Prise en charge de ce travail a été fourni par National Institutes of Health octroi # 108229 R01 HL, l&#39;American Heart Association Scientifique Development Grant # 0830352N, Howard Hughes Medical Institute Award de développement de carrière, Ventures Broadview, Programme de la Communauté européenne septième programme-cadre (FP7/2007-2013 titre de la subvention # 235689 accord), et l&#39;HGM William Schreyer Bourse.

Modèle animal in vivo: Génération de l&#39;athérosclérose aortique expérimentale 1) L&#39;angiographie de base et la dénudation ballon <ol><li> Avant d&#39;obtenir une angiographie de base et la dénudation ballon, un en Nouvelle-Zélande lapin blanc est alimenté un cholestérol élevé (1%) régime alimentaire pendant 1 semaine. Cet animal est utilisé pour la pertinence de translation que 1) les vaisseaux aorto-iliaques chez les lapins sont du même calibre que les artères coronaires humaines (2,5 à 3,5 mm) et 2) les hyperlipidémiques, ballon blessures modèle génère l&#39;athérosclérose enflammées portant similaires cellules inflammatoires (macrophages ) et les molécules (cathepsines) comme dans l&#39;athérosclérose humaine. </li><li> Suite à l&#39;alimentation de cholestérol, l&#39;animal est anesthésié avec du propofol et de kétamine. Une incision d&#39;un pouce ventrale ligne médiane du cou est faite en utilisant en utilisant une taille de 15 bistouri lame. En utilisant des techniques dissection, le muscle sous la aponévrose sur le côté droit de la trachée est exposée. Le muscle est séparé sternocephalicus gauche le long de sa jonction des tissus conjonctifs, et l&#39;artère carotide commune droite est exposée. L&#39;artère est séparé du nerf vague. Boucles de suture proximale et distale sont placés sur l&#39;artère afin de permettre la rétraction et l&#39;occlusion. A 1 à 2mm biseauté artériotomie est faite à travers lequel un 5 en français (diamètre extérieur 1.67mm) gaine vasculaire est inséré et l&#39;héparine (1000μ/mL, ~ 150units/kg) est administré par voie intra-artérielle de la gaine. </li><li> Colorant de contraste (Ultravist) est ensuite injecté (1 à 2 ml) sur une période de 2 secondes pour obtenir une angiographie de contrôle de l&#39;aorte distale et les deux artères iliaques. </li><li> Les artères ilio et l&#39;aorte sont alors blessés par une dénudation endothéliale. En utilisant des méthodes standard de radioscopie, une sonde de Fogarty 3FR embolectomie est placé dans l&#39;artère ilio-fémorale distale et gonflé avec 0,3 à 0,5 cc de produit de contraste (50% contrast/50% saline) ou de l&#39;air. Le cathéter est ensuite retiré proximalement dans son état gonflé une distance le long de l&#39;artère iliaque droite et l&#39;aorte distale jusqu&#39;à l&#39;envol de l&#39;artère rénale gauche. Suite à la dénudation ballon, l&#39;angiographie est répété à documenter la perméabilité des vaisseaux. Suite à l&#39;angiographie, tous les cathéters et les gaines sont retirés et la portion proximale artère carotide commune droite est ligaturé, le muscle et le fascia sont suturés par un surjet 4 / 0 résorbable, et l&#39;incision cutanée fermée avec un 4 / 0 non résorbable. </li><li> L&#39;animal est alors autorisé à récupérer à l&#39;administration d&#39;une dose d&#39;antibiotiques (céphazoline, 0,5 grammes de messagerie instantanée). Médicaments contre la douleur, y compris 0,01 IM mg / kg de buprénorphine (deux fois par jour selon les besoins). Les animaux sont ensuite poursuivi sur le cholestérol de 1% pour 4 semaines post-ballon dénudation. À la semaine 5, les animaux sont passés à régime riche en cholestérol de 0,3%. </li></ol> Multimodal intégré d&#39;imagerie du lapin athéromes 2) L&#39;étiquetage des plaques enflammées protéolytique actif à l&#39;aide nanocapteur injectables; angiographie, échographie intravasculaire (IVUS), et l&#39;imagerie in vivo intravasculaire NIRF de Lapin