Agradecemos a Daniel Bukala, Natalie Altmeyer e Funda Cay para excelente suporte técnico. Agradecemos Jonathan Cotton, Greg Bowden e Paul Soubiran para a edição do manuscrito. Este trabalho foi apoiado pela Werner Siemens-Foundation e da Faculdade de Medicina da Universidade Eberhard Karls de Tübingen ( &#39;&#39; Promotionskolleg &#39;&#39;) e pela DFG através da CRC 156 (C3 projeto).

Todos os procedimentos descritos neste documento, seguiu as diretrizes e normas internacionais do cuidado e uso de animais de laboratório e foram aprovados pelo Bem-Estar Animal e Comitê de Ética local do país Comissão Tuebingen, Alemanha. 8 - 12 semanas de idade BALB / c e C57BL / 6 murganhos foram mantidos num ciclo de 12 h: 12 h luz: ciclo escuro e foram alojados em IVCS e condições ambientais padronizadas a 22 ± 1 ° C em grupos de 2-5 com água e alimentos ad libitum acesso. 1. Preparação de Material <ol><li> Dilui-se o corante OI para imagiologia de fluorescência no infravermelho próximo de acordo com a respectiva folha de dados directamente antes da injecção. O corante OI activável (sonda comercial com uma excitação a 680 nm) para medir as MMPs in vivo, está pronto para usar, a uma concentração de 20 nmol em 1,5 mL de 1x PBS. Agitar suavemente a solução ou vortex antes da utilizao. OI corante pode ser armazenado a 2 - 8 ° C durante até 6 meses quando protegida da luz. </li&gt; <li> Para injecções intravenosas ( iv ) preparar um cateter venoso. Use uma seringa de insulina de 20 U (0,5 mL) cheia com solução salina a 0,9% (contendo 10 unidades de injeção de heparina em 50 mL) e uma agulha de calibre 30 conectada a um cateter de polietileno. Injectar a dose recomendada de 2 nmol por rato do corante OI. </li></ol> 2. Indução da Artrite Reumatóide e da Reação de Hipersensibilidade de Contato Crônico <ol><li> Artrite reumatóide: <ol><li> Diluir 100 μL de soro (obtido de ratinhos K / BxN 10 ) contendo anticorpos (AB) contra glucose-6-fosfato isomerase (GPI) 1: 1 com 1x PBS (200 μL) para induzir RA. Armazenar o soro diluído a -80 ° C. Os procedimentos detalhados para a indução da AR são descritos por Monach et al. 8 . </li><li> Para induzir RA, levantar cada rato suavemente pela sua cauda e injectar 200 μL do soro diluído intraperitoneal ( ip ) no dia 0 de tEle experimenta. Após a injeção, coloque o mouse diretamente em sua gaiola. </li><li> Opcionalmente, medir os diâmetros do tornozelo de cada tornozelo, antes de injeções de AB e definir uma &quot;pontuação artrítica&quot; ( Figura 1A ). Continuar a medição do inchaço do tornozelo diariamente até ao dia 6 após GPI-soro utilizando um dispositivo mecânico de medição (micrómetro). </li><li> Injectar o corante OI activável (passo 1.1 - 1.2), para medir a actividade de MMP in vivo iv na veia da cauda de ratinhos RA No dia 5 após injecção de GPI-soro e na veia da cauda de ratinhos de controlo. Realizar experiências de imagem óptica 24 h após a injeção da veia da cauda. </li></ol></li><li> Reacção de hipersensibilidade de contacto: <ol><li> Para a sensibilização de ratinhos C57BL / 6, preparar uma solução de TNCB a 5% dissolvida numa mistura 4: 1 de acetona / óleo. </li><li> Anestesiar ratos C57BL / 6 utilizando isoflurano a 1,5% vaporizado em oxigénio a 100% (1,5 L / min). Uma vez que os ratos são anestesiados, colocá-lo em um auto-louco<html> E um cone de nariz (uma seringa de polietileno cortada de 5 mL, ligada ao