Suporte para este trabalho foi fornecida pelo National Institutes of Health conceder # R01 HL 108229, American Heart Association Scientist Desenvolvimento Grant # 0830352N, Howard Hughes Medical Institute Award de Desenvolvimento de Carreira, Broadview Ventures, o Programa da Comunidade Europeia Quadro (FP7/2007-2013 sob concessão # 235689 acordo), e do MGH William Schreyer Fellowship.

In vivo Modelo Animal: Geração da Aterosclerose Experimental aortoilíaca 1) Linha de Base e Angiografia Denudation Balloon <ol><li> Antes da obtenção de linha de base angiografia e desnudação do balão, uma Nova Zelândia coelho branco é alimentado um colesterol elevado (1%) dieta por uma semana. Este animal é utilizado para a relevância de translação como: 1) os vasos aorto-ilíacas de coelhos são mesmo calibre de artérias coronárias humanas (2,5 3,5 mm) e 2) a hiperlipidemia, o modelo de balão de lesão gera aterosclerose inflamado tendo similares células inflamatórias (macrófagos ) e moléculas (catepsinas) como na aterosclerose humana. </li><li> Após a alimentação de colesterol, o animal é anestesiado com propofol e cetamina. A one-inch incisão cervical mediana ventral é feita usando usando um tamanho de 15 lâmina de bisturi. Usando técnicas de dissecção romba, os músculos debaixo da fáscia no lado direito da traquéia está exposto. O músculo sternocephalicus esquerdo é separado ao longo de sua junção do tecido conjuntivo, ea artéria carótida comum direita é exposta. A artéria é separado do nervo vago. Loops de sutura proximal e distal são colocados sobre a artéria para permitir a retração e oclusão. A 1 a 2mm chanfrado arteriotomia é feita através das quais a 5 (diâmetro externo 1,67 milímetros) Francês bainha vascular é inserido e heparina (1000μ/mL, ~ 150units/kg) é administrado via intra-arterial da bainha. </li><li> Corante de contraste (Ultravist) é então injetada (1 a 2 mL) durante um período de 2 segundos para obter um angiograma controle da aorta distal e ambas as artérias ilíacas. </li><li> As artérias femoral e aorta são, então, ferido por desnudação endotelial. Usando métodos padrão fluoroscopia, um cateter de embolectomia 3FR Fogarty é colocado na artéria femoral distal e inflado com 0,3 a 0,5 cc de contraste (50% contrast/50% salina) ou ar. O cateter é então retirado proximalmente em seu estado inflado uma distância ao longo da ilíaca direita e aorta distal até o take-off da artéria renal. Após a desnudação do balão, a angiografia é repetido para documentar a permeabilidade do vaso. Após a angiografia, todos os cateteres e bainhas são removidos ea artéria carótida proximal direito comum é ligada, o músculo e fáscia são suturada com fio de 4 / 0 absorvível, ea incisão na pele fechada com um 4 / 0 de sutura não absorvível. </li><li> O animal é então autorizado a recuperar com a administração de um antibiótico de dose (cefazolina, 0,5 gramas IM). Medicamentos para a dor, incluindo 0,01 mg / kg IM de buprenorfina (duas vezes por dia, conforme necessário). Os animais são então continuou sobre o colesterol 1% para 4 semanas desnudação pós-balão. Na semana 5, os animais são a transição para dieta de colesterol 0,3%. </li></ol> Imagens Multi-modal integrado de coelho ateromatosas 2 Labeling) de proteoliticamente ativa placa inflamado usando nanosensor injetável; angiografia, ultra-som intravascular (IVUS), e in vivo intravascular NIRF imagem do coelho ateroma <ol><li> Oito semanas após a lesão por balão e 24 horas antes da imagem, o coelho é injetado com intravenosa 500 nmol / kg Prosense/VM110 (PerkinElmer) pela veia da orelha. </li><li> Vinte e quatro horas após a injeção, os animais são anestesiados e acesso arterial é obtida através da artéria carótida comum esquerda (veja o passo 1.2). Intra-arterial de heparina é administrada (150 unidades / kg). Linha de base é obtida como a angiografia acima. </li><li> Um cateter ultra-som intravascular é carregado em um clínica da artéria coronária 0.014 capaz polegadas e inserido na bainha. Usando orientação fluoroscópica, a ponta do fio radiopaco está posicionado distalmente na artéria ilíaca direita. O cateter ultra-som intravascular é então avançado para a artéria ilíaca proximal usando um padrão de técnica monorail clínico. </li><li> A retirada de 100 mm é iniciado e as imagens são gravadas. Reconstrução longitudinal da embarcação é obtida e placa luminal é identificado. </li><li> O cateter NIRF 11,12 é carregado no fio de 0,014 polegadas (monorail do sistema), eo cateter é cuidadosamente inserido na bainha e cabeça de imagens está posicionado distalmente em artéria ilíaca direita. </li><li> Múltiplas pullbacks automatizado (1 mm / seg pullback longitudinal, 30 tiros por minuto) são realizadas e os sinais de fluorescência dentro das zonas de aterosclerose são anotados. As imagens são gravadas e posterior processamento, com escala adequada e janelas com o alcance do sinal é alcançado. </li></ol> Eutanásia 3) e isolamento de ex vivo de tecido aorto-ilíaca <ol><li> A eutanásia é realizada com 1cc de agente de eutanásia (390mg de sódio solução de pentobarbital sódico fenitoína e 50 mg), injeção, intravenosa única. </li><li> A árvore arterial é perfundido com solução salina 0,9% até a veia cava inferior