Il supporto per questo lavoro è stato fornito dal National Institutes of Health concedere R01 # 108229 HL, American Heart Association Scientist Development Grant # 0830352N, Howard Hughes Medical Institute Career Development Award, Ventures Broadview, Settimo programma quadro della Comunità europea (FP7/2007-2013 in borsa accordo # 235689), e il MGH William Schreyer Fellowship.

Nel modello animale in vivo: Generazione di aterosclerosi aortoiliaco Sperimentale 1) L&#39;angiografia basale e denudazione Balloon <ol><li> Prima di chiedere al basale angiografia e denudazione palloncino, un coniglio bianco neozelandese è nutriti con una colesterolo alto (1%) la dieta per 1 settimana. Questo animale è utilizzato per rilevanza traslazionale come 1) la aorto-iliache navi conigli sono dello stesso calibro come umano arterie coronarie (2,5-3.5mm) e 2) l&#39;, iperlipidemici pallone-infortunio modello genera aterosclerosi infiammato cuscinetto simili cellule infiammatorie (macrofagi ) e molecole (catepsine) come l&#39;aterosclerosi umana. </li><li> A seguito di alimentazione colesterolo, l&#39;animale viene anestetizzato con propofol e ketamina. Un one-inch linea mediana ventrale incisione del collo è realizzato con con una dimensione di 15-bisturi lama. Utilizzando tecniche di dissezione smussa, i muscoli sotto la fascia sul lato destro della trachea è esposto. Il muscolo sternocephalicus sinistra è divisa lungo il raccordo del tessuto connettivo, e il diritto arteria carotide comune è esposto. L&#39;arteria è separato dal nervo vago. Anelli di sutura prossimale e distale sono immessi sul arteria per consentire retrazione e occlusione. A 1 a 2mm smussati arteriotomia è fatto attraverso il quale un 5 Francese (diametro esterno 1,67 millimetri) guaina vascolare è inserita ed eparina (1000μ/mL, ~ 150units/kg) viene somministrato intra-arteriosa attraverso la guaina. </li><li> Mezzo di contrasto (Ultravist) viene quindi iniettato (da 1 a 2 ml) per un periodo di 2 secondi per ottenere un angiogramma controllo dell&#39;aorta distale ed entrambe le arterie iliache. </li><li> Le arterie e aorta ileofemorale vengono feriti da denudazione endoteliale. Utilizzando metodi di fluoroscopia standard, un catetere 3FR embolectomia di Fogarty è posizionato in arteria distale ileofemorale e gonfiato con 0,3-0,5 cc di contrasto (50% contrast/50% salina) o aria. Il catetere viene poi ritirato prossimalmente nel suo stato gonfiato a distanza lungo l&#39;iliaca destra e l&#39;aorta distale fino al decollo della arteria renale sinistra. Dopo la denudazione pallone, l&#39;angiografia è ripetuto per documentare pervietà del vaso. A seguito di angiografia, guaine e tutti i cateteri vengono rimossi e il prossimale dell&#39;arteria carotide comune è la legatura, il muscolo e fascia sono suturate con una sutura 4 / 0 riassorbibile, e l&#39;incisione cutanea è chiuso con un 4 / 0 non riassorbibile sutura. </li><li> L&#39;animale è poi permesso di recuperare con la somministrazione di una dose di antibiotico (cefazolina, 0,5 grammi IM). Farmaci per il dolore tra 0,01 mg / kg IM buprenorfina (due volte al giorno se necessario). Gli animali sono poi proseguito l&#39;1% del colesterolo per 4 settimane post-palloncino denudazione. Alla 5 ° settimana, gli animali sono passati ad una dieta colesterolo 0,3%. </li></ol> Integrato multi-modale Imaging di Coniglio Atheromata 2) Etichettatura di proteoliticamente attivo placca infiammata con nanosensori iniettabili, angiografia, ecografia intravascolare (IVUS), e in vivo intravascolare NIRF immagini di Coniglio Ateroma <ol><li> Otto settimane dopo un trauma palloncino e 24 ore prima di imaging, il coniglio viene iniettato per via endovenosa con 500 nmol / kg Prosense/VM110 (PerkinElmer) tramite vena orecchio. </li><li> Ventiquattro ore dopo l&#39;iniezione, gli animali sono anestetizzati e l&#39;accesso arterioso è ottenuto tramite sinistra arteria carotide comune (vedi punto 1.2). Intra-arteriosa eparina viene somministrato (150 unità / kg). Baseline angiografia è ottenuto come sopra. </li><li> Un catetere IVUS è caricato su una clinica in grado coronarica 0,014 filo pollici e inserito nel fodero. Utilizzando guida fluoroscopica, la punta radiopaca del filo è posizionato distalmente in arteria iliaca destra. Il catetere IVUS è poi avanzato in arteria iliaca prossimale utilizzando una tecnica standard clinico monorotaia. </li><li> A 100 pullback mm è iniziato e le immagini vengono registrate. Ricostruzione longitudinale della nave si ottiene e placca luminale è identificato. </li><li> Il catetere NIRF 11,12 viene caricato sul filo di 0,014 pollici (monorotaia sistema) e il catetere viene accuratamente inserito nella guaina e la testa di imaging è posizionato distalmente in arteria iliaca destra. </li><li> Multipli pullbacks automatizzati (1 mm / sec pullback longitudinale, 30 colpi al minuto) sono eseguite e segnali di fluorescenza all&#39;interno delle zone di aterosclerosi sono noti. Le immagini vengono registrate e il successivo trattamento con scala appropriata e finestre a seconda della distanza del segnale viene raggiunto. </li></ol> 3) L&#39;eutanasia e l&#39;isolamento di ex vivo aorto-iliaca del tessuto <ol><li> L&#39;eutanasia è compiuta con 1cc di agente eutanasia (soluzione di 390mg di sodio fenitoina sodio pentobarbital e 50 mg), per via endovenosa, iniezione singola. </li><li> L&#39;albero arterioso è perfuso con soluzione fisiologica 0,9% fino alla vena cava