Apoyo a este trabajo fue proporcionado por los Institutos Nacionales de Salud de subvención R01 HL # 108229, Asociación Americana del Corazón de Desarrollo Científico Subvención # 0830352N, Howard Hughes Medical Career Institute Premio al Desarrollo, empresas Broadview, Séptimo Programa Marco de la Comunidad Europea (FP7/2007-2013 cargo a la subvención acuerdo # 235 689), y el MGH William Schreyer Comunidad.

En el modelo animal in vivo: La generación de aterosclerosis aortoilíaca Experimental 1) Línea de Base angiografía balón y denudación <ol><li> Antes de obtener la angiografía basal balón y denudación, un conejo blanco de Nueva Zelanda se alimenta un colesterol alto (1%), la dieta durante 1 semana. Este animal se utiliza la relevancia de la traducción como: 1) la aorto-ilíacos buques en los conejos son del mismo calibre que las arterias coronarias humanas (2.5-3.5mm) y 2) la hiperlipemia, el globo de la lesión modelo genera inflamación de la aterosclerosis teniendo similares células inflamatorias (macrófagos ) y moléculas (catepsinas) como en la aterosclerosis humana. </li><li> Después de la alimentación de colesterol, el animal se anestesia con propofol y la ketamina. A una pulgada incisión ventral línea media del cuello se realiza mediante el uso de un tamaño de 15 bisturí hoja. Utilizando técnicas de disección roma, los músculos debajo de la fascia en el lado derecho de la tráquea está expuesto. El músculo sternocephalicus izquierda se separa a lo largo de su unión del tejido conectivo, y la arteria carótida común derecha está expuesto. La arteria se separa del nervio vago. Bucles de sutura proximal y distal se colocan en la arteria para permitir la retracción y la oclusión. A 1 a 2 mm biselado arteriotomía se hace a través del cual un 5 franceses (diámetro externo 1.67mm) vaina vascular se inserta y heparina (1000μ/mL, ~ 150units/kg) se administra por vía intraarterial a través de la vaina. </li><li> Medio de contraste (Ultravist) y luego se inyecta (1 a 2 ml) durante un período de dos segundos para obtener una angiografía de control de la aorta distal y ambas arterias ilíacas. </li><li> Las arterias iliofemoral y la aorta son entonces herido por denudación endotelial. Utilizando los métodos estándar de fluoroscopia, un catéter de Fogarty embolectomía 3FR se coloca en la arteria iliofemoral distal y se infla con 0,3 a 0,5 cc de contraste (50% contrast/50% de solución salina) o el aire. El catéter se retira proximal en el estado infla una distancia a lo largo de la ilíaca derecha y la aorta distal hasta el despegue de la arteria renal izquierda. Después de la denudación del globo, se repite la angiografía para documentar la permeabilidad del vaso. Después de la angiografía, todos los catéteres y las vainas se retiran y la porción proximal de la arteria carótida derecha común ligado es, el músculo y la fascia se sutura con una sutura 4 / 0 absorbible, y la incisión de la piel se cerró con un 4 / 0 sutura no absorbible. </li><li> El animal se deja recuperar con la administración de una dosis de antibióticos (cefazolina, 0,5 gramos de mensajería instantánea). Medicamentos para el dolor incluyendo IM 0,01 mg / kg de buprenorfina (dos veces al día, según sea necesario). Los animales son luego continuó el 1% de colesterol durante 4 semanas post-globo denudación. En la semana 5, los animales son la transición a la dieta de colesterol al 0,3%. </li></ol> Integrada multi-modal de imágenes de Rabbit Atheromata 2) El etiquetado de placa inflamada proteolíticamente activa con nanosensor inyectables, angiografía, ultrasonido intravascular (IVUS), y en vivo intravascular NIRF imágenes de ateroma de conejo <ol><li> Ocho semanas después de la lesión con balón y 24 horas antes de la imagen, el conejo se inyecta por vía intravenosa con 500 nmol / kg Prosense/VM110 (PerkinElmer) a través de vena de la oreja. </li><li> Veinticuatro horas después de la inyección, los animales son anestesiados y el acceso arterial se obtiene a través de la arteria carótida común (véase el paso 1.2). Intraarterial se administra heparina (150 U / kg). De referencia la angiografía se obtiene que el anterior. </li><li> Un catéter de IVUS se carga en una clínica coronaria capaz 0,014 pulgadas de alambre y se inserta en la vaina. Usando la guía fluoroscópica, la punta del hilo radiopaco se coloca distal a la arteria ilíaca derecha. El catéter de IVUS se avanzó entonces en la arteria ilíaca proximal utilizando una técnica estándar clínico monorraíl. </li><li> Un retroceso de 100 mm se inicia y las imágenes se registran. Reconstrucción longitudinal del buque se obtiene y la placa luminal se identifica. </li><li> El catéter NIRF 11,12 cargado en el alambre de 0,014 pulgadas (monorraíl del sistema), y el catéter se inserta cuidadosamente en la vaina y el cabezal de exposición está situado distalmente en la arteria ilíaca derecha. </li><li> Múltiples retrocesos automatizado (1 mm / seg retroceso longitudinal, 30 disparos por minuto) se llevan a cabo y las señales de fluorescencia dentro de las zonas de la aterosclerosis es anotada. Las imágenes se graban y el tratamiento posterior con la escala adecuada y ventanas en función del alcance de la señal se logra. </li></ol> 3) La eutanasia y el aislamiento de los ex vivo aorto-ilíacos tejido <ol><li> La eutanasia se lleva a cabo con 1 cc de agente de la eutanasia (solución de fenitoína sódica 390mg de sodio pentobarbital y 50 mg), la inyección intravenosa, sola. </li><li> El árbol arterial es perfundido con solución salina normal al 0,9% hasta la