il protocollo che segue illustra in dettaglio la preparazione degli animali, le misurazioni OI utilizzando una sonda OI Activatable all&#39;immagine MMP-2, -3, -9 e -13 attività in vivo e due modelli sperimentali di infiammazione (RA, CHR).

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			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
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			<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>
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			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</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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			<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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			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
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			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
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			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
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			<td>Optical imaging system</td>
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			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</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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			<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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			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
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			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
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			<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

In questo articolo si descrive un metodo non invasivo per l&#39;attività di matrice metalloproteinasi (MMP) di imaging mediante una sonda fluorescente attiva, tramite l&#39; ottica ottica (OI) in fluorescenza in vivo , in due diversi modelli di infiammazione: un artrite reumatoide (RA) e un contatto Modello di reazione di ipersensibilità (CHR). La luce con una lunghezza d&#39;onda nella finestra vicino a infrarossi (NIR) (650 - 950 nm) consente una penetrazione più profonda del tessuto e un assorbimento minimo del segnale rispetto alle lunghezze d&#39;onda inferiori a 650 nm. I principali vantaggi che usano l&#39;OI di fluorescenza è che è conveniente, veloce e facile da implementare in diversi modelli animali. Le sonde fluorescenti attivatabili sono otticamente silenziose nei loro stati inattivati, ma diventano altamente fluorescenti quando attivata da una proteasi. I MMP attivati ​​portano alla distruzione dei tessuti e svolgono un ruolo importante per la progressione della malattia nelle reazioni di ipersensibilità di tipo ritardato (DTHR) come RA e CHR. Inoltre, i MMP sonoLe proteasi chiave per la cartilagine e il degrado dell&#39;osso e sono indotti da macrofagi, fibroblasti e condrociti in risposta a citochine pro-infiammatorie. Qui usiamo una sonda che viene attivata dai MMP chiave come MMP-2, -3, -9 e -13 e descrivono un protocollo di imaging per l&#39;attività di fluorescenza vicino all&#39;infrarosso OI dell&#39;attività MMP in RA e nei topi di controllo 6 giorni dopo l&#39;induzione della malattia Come nei topi con l&#39;acuto (1x challenge) e la cronica (5x challenge) CHR sull&#39;orecchio destro rispetto alle orecchie sane.

Per indurre l&#39;artrite reumatoide (RA) in topi naive BALB / c, gli animali sono stati iniettati ip con auto-anticorpi (1: 1 diluizione con PBS 1x) contro GPI al giorno 0. L&#39;infiammazione massima (gonfiore alle caviglie) in questo GPI-siero indotta modello di RA è il giorno 6 dopo l&#39;iniezione 11. Pertanto, 2 nmol del colorante OI attivabile stata preparata ed iniettata iv nella vena caudale di topi artritici e animali sani di controllo...

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

Questo articolo spiega l&#39;applicazione dell&#39;immagine fluorescente utilizzando una sonda ottica attivabile per la visualizzazione dell&#39;attività in vivo delle metalloproteinasi di matrici chiave in due diversi modelli sperimentali di infiammazione.

Non invasivo<em&gt; In Vivo</em&gt; Fluorescenza ottica di imaging dell&#39;attività MMP infiammatoria utilizzando un agente fluorescente di imaging attivo

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

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Research

JoVE Journal

Immunology and Infection

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

8.7K Views.  Eberhard Karls University of Tübingen.  Questo articolo spiega l'applicazione dell'immagine fluorescente utilizzando una sonda ottica attivabile per la visualizzazione dell'attività in vivo delle metalloproteinasi di matrici chiave in due diversi modelli sperimentali di infiammazione. 

Video: 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

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Ricerca

Immunologia e infezioni

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

L&#39;uso di una trappola ottica per lo Studio delle interazioni ospite-patogeno per Dynamic Imaging Live Cell

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

Imaging bioluminescenza di attività NADPH ossidasi in diversi modelli animali

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

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Non invasiva Imaging di candidosi disseminata nelle larve Zebrafish

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Imaging batterica<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non invasiva di imaging ottico del sistema vascolare linfatico di un mouse

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo metodo di imaging per distinguere infiammazione acuta e cronica

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

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imaging non invasiva della risposta immunitaria innata in un modello di Zebrafish larvale<em&gt; Streptococcus iniae</em&gt; Infezione

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

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

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

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

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino linfociti Etichettatura da<sup&gt; 64</sup&gt; Cu-anticorpo recettore Targeting per<em&gt; In Vivo</em&gt; Cell Traffico da PET / CT

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

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Rilevamento della colonizzazione di Escherichia Coli enteroemorragica in murino Host di sistema Non invasivo In Vivo bioluminescenza

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

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