Este trabalho é apoiado pelo Laboratório Jackson Câncer Piloto Centro Grant e The Maine Cancer Foundation.

1. Protocolo <ol><li> Anestesiar o rato com cetamina e xilazina na dose de 0,1 mg de cetamina e 0,01 mg de xilazina por 1 g de peso corporal. Carprofeno (0,005 mg por 1 g de peso corporal) é utilizado para a analgesia e é administrado antes da cirurgia. </li><li> Todos os instrumentos cirúrgicos, incluindo a broca dental, são esterilizados a vapor em autoclave. Se cirurgias de lote devem ser realizados, as pontas dos instrumentos cirúrgicos devem ser re-esterilizado com uma esfera de vidro esterilizador (Fine Ciência Ferramentas FST 250) antes de cada cirurgia subsequente. </li><li> Uma vez que o rato atingiu um plano cirúrgico de anestesia, avaliada pela ausência de uma resposta a uma pitada dedo do pé, que é colocado sobre uma almofada de algodão, que é então colocado sobre uma almofada de aquecimento para manter a temperatura do corpo de ~ 37 ° C. A profundidade da anestesia é frequentemente monitorizado durante a cirurgia. </li><li> Pele é removida da cabeça do dorso, em uma área de aproximadamente triangular ~ 3 milímetros caudal para as narinas e estendendo-se caudalmente para as cervical vértebras. Pomada oftálmica é aplicada para os olhos para protegê-los de desidratação e irritação. </li><li> O rato é colocado na recumbancy ventral e a pele é desinfectada com iodo cirúrgico, etanol a 70%, e compressas estéreis. A 0,8 x 1,0 cm aba de pele que cobre a região dorsal dos ossos frontal e parietal do crânio é removida usando tesoura fina. </li><li> Um analgésico tópico, lidocaína a 0,5%, é aplicado localmente ao periósteo e os tecidos expostos na periferia da ferida. O periósteo é removida por raspagem suave do crânio com uma lâmina de bisturi, o que irá ajudar a cola de cianoacrilato para aderir ao osso. </li><li> A área cortical de interesse é delineada no crânio com um marcador. A área de interesse não deve ser localizado por cima das linhas de sutura do crânio, a fim de evitar danos para os grandes vasos subjacentes. </li><li> Uma fina camada de adesivo de cianoacrilato é aplicado às margens da ferida, para impedir a infiltração de fluido serosanguinolento, e ao crânio, excepto ema área de interesse. </li><li> Activado pela luz cimento dentário é utilizado no protocolo para a selagem do crânio. Antes da aplicação do cimento dental, tudo crânio exposta à excepção da área destinada ao desbaste é tratado com os iniciadores e subsequentemente com o agente de ligação do kit de cimento activado pela luz dental. Uma vez que o agente de ligação é seco, o cimento dental activado pela luz é aplicado para cobrir o crânio. Para estabilizar a cabeça do rato para cirurgia posterior e de imagem, uma porca hexagonal de estéril # 00-90 está incorporado no cimento dentário a uma distância da área a ser diluído. O uso da luz ativado cimento dental permite-nos preparar o crânio, sem ter que se apressar para terminar todo o procedimento. Uma vez que a preparação do cimento dental no crânio é completada, o cimento dentário é curado com luz visível. </li><li> Após o cimento dentário é curado, um suporte do dispositivo de capacete estereotáxica é montada na porca hexagonal na cabeça do rato utilizando um parafuso # 00-90. </li><li> A drop da temperatura ambiente, 0,9% de solução salina estéril é aplicada à área do crânio para ser diluído. Com o auxílio de um estereomicroscópio, uma broca de alta velocidade de micro-é utilizado para diluir uma área circular do crânio (tipicamente 4-5 mm ~ de diâmetro) através da região de interesse. Perfuração e a aplicação de uma solução salina para a área perfurada são realizados de forma intermitente durante o procedimento de desbaste para evitar lesão térmica do tecido cerebral subjacente. Salina absorve o calor e também ajuda a amaciar o osso. </li><li> O crânio do rato compreende duas camadas finas de osso compacto, imprensando uma espessa camada de osso esponjoso. A camada externa de osso compacto e a maior parte do osso esponjoso são removidos com um berbequim. </li><li> Depois de a maior parte do osso esponjoso é removida, as cavidades remanescentes dentro do osso esponjoso pode ser vista sob um microscópio de dissecação, o que indica que se aproxima a perfuração da camada interna do osso compacto. Nesta fase, a espessura crânio deve ainda ser mais do que 50 um. Crânio de