vena cava inferior es de la sangre. Las arterias ateroscleróticas aorta e ilíaca se identifican y se diseca de los tejidos circundantes. Además, las pequeñas piezas de 2 x 2 cm de liver, los riñones, el bazo y el corazón también se obtienen. </li><li> Ex vivo NIRF de imágenes con el catéter intravascular NIRF imágenes se puede hacer en esta etapa. El buque es alargada y el catéter NIRF se reinserta en la aorta proximal hasta que la cabeza de imágenes se coloca en la arteria ilíaca derecha o bifurcación. Múltiples retrocesos automatizados se llevan a cabo como anteriormente (véase 2.6). </li></ol> 4) ex vivo de fluorescencia de reflectancia de imágenes (FRI) de la aorta disecada y las arterias ilíacas <ol><li> Los tejidos disecados está situado en el 10-20 cc de solución fisiológica y se transportan para el análisis de vie (Kodak Image Pro Estación de 4000mm, Carestream Health, Inc.). </li><li> Aorta, vasos ilíacos son alargadas para aproximar en tiempo real longitudes y se obtienen imágenes en varias longitudes de onda [luz blanca, el canal verde fluorescente (ex 495 nm, em 515 nm), Cy5 (ex 565 nm, em 670 nm) y Cy7 (ex 650 nm, 760 nm em)] canales. Una serie de tiempos de exposición se utilizan para cada longitud de onda (0.1-30seg) y las imágenes obtenidas se exportan como DICOM o 16-bit TIFF sin escala para otros análisis. Como controles positivos y negativos, los órganos (hígado, bazo, riñón y corazón) son fotografiados en canales similares y tiempos de exposición. </li><li> Áreas de aumento de la señal en el canal infrarrojo cercano (780nm +) se observan en las arterias ateroscleróticas. </li></ol> 5) Incorporación de tejidos para el análisis de sección y de inmunohistoquímica <ol><li> Áreas de la normalidad (sin lesiones de tejidos, es decir, la arteria ilíaca izquierda) y zonas de la placa de identificación y pequeños anillos de 5-10 mm de tejido están incrustadas en octubre (la temperatura óptima de corte) los medios de comunicación. Los bloques se almacenan a -80 C hasta el corte. </li><li> Técnicas estándar para el análisis inmunohistoquímico de seccionamiento y se llevan a cabo. Hematoxilina y eosina, Ram-11 y la tinción de la catepsina B se llevan a cabo. </li></ol> Los análisis e integración de multi-modal imágenes (angiografía, IVUS, NIRF y FRI) 6) El procesamiento de imágenes NIRF y el FRI <ol><li> Archivos DICOM contienen datos de imágenes de NIRF y el FRI (tomada en cerca del canal infrarrojo 780 nm) retiradas se procesan utilizando MATLAB y software Osirix, respectivamente. Ventanas adecuado para mostrar amplia gama de intensidad de la señal se logra. Las imágenes finales se exportan como archivos TIFF. </li><li> Los archivos se importan en el software estándar de procesamiento de imágenes (Keynote se puede utilizar). Las imágenes se alinean sobre la base de puntos de referencia (es decir, las vértebras en la angiografía, la bifurcación ilíaca y la arteria renal). Las áreas de vaso normal y la placa se identifican. </li><li> Las regiones de interés (ROI) de forma manual trazado (por el tejido normal y áreas de la placa) y la media de intensidad de la señal son adquiridos mediante Osirix y MATLAB, respectivamente, para ambos vie e imágenes NIRF. Para guiar el seguimiento adecuado, la imagen IVUS longitudinal del buque se utiliza y la identificación de vaso normal y la placa son fácilmente identificables. </li><li> Al objetivo de fondo (RBT) ratios se calculan para las zonas de la placa. </li></ol> Los resultados representativos: Una vez finalizado el protocolo anterior, podemos identificar y caracterizar las áreas de la actividad aumentada de la proteasa catepsina en la placa inflamatoria en la aorta y los vasos ilíacos. La inyección de un nanosensor activable (Prosense/VM110) que nos permite identificar la placa proteolíticamente activa. Estos aparecen como zonas brillantes o señal intensa en imágenes utilizando vie en el canal infrarrojo cercano (750 nm). Los retrocesos NIRF se correlacionan con la intensidad de la señal aumentó FRI y alineaciones con IVUS que permiten el registro de las señales anatómicas NIRF. TBR calcula la placa, obtenidos a partir de vie y NIRF fueron similares (véase la Figura 3: media NIRF TBR 4.2, significa vie TBR 2,9). El análisis inmunohistoquímico de la placa brillante confirma la presencia intensa de RAM-11 y la actividad de la catepsina B en las áreas de la placa (datos no mostrados). <img alt="Figura 1" src="/files/ftp_upload/2257/2257fig1.jpg" /> Figura 1. Esquemática de 2D catéter NIRF Para ampliar el potencial clínico de un enfoque 1D NIRF detección 6, se construyó una novela en 2-D NIRF-catéter intravascular para la imagen. 11,12 El catéter costumbre-construido consta de una fibra óptica (125 micrones de diámetro ubicado en tubería de polietileno: 2.9F) que se ilumina con un láser de 750 nm fuente de excitación. La luz del láser es emitida en un ángulo de 90 grados respecto al eje de la fibra. El sistema utiliza dos motores automatizados (rotación y de traslación) para permitir imágenes concomitante de 360 ​​grados y retroceso longitudinal para obtener imágenes 2D verdad. Imágenes que se utilizan con permiso de referencia 11. <img alt="Figura 2" src="/files/ftp_upload/2257/2257fig2.jpg" /> Figura 2. Esquemático que demuestra la activación mediada por la proteasa del nanosensor, Prosense/VM110. Imagen que se utiliza con permiso de referencia 10. <img alt="Tabla 1" src = &quot;/ files/ftp_upload/2257/2257table1.jpg&quot; /&gt; Figura 3. En vivo y ex vivo TBRs placa (meta-a las relaciones de fondo)