é claro de sangue. As artérias aorta e ilíaca aterosclerótica são identificadas e dissecadas livre dos tecidos circundantes. Além disso, pequenos pedaços 2 x 2 cm de liver, rins, baço e coração também são obtidos. </li><li> Ex vivo NIRF imagem com o cateter intravascular imagem NIRF pode ser feito nesta fase. O navio é alongado eo cateter NIRF é re-inserido na aorta proximal até que a cabeça de imagem é posicionada na artéria ilíaca direita ou bifurcação. Múltiplas pullbacks automatizados são realizados como descrito acima (ver 2.6). </li></ol> 4) Ex vivo de fluorescência Reflectância Imaging (FRI) da aorta dissecada e artérias ilíacas <ol><li> Tecidos dissecados é colocado em 10-20 cc de soro fisiológico e transportados para análises sex (Imagem Kodak Estação 4000mm Pro, Carestream Health, Inc.). </li><li> Vasos aorta, ilíacas são alongadas para aproximar tempo real comprimentos e as imagens são obtidas em vários comprimentos de onda [luz branca, o canal verde fluorescente (ex 495 nm, empregando 515 nm), Cy5 (ex 565 nm, empregando 670 nm) e Cy7 (ex 650 nm, empregando 760 nm)] canais. Uma série de tempos de exposição são utilizados para cada comprimento de onda (0.1-30s) e imagens adquiridas são exportados como DICOM ou 16 bits TIFF arquivos fora de escala para análises posteriores. Como controles positivos e negativos, os órgãos (fígado, baço, rim e coração) são gravadas em canais similares e tempos de exposição. </li><li> Áreas de sinal aumentado no canal infravermelho próximo (780nm +) são anotadas nas artérias ateroscleróticas. </li></ol> 5) Incorporação de tecido para análise de corte e imuno-histoquímico <ol><li> Áreas de normal (sem lesão tecidual; artéria ilíaca ou seja, à esquerda) e áreas de placa são identificados e pequenos anéis de 5-10 mm de tecido são incorporados em outubro (Temperatura ideal de corte) de mídia. Blocos são armazenadas a -80 C até o corte. </li><li> Técnicas padronizadas para análises de corte e imuno-histoquímica são executadas. Hematoxilina e eosina, Ram-11 e catepsina B coloração são executadas. </li></ol> Análise e Integração de multi-modal Imagens (angiografia, IVUS, NIRF e sex.) 6) Processamento de imagens NIRF e sex. <ol><li> Arquivos DICOM que contém dados de imagem de NIRF e sex (tomadas no canal infravermelho próximo nm 780) pullbacks são processados ​​usando MATLAB e software OsiriX, respectivamente. Janelas adequado para mostrar gama completa de intensidade do sinal é alcançado. Imagens finais são exportados como arquivos TIFF. </li><li> Os arquivos são importados para o software de processamento de imagem padrão (Keynote pode ser usado). As imagens são alinhadas com base em pontos de referência (ou seja, vértebras na angiografia, bifurcação ilíaca, e da artéria renal). Áreas de navio normal e placa são identificados. </li><li> Regiões de interesse (ROI) são manualmente traçado (para tecido normal e áreas de placa) e média intensidades de sinal são adquiridos por meio OsiriX e MATLAB, respectivamente, para ambos os sex e imagens NIRF. Para orientar o rastreio adequado, a imagem longitudinal IVUS do navio é usado e identificação da embarcação normal e placa são facilmente identificados. </li><li> Alvo de fundo (TBR) rácios são calculados para as zonas de placa. </li></ol> Resultados representativos: Após a conclusão do protocolo acima, podemos identificar e caracterizar as áreas de atividade aumentada protease catepsina na placa inflamatória dentro da aorta e vasos ilíacos. Injeção de um nanosensor ativável (Prosense/VM110) nos permite identificar a placa proteoliticamente ativo. Estes aparecem como sinal luminoso ou zonas de intenso quando fotografada usando sex no canal infravermelho próximo (750 nm). O pullbacks NIRF se correlacionam com a intensidade de sinal aumentado em sex e alinhamentos com IVUS que permitem o registo de sinais anatômicos NIRF. TBR placa calculados, obtidos a partir de sex e NIRF foram semelhantes (ver Figura 3: média NIRF TBR 4.2, significa sex TBR 2.9). Análise imuno-histoquímica de placa luminosa confirma presença intensa de RAM-11 e atividade catepsina B em áreas de placa (dados não mostrados). <img alt="Figura 1" src="/files/ftp_upload/2257/2257fig1.jpg" /> Figura 1. Esquemática de 2D Cateter NIRF Para ampliar o potencial clínico de uma abordagem 1D sensoriamento NIRF 6, construímos um romance 2-D NIRF cateter para imagens intravascular. 11,12 cateter A custom-built consiste em uma fibra óptica (125 mícron de diâmetro alojada em tubos de polietileno: 2.9F) que ilumina usando uma fonte de excitação 750 nm laser. Luz laser é emitido em um ângulo de 90 graus em relação ao eixo da fibra. O sistema utiliza dois motores automatizados (rotação e translação) para permitir a imagem grau concomitante 360 ​​e recuo longitudinal para a obtenção de imagens 2D verdade. Imagens usadas com permissão da referência 11. <img alt="Figura 2" src="/files/ftp_upload/2257/2257fig2.jpg" /> Figura 2. Esquemático demonstrando a ativação da protease mediada do nanosensor, Prosense/VM110. Imagem usada com permissão da referência 10. <img alt="Tabela 1" src = &quot;files/ftp_upload/2257/2257table1.jpg /&quot; /&gt; Figura 3. In vivo e ex vivo TBRs placa (alvo de relações de fundo)