Athérome <ol><li> Huit semaines après la blessure du ballon et 24 heures avant l&#39;imagerie, le lapin est injecté par voie intraveineuse de 500 nmol / kg Prosense/VM110 (PerkinElmer) via veine de l&#39;oreille. </li><li> Vingt-quatre heures après l&#39;injection, les animaux sont anesthésiés et l&#39;accès artériel est obtenue via l&#39;artère carotide commune gauche (voir étape 1.2). Intra-artérielle héparine est administrée (150 unités / kg). Base angiographie est obtenu comme ci-dessus. </li><li> Un cathéter IVUS est chargé sur une artère coronarienne cliniques capables 0,014 pouces de fil et inséré dans la gaine. Utiliser guidage fluoroscopique, le bout du fil radio-opaque est positionné distalement dans l&#39;artère iliaque droite. Le cathéter IVUS est alors avancé dans l&#39;artère iliaque proximale en utilisant une norme technique clinique monorail. </li><li> Un recul de 100 mm est lancé et les images sont enregistrées. La reconstruction longitudinale du navire est obtenue et la plaque luminale est identifié. </li><li> Le cathéter NIRF 11,12 est chargé sur le fil 0,014 pouces (monorail), et le cathéter est soigneusement inséré dans la gaine et la tête d&#39;imagerie est positionné distalement dans l&#39;artère iliaque droite. </li><li> Multiples retraits automatiques (1 mm / sec retrait longitudinal, 30 coups par minute) sont réalisées et les signaux de fluorescence dans les zones de l&#39;athérosclérose sont notées. Les images sont enregistrées et le traitement ultérieur avec mise à l&#39;échelle appropriée et de fenêtrage basé sur l&#39;étendue du signal est réalisée. </li></ol> 3) L&#39;euthanasie et l&#39;isolement des ex vivo aorto-iliaque tissus <ol><li> L&#39;euthanasie est accompli avec 1cc d&#39;agent euthanasie (solution de sodium phénytoïne sodique 390mg pentobarbital et 50mg), intraveineuse, seule injection. </li><li> L&#39;arbre artériel est perfusé avec une solution saline normale à 0,9% jusqu&#39;à la veine cave inférieure est clair de sang. Les artères iliaques athérosclérose aortique et sont identifiés et disséqués par les tissus environnants. En outre, les petits morceaux de 2 x 2 cm de liVer, du rein, la rate et le coeur sont également obtenus. </li><li> Ex vivo NIRF imagerie avec le cathéter intravasculaire NIRF imagerie peut être fait à ce stade. Le navire est allongé et le cathéter est NIRF ré-inséré dans l&#39;aorte proximale jusqu&#39;à la tête d&#39;imagerie est positionné à l&#39;artère iliaque droite ou de bifurcation. Multiples retraits automatiques sont effectués comme ci-dessus (voir 2.6). </li></ol> 4) ex vivo par fluorescence Réflectance Imaging (FRI) de l&#39;aorte et des artères iliaques disséqué <ol><li> Tissus disséqués est placé dans 10-20 cc de solution saline normale et transportés pour analyse VEN (Kodak Image Station 4000mm Pro, Carestream Health, Inc.) </li><li> Aorte vaisseaux iliaques, sont allongés à rapprocher en temps réel les longueurs et les images sont obtenues à multiples longueurs d&#39;onde [lumière blanche, vert fluo canaux (ex 495 nm, em 515 nm), Cy5 (ex 565 nm, em 670 nm) et Cy7 (ex 650 nm, em 760 nm)] canaux. Une série de temps d&#39;exposition sont utilisés pour chaque longueur d&#39;onde (0,1-30sec) et les images acquises sont exportés sous forme de DICOM ou 16-bit unscaled fichiers TIFF pour des analyses ultérieures. Comme les contrôles positifs et négatifs, les organes (foie, rate, des reins et du cœur) sont imagées à des canaux similaires et temps d&#39;exposition. </li><li> Domaines d&#39;augmentation du signal dans le canal proche infrarouge (780nm +) sont notées dans les artères athérosclérotiques. </li></ol> Enrobage des tissus 5) pour l&#39;analyse de sectionnement et immunohistochimique <ol><li> Domaines de la normale (non blessés tissus, à savoir l&#39;artère iliaque gauche) et les zones de la plaque sont identifiés et petits