inferiore è chiaro di sangue. Aterosclerotica delle arterie aorta e iliache sono identificati e sezionati libero dai tessuti circostanti. Inoltre, piccoli pezzi di 2 x 2 cm di liver, rene, milza e cuore sono anche ottenuti. </li><li> Ex vivo NIRF imaging con il catetere intravascolare NIRF immagini può essere fatto in questa fase. La nave è allungata e il catetere NIRF è reinserito in aorta prossimale fino a quando la testa di imaging è posizionato a livello dell&#39;arteria iliaca destra o di biforcazione. Multipli pullbacks automatici vengono eseguiti come sopra (vedi 2.6). </li></ol> 4) ex vivo fluorescenza Riflettanza Imaging (FRI) di aorta e le arterie iliache sezionato <ol><li> Tessuto sezionato viene inserito in 10-20 cc di soluzione fisiologica e trasportati per le analisi ven (Kodak Image Stazione 4000MM Pro, Carestream Health, Inc.). </li><li> Aorta, vasi iliaci sono allungate per approssimare in tempo reale lunghezze e le immagini sono ottenute a più lunghezze d&#39;onda [luce bianca, canale verde fluorescente (ex 495 nm, em 515 nm), Cy5 (ex 565 nm, em 670 nm) e Cy7 (ex 650 nm, em 760 nm)] canali. Una serie di tempi di esposizione sono utilizzati per ogni lunghezza d&#39;onda (0.1-30sec) e le immagini acquisite vengono esportati come DICOM o 16-bit TIFF i file non in scala per ulteriori analisi. Come controlli positivi e negativi, organi (fegato, milza, rene e cuore) sono esposte a canali simili e tempi di esposizione. </li><li> Aree di segnale aumentato nel vicino infrarosso canale (780nm +) sono noti aterosclerotiche nelle arterie. </li></ol> 5) Integrazione del tessuto per l&#39;analisi Sezioni e immunoistochimica <ol><li> Aree di normale (non feriti tessuto, cioè l&#39;arteria iliaca sinistra) e le aree di placca sono identificati e piccoli anelli di 5-10 mm di tessuto sono incorporati in ottobre (temperatura di taglio ottimale) dei media. I blocchi sono conservati a -80 C fino al sezionamento. </li><li> Le tecniche standard per le analisi immunoistochimiche e di sezionamento vengono eseguite. Ematossilina ed eosina, Ram-11 e catepsina B colorazione vengono eseguite. </li></ol> Analisi e integrazione di immagini multimodali (angiografia, IVUS, NIRF e ven.) 6) Elaborazione di immagini NIRF e VEN <ol><li> File DICOM contenenti dati di immagini da NIRF e VEN (prese al vicino canale dell&#39;infrarosso da 780 nm) pullback sono trattati con MATLAB e software OsiriX, rispettivamente. Windowing corretto per visualizzare gamma completa di intensità del segnale è ottenuto. Immagini finali sono esportati come file TIFF. </li><li> I file vengono importati nel software standard di elaborazione delle immagini (Keynote può essere utilizzato). Le immagini sono allineati sulla base di punti di riferimento (cioè vertebre su angiogramma, biforcazione iliaca, e dell&#39;arteria renale). Aree di nave normale e placca sono identificati. </li><li> Regioni di interesse (ROI) sono tracciate manualmente (per il tessuto normale e aree di placca) e media intensità di segnale sono acquisite con OsiriX e MATLAB, rispettivamente, sia per ven e immagini NIRF. Per guida appropriata tracciamento, l&#39;immagine IVUS longitudinale della nave viene utilizzato e l&#39;identificazione della nave normale e targa sono facilmente identificabili. </li><li> Obiettivo da sfondo (TBR) rapporti sono calcolati per le zone della placca. </li></ol> Rappresentante dei risultati: Al termine del protocollo di cui sopra, siamo in grado di identificare e caratterizzare le aree di attività aumentata della proteasi catepsina nella placca infiammatoria all&#39;interno della aorta e vasi iliaci. L&#39;iniezione di un nanosensore attivabili (Prosense/VM110) ci permette di identificare la placca proteoliticamente attivo. Questi appaiono come segnali luminosi o zone di intensa quando ripreso utilizzando ven nel canale vicino infrarosso (750 nm). Il pullback NIRF correlazione con l&#39;intensità del segnale è aumentato del venerdì e allineamenti con IVUS anatomiche che permettono di registrazione dei segnali NIRF. TBR targa calcolato è ottenuto da venerdì e NIRF erano simili (vedi Figura 3: media NIRF TBR 4.2, significa ven TBR 2,9). Analisi immunoistochimica della placca luminosa conferma la presenza intensa di RAM-11 e l&#39;attività della catepsina B in aree di placca (dati non riportati). <img alt="Figura 1" src="/files/ftp_upload/2257/2257fig1.jpg" /> Figura 1. Schematica del catetere 2D NIRF Per estendere il potenziale clinico di un approccio 1D NIRF rilevamento 6, abbiamo costruito un romanzo in 2-D NIRF-catetere per l&#39;imaging intravascolare. 11,12 La fuoriserie catetere è costituito da una fibra ottica (125 micron di diametro alloggiati in tubo di polietilene: 2.9F), che illumina con una sorgente laser a 750 nm di eccitazione. La luce laser è emessa ad un angolo di 90 gradi rispetto all&#39;asse della fibra. Il sistema utilizza due motori automatici (rotazionale e traslazionale) per consentire la concomitante immagini a 360 gradi e pullback longitudinale per ottenere vere immagini 2D. Le immagini utilizzate con il permesso di riferimento 11. <img alt="Figura 2" src="/files/ftp_upload/2257/2257fig2.jpg" /> Figura 2. Schematico dimostrando proteasi-mediata attivazione dei nanosensori, Prosense/VM110. Immagine usata con il permesso di riferimento 10. <img alt="Tabella 1" src = &quot;/ files/ftp_upload/2257/2257table1.jpg&quot; /&gt; Figura 3. In vivo ed ex TBRs placca in vivo (target a rapporti di sfondo)