desbaste deve sercontinuou com cuidado para se obter uma preparação muito fina (~ 20 mm) e liso. </li><li> Uma vez que a espessura desejada do crânio é atingida (~ 20 mm), o crânio é primeiramente polida usando um chicote de silicone feito por medida com pasta de diamante (6-15 mm) para cerca de 10 min. O polimento de diamantes colar serve a dois propósitos. Primeiro, ele ainda dilui o crânio, sem a aplicação de pressão sobre o crânio diluído, evitando danos acidentais no tecido cerebral subjacente. Em segundo lugar, serve como um passo inicial para o polimento do crânio diluído antes do óxido de estanho de grão fino é utilizado para o polimento crânio adicional. </li><li> Seguindo o polimento pasta de diamante, o crânio diluído é polido com óxido de estanho por aproximadamente 10 min. Uma vez que o crânio é polido, o óxido de estanho é lavada com solução salina fora do crânio até diluído aparece limpo. </li><li> Neste passo, se a janela craniana é para ser utilizado para imagem in vivo longitudinal, a área limpa crânio fino é seco. Uma pequena gota de claracola de cianoacrilato é aplicado a vedar a janela craniana. A camada de cola de cianoacrilato entre o crânio e diluído a lamela deverá ser tão fino quanto possível. </li><li> Se uma injecção está a ser realizado após a criação de uma janela craniana portos, neste passo, uma agulha de seringa de calibre 26 é usado para criar uma pequena abertura de um lado da janela do crânio fino polido. Esta abertura é utilizada para a injecção das células de GBM. </li><li> Uma pipeta de vidro esterilizada com uma ponta de abertura de cerca de 50 um de diâmetro, é puxada através de um extractor de pipeta. Esta pipeta será usado para a injecção de células de GBM, e é carregado em um micromanipulador. A pipeta é carregado com um ângulo de 30 graus de modo que o local da injecção das células será GBM para longe do local de imagem final. A ponta da pipeta é removida sob um microscópio de dissecação. A suspensão de células de GBM é concentrada na fase inicial para a pipeta por sucção através de uma bomba de seringa. </li><li> Uma vez que a pipeta de injeção é carregado com células GBM, o mouse é movido para trás em pomicroscópio e. A pipeta de injecção é reduzida para a pequena abertura na janela craniana e, finalmente, inserido no cérebro através da abertura usando um micromanipulador. 1 ul de uma suspensão de células de GBM é injectado no cérebro 100-200 ^ M a partir da superfície do cérebro (velocidade: 0,1 ul / min, a duração: 10 min). </li><li> O crânio é seco, uma pequena gota de cola de cianoacrilato claro é aplicado ao crânio seco, e uma peça de 3 mm de tamanho # 1 lamela é aderida à superfície do crânio fino e polido. O espaço entre as lamelas e cimento dental adjacente é selada com cola de cianoacrilato. </li><li> Após a cirurgia, o rato é injectado subcutaneamente com 1 ml de solução salina estéril quente e provido de calor suplementar para manter a temperatura corporal até totalmente recuperado da anestesia. </li><li> Para imagiologia, os ratos são anestesiados com cetamina e xilazina na dose de 0,1 mg de cetamina e 0,01 mg de xilazina por 1 g de peso corporal. Ratos são imobilizadas utilizando uma moldura estereotáxica personalizadosob o microscópio. Se repetitivo imagem in vivo é realizada em base diária, isofluorano vai ser uma opção melhor, como os ratos mostram sinais de dependência com o uso repetitivo da cetamina. </li></ol> 2. Resultados representativos Um sucesso portos cirurgia craniana janela permite que a janela craniana permanecer claro durante semanas a meses. Figura 1 mostra a vasculatura debaixo de uma janela Portas craniana como visualizado utilizando luz retroespalhados. Estas imagens vasculatura foram usados ​​como pontos de referência para localizar as mesmas áreas do cérebro para imagens repetitivo. Para visualizar tanto as células endoteliais e células vasculatura GBM, atravessamos a B6.Cg-Tg (Tek-CRE) 1Ywa / J mouse, que rotula células endoteliais vasculares com o B6.Cg-Gt (ROSA) 26Sor tm9 (CAG-tdTomato ) HZE / J rato e ratos gerados com células endoteliais vasculares relacionados com a tdTomato proteína fluorescente vermelha. Em seguida, realizou o procedimento de craniotomia portas nestes milhasce, e injectado GFP marcadas com células de GBM em seus cérebros através de uma pequena abertura no lado de uma janela craniana. Procedeu-se então