anneaux 5-10 mm de tissu sont intégrés dans de l&#39;OCT (Température de coupe optimale) des médias. Les blocs sont stockés à -80 C jusqu&#39;à la coupe. </li><li> Les techniques standard pour les analyses de sectionnement et immunohistochimiques sont effectuées. Hématoxyline et éosine, Ram-11 et de la cathepsine B coloration sont effectuées. </li></ol> Analyses et l&#39;intégration des images multi-modales (angiographie, échographie intravasculaire, NIRF et ven.) 6) Le traitement des images et des NIRF ven. <ol><li> Fichiers contenant des données d&#39;imagerie DICOM à partir NIRF et ven. (prise au proche infrarouge 780 nm de canal) pullbacks sont traitées en utilisant MATLAB et logiciels Osirix, respectivement. Fenêtrage approprié à afficher gamme complète de l&#39;intensité du signal est réalisée. Images finales sont exportés sous forme de fichiers TIFF. </li><li> Les fichiers sont importés dans le logiciel de traitement d&#39;image standard (Keynote peut être utilisé). Les images sont alignées en fonction des points de référence (c&#39;est à dire sur les vertèbres angiographie, bifurcation iliaque et l&#39;artère rénale). Domaines d&#39;vaisseau normal et la plaque sont identifiés. </li><li> Régions d&#39;intérêt (ROI) sont tracées manuellement (pour les tissus normaux et les zones de la plaque) et la moyenne des intensités du signal sont acquis à l&#39;aide Osirix et MATLAB, respectivement pour les deux ven. et images NIRF. Pour guider le traçage appropriés, l&#39;image IVUS longitudinale du navire est utilisé et l&#39;identification des navires et la plaque normale sont facilement identifiés. </li><li> Cible à fond (TBR) ratios sont calculés pour les zones de la plaque. </li></ol> Les résultats représentatifs: À la fin du protocole ci-dessus, nous pouvons identifier et caractériser les zones d&#39;activité augmentée protéase cathepsine inflammatoires dans la plaque dans l&#39;aorte et les vaisseaux iliaques. Injection d&#39;une nanocapteur activable (Prosense/VM110) nous permet d&#39;identifier la plaque protéolytique actif. Celles-ci apparaissent aussi lumineux ou un signal intense lorsque les zones imagées par MRF dans le canal proche infrarouge (750 nm). Les retraits NIRF en corrélation avec l&#39;intensité du signal a augmenté de FRI et alignements avec IVUS qui permettent d&#39;inscription anatomiques des signaux NIRF. TBR plaque Calculé obtenue à partir FRI et NIRF étaient similaires (voir Figure 3: moyenne NIRF TBR 4.2, signifie ven. TBR 2,9). L&#39;analyse immunohistochimique de la plaque lumineuse confirme la présence intense de RAM-11 et de la cathepsine B dans les domaines d&#39;activité de la plaque (données non présentées). <img alt="Figure 1" src="/files/ftp_upload/2257/2257fig1.jpg" /> Figure 1. Schéma de la 2D NIRF cathéter pour élargir le potentiel clinique d&#39;une approche 1D NIRF détection 6, nous avons construit un roman en 2-D NIRF-cathéter pour l&#39;imagerie intravasculaire. 11,12 Le cathéter sur mesure se compose d&#39;une fibre optique (125 microns de diamètre logé dans les tuyaux en polyéthylène: 2.9F) qui éclaire en utilisant une source laser à 750 nm d&#39;excitation. La lumière laser est émis à un angle de 90 degrés par rapport à l&#39;axe de la fibre. Le système utilise deux moteurs automatisés (rotation et de translation) pour permettre à l&#39;imagerie simultanée de 360 ​​degrés et pullback longitudinale pour obtenir vraie imagerie 2D. Les images utilisées avec la permission de la référence 11. <img alt="Figure 2" src="/files/ftp_upload/2257/2257fig2.jpg" /> Figure 2. Schéma montrant la protéase médiée par l&#39;activation de la nanocapteurs, Prosense/VM110. Image utilisée avec la permission de la référence 10. <img alt="Tableau 1" src = &quot;/ files/ftp_upload/2257/2257table1.jpg&quot; /&gt; Figure 3. In vivo et in vivo RBT ex plaque (la cible à des ratios de fond)