<ol>
	<li>Andersson, J., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+immunity+and+atherosclerosis.">Adaptive immunity and atherosclerosis.</a> Clin Immunol. 134, 33-46 (2010).</li><li>Calfon, M. A., Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+near-infrared+fluorescence+molecular+imaging+of+atherosclerosis:+toward+coronary+arterial+visualization+of+biologically+high-risk+plaques.">Intravascular near-infrared fluorescence molecular imaging of atherosclerosis: toward coronary arterial visualization of biologically high-risk plaques.</a> Journal of Biomedical Optics. 15, 011107-011107 (2010).</li><li>Chen, J., Tung, C. -. H. <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+Proteolytic+Activity+in+Atherosclerosis.">In Vivo Imaging of Proteolytic Activity in Atherosclerosis.</a> Circulation. 105, 2766-2771 (2002).</li><li>Jaffer, F. A., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Cardiovascular+Disease.">Molecular Imaging of Cardiovascular Disease.</a> Circulation. 116, 1052-1061 (2007).</li><li>Jaffer, F. A., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+Multimodality+Molecular+Imaging:+Insights+Into+Atherosclerosis.">Optical and Multimodality Molecular Imaging: Insights Into Atherosclerosis.</a> Arterioscler Thromb Vasc Biol. 29, 1017-1024 (2009).</li><li>Jaffer, F. A., Vinegoni, C. <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+Catheter+Molecular+Sensing+of+Inflammation+in+Proteolytically+Active+Atherosclerosis.">Real-Time Catheter Molecular Sensing of Inflammation in Proteolytically Active Atherosclerosis.</a> Circulation. 118, 1802-1809 (2008).</li><li>Kim, D. -. E., Kim, J. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease+Imaging+of+Human+Atheromata+Captures+Molecular+Information+of+Atherosclerosis,+Complementing+Anatomic+Imaging.">Protease Imaging of Human Atheromata Captures Molecular Information of Atherosclerosis, Complementing Anatomic Imaging.</a> Arterioscler Thromb Vasc Biol. 30, 449-456 (2010).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420, 868-874 (2002).</li><li>Naghavi, M., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Vulnerable+Plaque+to+Vulnerable+Patient:+A+Call+for+New+Definitions+and+Risk+Assessment+Strategies:+Part+I.">From Vulnerable Plaque to Vulnerable Patient: A Call for New Definitions and Risk Assessment Strategies: Part I.</a> Circulation. 108, 1664-1672 (2003).</li><li>Weissleder, R., Tung, C. -. H. <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 Biotech. 17, 375-375 (1999).</li><li>Razansky, R. N., Rosenthal, A. <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+fluorescence+catheter+system+for+two-dimensional+intravascular+imaging+in+vivo.">Near-infrared fluorescence catheter system for two-dimensional intravascular imaging in vivo.</a> Optics Express. 18, 11372-11381 (2010).</li><li>Jaffer, F. A., Calfon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Dimensional+Intravascular+Near-Infrared+Fluorescence+Molecular+Imaging+of+Inflammation+in+Atherosclerosis+and+Stent-Induced+Vascular+Injury.">Two-Dimensional Intravascular Near-Infrared Fluorescence Molecular Imaging of Inflammation in Atherosclerosis and Stent-Induced Vascular Injury.</a> Journal of the American College of Cardiology. 57, 2516-2526 (2011).</li></ol>