tubo de isoflurano a 1,5% em volume) e manter a anestesia. Aplique pomada oftálmica veterinária em animais anestesiados para evitar a secura dos olhos durante a anestesia. </li><li> Raspar o abdômen cuidadosamente (2 x 2 cm) usando um pequeno aparador de pêlos de animais. Evite ferir a pele, pois isso pode levar a sinais fluorescentes inespecíficos dentro do animal e pode influenciar os resultados do estudo. </li><li> Aplique 80 μL da solução de TNCB a 5% lentamente na área do abdome raspado, utilizando uma pipeta de 100 μL para sensibilizar o animal. Coloque o mouse suavemente de volta em sua gaiola tendo o cuidado de garantir que os olhos do mouse não entrar em contato com a cama para evitar lesões da córnea e evitar baixas temperaturas corporais durante a fase de recuperação. </li><li> Seis dias após a sensibilização, preparar uma solução de TNCB a 1% (dissolvida numa mistura 9: 1 de acetona / óleo) e aplicar 20 μL na orelha direita utilizando uma pipeta para provocar CHSR aguda e crónica.&quot;&gt; 1 <ol><li> Comece com o primeiro desafio em ambos os lados da orelha direita com 20 μL de solução de TNCB a 1%, utilizando uma pipeta de 100 μL e repita o desafio a cada dois dias até cinco vezes (dia 15 após a sensibilização) para obter CHR ( Figura 1B ) . Medir a espessura da orelha diariamente, usando um dispositivo de medição micrômetro. </li></ol></li><li> Injectar o corante OI activável (uma sonda comercial com excitação a 680 nm) (passo 1.1-1.2) para medir a actividade de MMP in vivo em ratinhos CHR 12 h após a provocação. Realizar experimentos de imagem óptica 24 h após a injeção da veia da cauda ( iv ). </li></ol></li></ol> 3. Preparação de animais para imagens ópticas <ol><li> Mudar o chow do rato (pelo menos 3 dias antes da imagem) para comida baixa ou não fluorescente ( por exemplo , livre de manganês) para evitar a interferência da auto-fluorescência (cerca de 700 nm) com o sinal de fluorescência dos agentes OI utilizados. </li><li> Coloque o animal empara uma caixa de anestesia e anestesiar utilizando 1,5 vol% de isoflurano vaporizado com oxigénio (1,5 L / min) (de oxigénio pode ser substituído por ar, mas deve ser normalizados para cada um experimento). </li><li> Quando o rato é visivelmente anestesiados, colocá-lo em um cone de nariz de auto-fabricados (por construir um corte de 5 mL de uma seringa de polietileno, ligado ao tubo de isoflurano 1,5% vol) e manter a anestesia. </li><li> Raspar o animal cuidadosamente no local alvo (2 cm por 2 cm), utilizando um pequeno aparador de pêlos. Evitar ferir a pele, pois isso pode levar a sinais fluorescentes inespecíficos dentro do animal e pode influenciar os resultados do estudo. NOTA: Os cabelos podem absorver o sinal de fluorescência durante OI (dependente da estirpe de ratinho, mais ou menos absorção é observado), dependendo da região de interesse (ROI). </li><li> Para injecção iv, colocar a cauda do rato em água aquecida, para induzir a vasodilatação, limpar suavemente a pele no local da injecção com álcool, e começar por colocar o cateter emo local distal da cauda. Coloque a &quot;cortar&quot; aresta da agulha em, a um ângulo de 20 ° na veia da cauda e testar a colocação correcta do cateter por re-suspensão da seringa. </li><li> Se o cateter está colocado correctamente, substituir a seringa e injectar a sonda OI (2 nmol). Após a injecção, substituir a seringa e injectar 25 mL de