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	<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-consultor, VisEn Médica; honorários, Boston Scientific

Inflamado placas de alto risco ou vulneráveis ​​são provavelmente responsáveis ​​para a maioria dos infartos do miocárdio. A identificação das placas, antes da aparecimento dos sintomas tem importantes implicações clínicas tanto na previsão de resultados e orientar a terapia médica. Convencionais coronária arterial modalidades de imagem, como raio-x angiografia geralmente se concentram na caracterização dos estreitamentos luminais ao invés de iluminar os perfis biológicos subjacentes de alto risc...

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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

A resposta vascular a lesão é uma resposta bem orquestrada inflamatório desencadeado pelo acúmulo de macrófagos na parede dos vasos, levando a um acúmulo de lipídios-laden placa intra-luminal, a proliferação de células musculares lisas e estreitamento progressivo da luz do vaso. A formação de tais placas vulneráveis ​​propensos a ruptura subjacente à maioria dos casos de infarto agudo do miocárdio. A cascata molecular e celular inflamatória complexa é orquestrado pelo recrutamento de linfócitos T e macrófagos e seus efeitos parácrinos em células endoteliais e musculares lisas 1. A imagem molecular na aterosclerose tem evoluído para uma importante ferramenta clínica e de pesquisa que permite visualização em vivo de inflamação e outros processos biológicos. Diversos exemplos recentes demonstram a capacidade de detectar placas de alto risco nos pacientes e avaliar os efeitos da farmacoterapia na aterosclerose. 4 Enquanto uma série de abordagens de imagem molecular (em especial de ressonância magnética e PET) podem aspectos biológicos imagem de grandes vasos como a carótida artérias, existem opções escassas para geração de imagens das artérias coronárias. 2 O advento de alta resolução estratégias de imagem óptica, de fluorescência do infravermelho próximo particular (NIRF), juntamente com ativável sondas fluorescentes, têm maior sensibilidade e levou ao desenvolvimento de novas estratégias intravascular para melhorar a imagem biológica de aterosclerose coronária humana. Infravermelho próximo de fluorescência (NIRF) imagem molecular utiliza luz de excitação com uma largura de banda definida (650-900 nm) como fonte de fótons que, quando entregues a um agente de contraste óptico ou sonda fluorescente, emite fluorescência na janela NIR que pode ser detectado utilizando um filtro de emissão adequadas e uma alta sensibilidade da câmera charge-coupled. Em oposição à luz visível, luz NIR penetra profundamente no tecido, é marcadamente menos atenuada pela absorção de fótons de lipídeos endógenos como a hemoglobina, e água, e permite alta-alvo para background-razões, devido à reduzida autofluorescência na janela NIR. Imagem dentro de &quot;janela&quot; do NIR pode melhorar substancialmente o potencial de imagem em vivo. 2,5 Cisteína proteases inflamatórios têm sido bem estudada usando sondas ativável NIRF 10, e desempenham um papel importante na aterogênese. Via degradação da matriz extracelular, proteases de cisteína contribuem de forma importante para a progressão e as complicações da aterosclerose 8. Em particular, a protease cisteína, catepsina B, é altamente expressa e colocalizes com macrófagos em camundongos experimental, coelho, e ateromatosas humana. 3,6,7 Além disso, a atividade catepsina B em placas podem ser detectados in vivo utilizando um descrito anteriormente um -D intravascular tecnologia do infravermelho próximo de fluorescência 6, em conjunto com um agente injetável nanosensor que consiste de uma espinha dorsal do polímero poli-lisina derivatizado com fluorocromos múltiplas NIR (VM110/Prosense750, ex / em 750/780nm, VisEn Medical, Woburn, MA) que resulta na extinção intramolecular forte no início. 10 Após alvo clivagem enzimática de proteases de cisteína, como catepsina B (conhecido por colocalize com os macrófagos da placa), a fluorocromos distintos, resultando em amplificação substancial do sinal NIRF. Detecção intravascular de NIR sinal de fluorescência pelo romance 2D utilizado cateter intravascular NIRF agora permite alta resolução, geometricamente precisos na detecção in vivo de atividade catepsina B na placa inflamado. In vivo de imagem molecular da aterosclerose utilizando cateter-baseado 2D NIRF de imagem, em oposição a uma prévia 1 D-abordagem espectroscópicas, 6 é um romance e promissora ferramenta que utiliza a atividade protease aumentada em macrófagos ricos em placa para detectar a inflamação vascular 11,12. O protocolo de pesquisa a seguir descreve o uso de um cateter intravascular NIRF 2-dimensional para a imagem e caracterizar a estrutura da placa utilizando os principais aspectos da biologia placa. É uma plataforma traduzíveis que, quando integradas com as actuais tecnologias de imagem clínica, incluindo angiografia e ultra-som intravascular (IVUS), oferece uma única e nova técnica de imagem molecular multimodal integrado que distingue ateromatosas inflamatórias, e permite a detecção de sinais intravascular NIRF em humanos porte artérias coronárias .