FAJ - Consulente Ex, VisEn medica; onorari, Boston Scientific

Infiammata placche ad alto rischio o vulnerabili sono probabilmente responsabili della maggior parte degli infarti del miocardio. L&#39;identificazione delle targhe precedenti l&#39;inizio dei sintomi ha importanti implicazioni cliniche, sia nel predire gli esiti e guidare la terapia medica. Tradizionali modalità di imaging delle arterie coronariche, come l&#39;angiografia a raggi X in genere si concentrano sulla caratterizzazione dei restringimenti del lume, piuttosto che illumina i profili biologici sottostanti ad al...

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<td>Prosense 750</td>
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in vivo near infrared fluorescence (nirf) intravascular molecular imaging of inflammatory plaque, a multimodal approach to imaging of atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

Watch this Scientific Journal Video about In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis at JoVE.co

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

Noi dettaglio un nuovo fluorescenza nel vicino infrarosso (NIRF) catetere per 2-dimensionale di imaging intravascolare molecolare della biologia della placca<em&gt; In vivo</em&gt;. Il catetere NIRF può visualizzare fondamentali processi biologici come l&#39;infiammazione segnalazione sulla presenza di fluorocromi attivabili e mirata placca avido NIR. Il catetere utilizza clinica requisiti di progettazione e di potere, ed è mirato per applicazioni in arterie coronarie umane. Lo studio di ricerca che segue descrive una strategia di imaging multimodale che utilizza un romanzo<em&gt; In vivo</em&gt; NIRF catetere intravascolare di immagine e quantificare placca infiammatoria nel proteoliticamente attivo atheromata coniglio infiammata.

In vivo Near Infrared fluorescenza (NIRF) intravascolare Imaging Molecolare della placca infiammatoria, un approccio multimodale per Imaging di aterosclerosi

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

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In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

17.9K Views.  Harvard Medical School. Noi dettaglio un nuovo fluorescenza nel vicino infrarosso (NIRF) catetere per 2-dimensionale di imaging intravascolare molecolare della biologia della placca In vivo. Il catetere NIRF può visualizzare fondamentali processi biologici come l'infiammazione segnalazione sulla presenza di fluorocromi attivabili e mirata placca avido NIR. Il catetere utilizza clinica requisiti di progettazione e di potere, ed è mirato per applicazioni in arterie coronarie u...

Video: In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo doppio substrato Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

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Medicina

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Impianto di un bracciale carotidea per innescare Shear-stress aterosclerosi indotta in topi

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

In vivo imaging dell&#39;angiogenesi tumorale mediante fluorescenza confocale Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Un umano<em&gt; Ex Vivo</em&gt; Placca aterosclerotica modello per studiare Lesione Biologia

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Tissue-simulando Phantoms per valutare il potenziale vicino infrarosso applicazioni di imaging di fluorescenza nella chirurgia del cancro al seno

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Cerenkov Luminescenza Imaging di interscapolare Brown Tessuto adiposo

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Murino Endoscopia per<em&gt; In Vivo</em&gt; Guarigione Imaging Multimodale di Cancerogenesi e valutazione della ferita intestinale e infiammazione

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validazione di Nanobody e anticorpi Based<em&gt; In Vivo</em&gt; Esperimenti tumore xenotrapianto NIRF imaging nei topi mediante<em&gt; Ex Vivo</em&gt; Citometria di flusso e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Imaging fluorescente combinata vicino infrarosso e la tomografia micro-computerizzata per Direttamente Visualizzazione cerebrale tromboembolie

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...