<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 - Ancien consultant, VISEN médicale; honoraires, Boston Scientific

Enflammé plaques à haut risque ou vulnérables sont probablement responsables de la majorité des infarctus du myocarde. L&#39;identification de ces plaques avant l&#39;apparition des symptômes a d&#39;importantes implications cliniques à la fois dans la prédiction des résultats et de guider le traitement médical. Coronaire conventionnelle modalités d&#39;imagerie artérielle tels que x-ray angiographie mettent généralement l&#39;accent sur la caractérisation des rétrécissements luminale plutôt éclairant ...

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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

Nous détaillons un nouveau proche infrarouge fluorescence (NIRF) cathéter pour deux dimensions d&#39;imagerie moléculaire de la biologie de plaque intravasculaire<em&gt; In vivo</em&gt;. Le cathéter NIRF pouvez visualiser les principaux processus biologiques tels que l&#39;inflammation par des rapports sur la présence de la plaque-avides fluorochromes NIR activable et ciblée. Le cathéter utilise l&#39;ingénierie des exigences cliniques et de la puissance et est ciblé pour des applications dans les artères coronaires humaines. L&#39;étude qui suit décrit une stratégie d&#39;imagerie multimodale qui utilise un roman<em&gt; In vivo</em&gt; Intravasculaire NIRF cathéter à l&#39;image et de quantifier la plaque inflammatoire dans athéromes lapin protéolytique actif enflammée.

In vivo par fluorescence dans le proche infrarouge (NIRF) intravasculaire d&#39;imagerie moléculaire de la plaque inflammatoire, une approche multimodale d&#39;imagerie de l&#39;athérosclérose

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

17.8K vues.  Harvard Medical School. Nous détaillons un nouveau proche infrarouge fluorescence (NIRF) cathéter pour deux dimensions d'imagerie moléculaire de la biologie de plaque intravasculaire In vivo. Le cathéter NIRF pouvez visualiser les principaux processus biologiques tels que l'inflammation par des rapports sur la présence de la plaque-avides fluorochromes NIR activable et ciblée. Le cathéter utilise l'ingénierie des exigences cliniques et de la puissance et est ci...

Vidéo: 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 double Substrat 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 ...

Recherche

Médecine

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.

Implantation d&#39;une manchette carotidienne pour le déclenchement de cisaillement-stress athérosclérose induite chez la souris

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.

L&#39;imagerie moléculaire à la cible transplanté des cellules progénitrices musculaires

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.

Dans l&#39;imagerie in vivo de angiogenèse tumorale en utilisant la fluorescence confocale vidéomicroscopie

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 humain<em&gt; Ex Vivo</em&gt; Plaque athéroscléreuse modèle pour étudier la biologie de la lésion

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.

Phantoms simulant un tissu pour l&#39;évaluation du potentiel proche infrarouge Applications d&#39;imagerie de fluorescence en chirurgie du cancer du sein

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

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.

Murin endoscopie pour<em&gt; In Vivo</em&gt; Guérison multimodale d&#39;imagerie de la cancérogenèse et de l&#39;évaluation de la plaie intestinale et l&#39;inflammation

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.

Validation des Nanobodies et Anticorps Based<em&gt; In Vivo</em&gt; Expériences tumeur xénogreffe NIRF-imagerie dans des souris en utilisant<em&gt; Ex Vivo</em&gt; Cytométrie en flux et microscopie

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.

Combiné proche infrarouge imagerie fluorescente et la tomographie par Micro-calculé pour Directement Visualizing cérébrale thromboembolies

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

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induction de l’athérosclérose accélérée chez les souris : le modèle « Wire-Injury »

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...