solução salina a 0,9% para totalmente claro o volume morto do tubo de polietileno. NOTA: O tecido tempo de meia-vida do corante usado OI (uma sonda comercial com excitação a 680 nm) para medir a actividade de MMP in vivo é de 72 h. Para garantir uma eliminação completa, a re-injeção do corante não é recomendado mais cedo do que 7 dias após a injecção antes. </li></ol> 4. Imagem Óptico <ol><li> Coloque um plástico preto ou folha de papel na caixa preta do OI-scanner no centro do campo de visão (FOV). </li><li> Defina-se um protocolo de medição e escolher o comprimento de onda direita (excitação: 680 ± 10 nm e eMissão: 700 ± 10 nm) e parâmetros de imagem. NOTA: Em alguns sistemas OI, a configuração para vários corantes de imagem está pré-definida. </li><li> Para escolher o protocolo correto para a imagem de fluorescência, abra o software de criação de imagens (fornecido pelo fabricante) e inicialize o sistema. A maioria das câmeras CCD precisa esfriar a sua temperatura de trabalho, e isso pode levar 10 minutos. Para resultados confiáveis, aguarde até que o sistema esteja pronto. </li><li> Observe o painel de controle de aquisição do sistema de imagem in vivo pop up e cada par de filtros selecionado irá representar uma imagem na seqüência. Neste caso, adquira uma imagem com os pares de filtros para o corante comercial com um comprimento de onda de excitação e emissão de 680 ± 10 nm e 700 ± 10 nm, respectivamente, e inicie a medição (Pressione &quot;Acquire sequence&quot;). Para obter instruções mais detalhadas, consulte o manual do fabricante. </li><li> Rotule adequadamente as imagens e salve as informações na seção &quot;Editar imagemLabels &quot;janela, que irá aparecer após a&quot; seqüência de aquisição &quot;. </li><li> Tome uma varredura basal de cada animal antes da injeção do corante OI, ou use animais de controle ingênuos para diferenciar o sinal de fundo. </li><li> H após a injeção da veia da cauda do corante OI, colocar os animais no centro do FOV, em uma posição para medir o sinal mais alto no sistema OI, e iniciar as medições. NOTA: Importante: A hipotermia pode influenciar significativamente a distribuição de agentes de imagem. Certifique-se que o estágio é aquecido até 37 ° C para evitar a hipotermia do animal. A imagiologia pode ser realizada simultaneamente com animais em grupos de 1 a 5 ratinhos. Escolha o tamanho do FOV dependendo do número de animais a medir ao mesmo tempo. Você pode tirar uma imagem de campo brilhante para verificar se cada mouse está visível. </li></ol> 5. Análise de Dados NOTA: Execute a análise de dados usando o software de imagemProtocolo do fabricante. <ol><li> Para medições de RA, use a unidade calibrada da emissão de fótons como mostrado na Figura 2 , enquanto que a CHSR crônica é representada como a intensidade do sinal em porcentagem (eficiência). </li><li> Para desenhar ROI manualmente, use a placa de ferramentas. Para a análise das imagens RA usar um círculo padronizado, colocado em torno do sinal mais alto em todos os tornozelos e patas de cada animal. Para analisar o CHSR, coloque o ROIs em torno de toda a orelha direita e esquerda de acordo com a imagem do campo brilhante. </li><li> Para medir a emissão de fótons ou os valores de intensidade do sinal no ROI específico, pressione &quot;measure&quot;. O sistema fornecerá valores no ROI desenhado para análise estatística descritiva. </li></ol>