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

Detalhamos um novo infravermelho próximo de fluorescência cateter (NIRF) para 2-dimensional imagem intravascular molecular da biologia placa<em&gt; In vivo</em&gt;. O cateter NIRF pode visualizar os principais processos biológicos, tais como inflamação, informando sobre a presença de placa-avid fluorocromos ativável e orientada NIR. O cateter utiliza requisitos clínicos de engenharia e energia e é direcionada para aplicação em artérias coronárias humanas. A pesquisa a seguir descreve uma estratégia de imagem multimodal que utiliza um romance<em&gt; In vivo</em&gt; Cateter intravascular NIRF para a imagem e quantificar placa inflamatória no ateromatosas coelho proteoliticamente ativa inflamado.

In vivo Near Infrared fluorescência de imagem intravascular (NIRF) Molecular da placa inflamatória, uma Abordagem Multimodal para imagem da Aterosclerose

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 visualizações.  Harvard Medical School. Detalhamos um novo infravermelho próximo de fluorescência cateter (NIRF) para 2-dimensional imagem intravascular molecular da biologia placa In vivo. O cateter NIRF pode visualizar os principais processos biológicos, tais como inflamação, informando sobre a presença de placa-avid fluorocromos ativável e orientada NIR. O cateter utiliza requisitos clínicos de engenharia e energia e é direcionada para aplicação em artérias coronárias humanas. A p...

Vídeo: 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 dupla Substrato Bioluminescent imagem

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

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.

Implantação de um Cuff Carotid para Triggering Shear-estresse aterosclerose induzida em camundongos

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.

Imagem Molecular para atingir as células progenitoras transplantadas 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.

Imagem in vivo da angiogênese tumoral utilizando fluorescência 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.

A Human<em&gt; Ex Vivo</em&gt; Placa aterosclerótica modelo para estudar Lesão Biologia

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

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

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

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

Phantoms Tissue-simulação para avaliação de potenciais aplicações de fluorescência de imagem infravermelho próximo na cirurgia do câncer 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; In Vivo</em&gt; Cura Multimodal imagem de Carcinogênese e Avaliação de Intestinal ferida e inflamação

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.

Validação de nanocorpo e Antibody Based<em&gt; In Vivo</em&gt; Experimentos Tumor Xenoenxerto NIRF-imagem em ratos usando<em&gt; Ex Vivo</em&gt; Citometria de fluxo e microscopia

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

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

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

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

Combinada Near-infrared imagens fluorescentes e tomografia Micro-computado para Diretamente Visualizando Cerebral tromboêmbolos

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.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

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