in vivo de dois fotões imaging longitudinalmente sobre os ratos injectados. O comprimento de onda de excitação de laser foi de 910 nm, o que resultou na excitação de GFP e tdTomato ambos. Dois fótons de imagem foi realizada num construído sob encomenda de dois fotões microscópio de varredura a laser com dois detectores para a capacidade de detecção simultânea verde / vermelho de fluorescência. Figura 2 mostra um exemplo em vivo de iniciação a partir de células de glioma de GBM injetados perto da vasculatura. A janela Portas craniana também é uma excelente opção para crônica lapso de tempo em imagem in vivo. Figura 3 mostra coloração GFAP de seções corticais de um mouse sem cirurgia portas, um rato 7 dias após a cirurgia portos, e um rato 7 dias após a cirurgia portos e injecção de solução salina. É evidente a partir das imagens que o mouse sem cirurgia e do rato com PORTS cirurgia não tem gliose, enquanto que após a injecção de soro fisiológico, gliose é observada no tecido do cérebro do rato, devido ao pipetar penetração e injecção de soro fisiológico no cérebro. Este resultado demonstra que gliose não ocorre sob a janela Portas craniana, proporcionando validação independente de que as portas janela craniana descritos aqui não levar à gliose que é tipicamente associada com um &quot;crânio aberto&quot; janela crônica craniano. Figura 4 mostra de alta resolução imagem de espinhas dendríticas do mesmo mouse no dia da cirurgia (dia 0) e 32 dias após a criação da janela Portos (dia 32), demonstrando que a janela de portas também é uma excelente opção para alta resolução de imagem in vivo. <img alt="Figura 1" src="/files/ftp_upload/4201/4201fig1.jpg" /> Figura 1. Visualização dos vasos sob uma janela crânio polido e reforçado diluído craniana usando luz retroespalhados. Dias representam t<html> ele o número de dias após a cirurgia, e as imagens foram tiradas com início no dia da cirurgia, imediatamente após a conclusão do GBM injecção das células. Barra de escala:. 0,5 milímetros <a href="http://www.jove.com/files/ftp_upload/4201/4201fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="Figura 2" src="/files/ftp_upload/4201/4201fig2.jpg" /> Figura 2. Visualização de células injectadas GBM e vasculatura in vivo. Camundongos portadores tanto Tek-Cre e Rosa26 CAG-tdTomato foram usados ​​para injeção de células GBM. As células endoteliais vasculares foram marcadas com tdTomato (vermelho), e as células foram marcadas com GBM GFP (verde). As imagens foram adquiridas começando 24 horas após a injecção das células de GBM. Um total de 10-12 campos adjacentes de vista foram colhidas e foram montadas adjacentes uma à outra, com as células identificadas GBM injectadas no centro do campo total, para produzir as imagens finais. Dias representam o número de dias após a cirurgia. Barra de escala: 100 m.href = &quot;http://www.jove.com/files/ftp_upload/4201/4201fig2large.jpg&quot; target = &quot;_blank&quot;&gt; Clique aqui para ver maior figura. <img alt="Figura 3" src="/files/ftp_upload/4201/4201fig3.jpg" /> Figura 3. Gliose não ocorrer após a cirurgia portas janela craniano. Painéis superiores: seção Rato cérebro coronal em baixa ampliação. Painel esquerdo: um rato sem qualquer cirurgia janela craniana; nenhuma activação GFAP é observado no tecido cerebral. Painel do meio: um rato com uma janela craniana portos gerado no lado direito do cérebro, sem activação GFAP é observado no tecido cerebral. Painel da direita: um mouse com uma janela Portas craniano e injeção de solução salina do lado da janela Portas. Activação GFAP é observado do lado da janela portas, mas não sobre o lado do tecido do cérebro intacto. Barra de escala: 1 mm. Painel inferior: maior potência imagens dos tecidos cerebrais em áreas como indicado na caixa em branco das imagens superior do painel. Barra de escala: 100 m. <ahref = &quot;http://www.jove.com/files/ftp_upload/4201/4201fig3large.jpg&quot; target = &quot;_blank&quot;&gt; Clique aqui para ver maior figura. <img alt="Figura 4" src="/files/ftp_upload/4201/4201fig4.jpg" /> Figura 4. Imagens de alta resolução de espinhos dendríticos de Thy-1 ratos YFPH. As imagens foram adquiridas no dia da cirurgia portos, e 32 dias após a cirurgia portas. É evidente a partir das imagens que a maioria dos espinhos apresentam no dia 0 são claramente visíveis 32 dias após a cirurgia portas. Barra de escala: 2 m.