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OI é uma ferramenta muito útil, rápida e barata para imagens moleculares in vivo não invasivas em pesquisas pré-clínicas. Uma força particular de OI é a capacidade de monitorar processos altamente dinâmicos como respostas inflamatórias. Além disso, OI permite que se siga o curso de uma doença por um período de tempo prolongado, variando de dias a semanas. OI tem várias vantagens em relação a outras modalidades de imagem in vivo , tais como a tomografia por e...

doenças auto-imunes tais como artrite reumatóide (AR) ou psoríase vulgar são classificadas como reacções de hipersensibilidade do tipo retardado (DTHRs). 1 AR é uma doença auto-imune comum caracterizada por sinovite erosiva e destruição da articulação. 2 articulações artríticas inflamadas demonstrar a infiltração e a proliferação de células inflamatórias, um aumento da expressão de células pró-inflamatórias que conduzem à formação de pannus, cartilagem e osso destruições. 3, 4 A clivagem de moléculas da matriz extracelular, tal como colagio por metaloproteinases de matriz (MMPs), é essencial para a conversão de tecido e a angiogénese e provoca destruição de tecidos. 5, 6 de contato reacções de hipersensibilidade (CHR) são caracterizadas pela agregação de neutrófilos que conduzem a uma explosão oxidativa. 7 Semelhante ao RA, MMPs em CHR são involved na conversão de tecido, a migração celular e a angiogénese, a fim de estabelecer a inflamação crónica. Para investigar a AR, foi usada a isomerase de glucose-6-fosfato (GPI) modelo de ratinho injecção -serum. 8 O soro de ratinhos K / BXN transgénicas contendo anticorpos contra a GPI, foi injectada em ratinhos Balb / c, após o que a inflamação reumática começaram a desenvolver dentro de 24 h com um máximo de tornozelo inchaço no dia 6 após a injecção GPI-soro (ver 1.1). Para analisar CHR crónica, ratinhos C57BL / 6 foram sensibilizados com trinitroclorobenzeno (TNCB) no abdómen. A orelha direita foi desafiada até 5 vezes a partir de 1 semana após a sensibilização (ver também 1,1 e 1,2). Não invasiva pequena OI animal é uma técnica baseada na investigação in vivo de Fluorescente, chemiluminescent- e bioluminescentes-sinais, que são usados principalmente em estudos pré-clínicos. Os dados semi-quantitativa adquiriu dá insights sobre o MolecMecanismos moleculares nos órgãos e tecidos de modelos animais saudáveis ​​e doentes, e permite medições de seguimento longitudinal ( por exemplo, para avaliar os perfis de resposta terapêutica in vivo ). Uma grande vantagem dos estudos longitudinais é a redução do número de animais, uma vez que os mesmos animais podem ser medidos em estudos de acompanhamento em vários pontos de tempo, em vez de usar ratos diferentes por ponto de tempo. A resolução de OI permite imagens funcionais detalhadas de órgãos e estruturas de tecido ainda menores em animais experimentais. O uso de filtros específicos de excitação e emissão com um espectro de transmissão estreito, uma proteção contra a luz dispersa por uma &quot;caixa escura&quot; à prova de luz e uma câmera sensível de acoplamento de carga (CCD), que é resfriada em muitos dispositivos até -70 ° C , Permite medidas altamente específicas e sensíveis de sinais de fluorescência. Utilizando agentes fluorescentes comEspectro de emissão na janela de fluorescência do infravermelho próximo (650 - 950 nm), as relações sinal / ruído podem ser melhoradas significativamente. A janela de fluorescência no infravermelho próximo é caracterizada por uma absorção relativamente baixa do sinal por hemoglobina e água, bem como uma auto-fluorescência de baixo fundo. 9 Isso permite uma profundidade de penetração de até 2 cm no tecido de pequenos animais. As sondas OI podem dirigir-se directamente a um alvo ( por exemplo, por um anticorpo marcado com fluorescï¿½cia) ou podem ser activadas no tecido alvo ( por exemplo, por proteases). As sondas OI activáveis ​​são opticamente silenciosas na sua forma inactivada devido à transferência de energia de ressonância de Förster (FRET) para uma porção de extinção, que transfere a energia de excitação dentro da molécula para outro domínio. Se o corante é clivado (por uma protease, por exemplo) a energia não é mais transferida dentro da molécula e um sinal fluorescente pode ser detectado por OI. Isto permite a concepção de sondas OI com alta especificidadey para os processos biológicos distintos e excelentes Noise-relações sinal-para-. O protocolo que se segue explica em pormenor a preparação dos animais, as medições OI OI utilizando uma sonda Activatable a imagem de MMP-2, -3, -9 e -13 actividade in vivo e dois modelos experimentais de inflamação (RA, CHR).