<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 - Ex consultora, VisEn médica; honorarios, Boston Scientific

Inflamado placas de alto riesgo o vulnerables son probablemente responsables de la mayoría de los infartos de miocardio. La identificación de estas placas antes de la aparición de los síntomas tiene importantes implicaciones clínicas, tanto en la predicción de los resultados y guiar el tratamiento médico. Modalidades convencionales de imagen coronaria arterial, tales como la angiografía de rayos X normalmente se centran en la caracterización de estrechamiento luminal en lugar de iluminar los perfiles biológico...

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<td>Prosense 750</td>
<td>&nbsp;</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

Se detalla una nueva en el infrarrojo cercano fluorescencia (NIRF) catéter de dos dimensiones de imagen intravascular molecular de la biología de la placa<em&gt; In vivo</em&gt;. El catéter NIRF pueden visualizar procesos biológicos clave, como la inflamación de la presentación de informes sobre la presencia de placa-ávido fluorocromos NIR activable y selectiva. El catéter se utiliza requisitos de ingeniería clínica y el poder y está destinada a la aplicación de las arterias coronarias humanas. El estudio de investigación A continuación se describe una estrategia de imagen multimodal que utiliza una novela<em&gt; In vivo</em&gt; NIRF intravascular del catéter a la imagen y cuantificación de placa inflamatoria en atheromata conejo proteolíticamente activos inflamados.

En vivo en el infrarrojo cercano fluorescencia (NIRF) imagen intravascular molecular de placa inflamatoria, un enfoque multimodal de imágenes de la aterosclerosis

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

in-vivo-near-infrared-fluorescence-nirf-intravascular-molecular

Research

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Medicine

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

17.8K vistas.  Harvard Medical School. Se detalla una nueva en el infrarrojo cercano fluorescencia (NIRF) catéter de dos dimensiones de imagen intravascular molecular de la biología de la placa In vivo. El catéter NIRF pueden visualizar procesos biológicos clave, como la inflamación de la presentación de informes sobre la presencia de placa-ávido fluorocromos NIR activable y selectiva. El catéter se utiliza requisitos de ingeniería clínica y el poder y está destinada a la aplicación ...

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.

En vivo doble sustrato bioluminiscentes imagen

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

Investigación

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.

Implantación de un manguito de la carótida para provocar esfuerzo cortante aterosclerosis inducida en ratones

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.

Imagen Molecular a Target trasplantó células progenitoras musculares

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.

Imágenes in vivo de la angiogénesis tumoral usando fluorescencia confocal videomicroscopia

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 humano<em&gt; Ex Vivo</em&gt; Placa aterosclerótica modelo para estudiar la lesión Biología

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 Tissue-simulación para la evaluación de las posibles aplicaciones de imágenes de fluorescencia en el infrarrojo cercano en la cirugía del cáncer de mama

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.

Murino Endoscopia para<em&gt; En Vivo</em&gt; Healing Multimodal Imaging de Carcinogénesis y Evaluación de la herida y la inflamación intestinal

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.

Validación de nanocuerpo y basados ​​en anticuerpos<em&gt; En Vivo</em&gt; Experimentos tumor de xenoinjerto NIRF-de formación de imágenes en ratones utilizando<em&gt; Ex Vivo</em&gt; Citometría de flujo y microscopía

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.

Imagen Fluorescente combinado del infrarrojo cercano y la tomografía computarizada-Micro para visualizar de manera directa tromboembolia cerebral

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.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

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