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			<td>2-Chloro-1,3,5-trinitrobenzene</td>
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			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
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			<td>Miglyol 812</td>
			<td>SASOL</td>
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			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
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			<td>Sigma Aldrich GmbH</td>
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			<td>For dilution of the RA serum&#160;</td>
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			<td>Eppendorf&#160;</td>
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non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

Este trabalho descreve um método não invasivo para a formação de imagens de metaloproteinases de matriz (MMP) por meio de uma sonda fluorescente ativável, através de imagens ópticas de fluorescência in vivo (OI), em dois modelos diferentes de inflamação: uma artrite reumatóide (RA) e um contato Reacção de hipersensibilidade (CHR). A luz com um comprimento de onda na janela do infravermelho próximo (NIR) (650 - 950 nm) permite uma penetração mais profunda do tecido e uma absorção de sinal mínima em comparação com comprimentos de onda inferiores a 650 nm. As principais vantagens da utilização de fluorescência OI é que é barato, rápido e fácil de implementar em diferentes modelos animais. As sondas fluorescentes activáveis ​​são opticamente silenciosas nos seus estados inactivados, mas tornam-se altamente fluorescentes quando activadas por uma protease. As MMPs activadas conduzem à destruição dos tecidos e desempenham um papel importante na progressão da doença em reacções de hipersensibilidade de tipo retardado (DTHRs) tais como RA e CHR. Além disso, as MMPs sãoas proteases essenciais para a cartilagem e osso e são degradação induzida por macrófagos, fibroblastos e condrócitos em resposta a citoquinas pró-inflamatórias. Aqui nós usar uma sonda que é activada pelas MMPs chave como a MMP-2, -3, -9 e -13 e descrever um protocolo de imagiologia para perto OI fluorescência no infravermelho de actividade de MMP na AR e ratinhos de controlo 6 dias após a indução da doença bem como em ratos com aguda (1x desafio) e crônica (5x desafio) CHR na orelha direita em comparação com os ouvidos saudáveis.

Para induzir a artrite reumatóide (RA) em ratinhos BALB / c nativos, os animais foram injectados ip com auto-anticorpos (diluição 1: 1 com 1x PBS) contra GPI no dia 0. A inflamação máxima (inchamento do tornozelo) induzida neste soro GPI O modelo RA está no dia 6 pós-injeção 11 . Por conseguinte, preparou-se 2 nmol do corante OI activável e injectou-se iv na veia da cauda de murganhos artríticos e animais de controlo saudáveis ​​...

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

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

Este documento explica a aplicação de imagem fluorescente usando uma sonda de imagiologia óptica activável para visualizar a actividade in vivo de metaloproteinases de matriz chave em dois modelos experimentais diferentes de inflamação.

Non-invasive<em&gt; In Vivo</em&gt; Fluorescência de imagem óptico de Inflamatória MMP Atividade Usando um Agente de Imaging Activatable fluorescente

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

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

Research

JoVE Journal

Immunology and Infection

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

8.7K visualizações.  Eberhard Karls University of Tübingen.  Este documento explica a aplicação de imagem fluorescente usando uma sonda de imagiologia óptica activável para visualizar a actividade in vivo de metaloproteinases de matriz chave em dois modelos experimentais diferentes de inflamação. 

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</em

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

Imunologia e Infecção

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

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

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

Uso de uma armadilha óptica de Estudos de Interações Hospedeiro-Patógeno para imagens de células dinâmica ao vivo

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

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

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

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

Imagem de bioluminescência de NADPH oxidase em diferentes modelos animais

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

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

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

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

Não-invasivos de imagem de candidíase disseminada em larvas do peixe

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

Bioluminescent Bacterial Imaging In Vivo

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

Bioluminescent Bacterial Imaging In Vivo

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

Imagem bacteriana Bioluminescent<em&gt; Em Vivo</em

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

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

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

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

Não-invasiva de Imagem Óptico da vasculatura linfática de um rato

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

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

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

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

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

No método de imagem in vivo Distinguir inflamação aguda e crônica

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

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

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

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

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

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval de<em&gt; Streptococcus iniae</em&gt; Infection

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

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

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

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

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

Murino Linfócitos Rotulagem por<sup&gt; 64</sup&gt; Cu-Antibody Receptor Segmentação para<em&gt; In Vivo</em&gt; Tráfico celular por PET / CT

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

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

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

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

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

Deteção de colonização Enterohemorrhagic Escherichia Coli murino Host pelo sistema não-invasivo na Vivo bioluminescência

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