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A chave para uma janela portuária craniana é o desbaste e polimento. Enquanto o desbaste inicial pode ser realizada rapidamente, deve ser tomado cuidado para assegurar homogeneidade adelgaçamento do crânio sobre uma grande área. Nós tipicamente aplicar uma camada fina de solução salina ao crânio, e depois dilua o crânio uma passagem de cada vez, de tal modo que a solução salina se evapora logo após a microdrill passou sobre o crânio de uma só vez. Isto permite-nos lentamente, mas de forma homogênea mais ...

<table border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> DMEM/F12 (1:1), com piruvato de sódio </td><td> Thermo Sci Hyclone </td><td> SH3026101 </td><td></td></tr><tr><td> B-27 Suplemento livre de soro (50x) </td><td> Invitrogen </td><td> 17504-044 </td><td></td></tr><tr><td> GlutaMax-1 Suplemento </td><td> Invitrogen </td><td> 35050061 </td><td></td></tr><tr><td> Penicilina-Estreptomicina Solução </td><td> Thermo Sci Hyclone </td><td> SV30010 </td><td></td></tr><tr><td> Accutase Enzyme-Médio Destacamento celular </td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> T25 frascos-vent tampa verde </td><td> SARSTEDT </td><td> 83.1810.502 </td><td></td></tr><tr><td> Álcool a 70% </td><td> JAX SCAF </td><td></td><td></td></tr><tr><td> 10% de solução de iodo-povidona tópica </td><td> JAX SCAF </td><td></td><td></td></tr><tr><td> Ketamina HCl </td><td> Butler Abastecimento Saúde Animal </td><td> NDC # 11695-0550-1 </td><td></td></tr><tr><td> Xilazina </td><td> Akorn, Inc. </td><td> NADA # 139-236 </td><td></td></tr><tr><td> Carprofeno </td><td> JAX SCAF </td><td></td><td></td></tr><tr><td> Pomada oftálmica </td><td> Produtos Veterinários Dechra </td><td> 17033-211-38 </td><td></td></tr><tr><td> 0,5% de lidocaína HCl </td><td> REGENT, Inc. </td><td> NDC 0517-0625-25 </td><td></td></tr><tr><td> Cola de cianoacrilato </td><td> Henkel Corp </td><td> 46551 </td><td></td></tr><tr><td> Swabs estéreis </td><td> Fisher Scientific </td><td> 23-400-114 </td><td></td></tr><tr><td> Pasta de diamante </td><td> Fonte Widget </td><td> BBE60 </td><td></td></tr><tr><td> Óxido de estanho </td><td> LORTONE, Inc. </td><td> 591-038 </td><td></td></tr><tr><td> Forro de Bond 2V </td><td> KURARAY Medical Inc. </td><td> 1921-KA </td><td></td></tr><tr><td> Clearfil AP-X </td><td> KURARAY Medical Inc. </td><td> 1721-KA </td><td></td></tr><tr><td> Salina </td><td> JAX SCAF </td><td></td><td></td></tr><tr><td> Tampa de vidro </td><td> Warner Instruments </td><td> 64-0720 </td><td></td></tr><tr><td> Porca Hex </td><td> Peças pequenas, Inc. </td><td> HNX-0090-C </td><td></td></tr><tr><td> Bomba de seringa </td><td> Syringepump.com </td><td> NE-1000 </td><td></td></tr><tr><td> Two-Photon sistema de imagem </td><td> Personalizado construído (Qualquer sistema comercial seriatrabalhar) </td><td></td><td></td></tr></table>

imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window

Glioma é a uma das formas mais mortais de cancro humano. A terapia mais eficaz para glioma a data da cirurgia, seguida de radioterapia oferece apenas pacientes benefícios modestos, como a maioria dos pacientes não sobrevivem mais de cinco anos após o diagnóstico, devido à glioma 1,2 recaída. A descoberta de células-tronco cancerosas em tumores cerebrais humanos tem a promessa de ter um enorme impacto sobre o desenvolvimento de novas estratégias terapêuticas para glioma 3. As células-tronco cancerosas são definidas pela sua capacidade tanto de se auto-renovar e diferenciar, e acredita-se ser as únicas células em um tumor que têm a capacidade de iniciar novos tumores 4. Glioma recaída radioterapia seguinte é pensado para surgir a partir da resistência de células estaminais de glioma (GSCS) à terapia 5-10. In vivo, GSCS são mostrados a residir num nicho perivascular, que é importante para manter as suas células estaminais características semelhantes a 11-14 . Central para a organizaçãozação do nicho SGC são células endoteliais vasculares 12. Evidências sugerem que o GSCS e sua interação com as células endoteliais vasculares são importantes para o desenvolvimento do tumor e identificar GSCS e sua interação com células endoteliais como importantes alvos terapêuticos para glioma. A presença de GSCS é determinado experimentalmente pela sua capacidade de iniciar novos tumores após transplante ortotópico 15. Isto é normalmente conseguido através da injecção de um número específico de células de GBM isoladas a partir de tumores humanos no cérebro de severamente imunodeficientes ratos, ou de células de rato GBM em cérebros de ratinhos congénicos hospedeiras. Os ensaios para o crescimento do tumor são, em seguida, realizada de acordo com o tempo suficiente para permitir que GSCS entre as células injectadas GBM para dar origem a novos tumores, tipicamente várias semanas ou meses. Assim, os ensaios existentes não permitem a análise do processo patológico de iniciação do tumor de GSCS individuais in vivo. Conseqüentemente, essencial insights para as funções específicas de GSCS e sua interação com as células endoteliais vasculares nos primeiros estágios de iniciação do tumor estão faltando. Tais revelações são críticos para o desenvolvimento de novas estratégias terapêuticas para glioma, e terá grandes implicações para a prevenção de recaídas em pacientes glioma. Aqui nós adaptamos as portas procedimento de janela craniana 16 e in vivo de dois fótons de microscopia para permitir a visualização de iniciação do tumor a partir de células de GBM injetadas no cérebro de um rato vivo. Nossa técnica irá pavimentar o caminho para futuros esforços para elucidar os principais mecanismos de sinalização entre GSCS e células endoteliais vasculares durante o início glioma.

Watch this Scientific Journal Video about Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window at JoVE.com

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Ao combinar um polido e reforçado crânio-fino (portas) craniana janela e injeção de células do glioblastoma (GBM), podemos observar glioma iniciação e crescimento de células GBM injetadas no cérebro de um rato vivo longitudinalmente.

Imagem Glioma Iniciação<em&gt; Em Vivo</em&gt; Através de um polido e reforçado Janela crânio-Thin craniana

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

imaging-glioma-initiation-vivo-through-polished-reinforced-thin-skull

Research

JoVE Journal

Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

13.0K visualizações.  The Jackson Laboratory. Ao combinar um polido e reforçado crânio-fino (portas) craniana janela e injeção de células do glioblastoma (GBM), podemos observar glioma iniciação e crescimento de células GBM injetadas no cérebro de um rato vivo longitudinalmente.

Vídeo: Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

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

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

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

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

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 ...

Medicina

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 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotípica tridimensional<em&gt; In Vitro</em&gt; Modelagem de Estroma-epitelial de ovário Interações durante o início e progressão do câncer

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Utilizando uma janela craniana para Visualizar a artéria cerebral média Durante endotelina 1 Induzida por oclusão da artéria cerebral média

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-neuronavegação<em&gt; In Vivo</em&gt; Através do cérebro de um paciente durante uma espontânea enxaquecas

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Seqüente<em&gt; In vivo</em&gt; Imagem de osteogênicas células-tronco / progenitoras durante o reparo de fratura

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

Estado da Arte Cranial Ecografia em recém-nascidos

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria Intrauterine medir Rato contrátil Pressão<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

In Vivo Imaging and Rastreamento de Tecnécio-99m Rotulado de Medula Óssea Células-Tronco Mesenquimais em Equine Tendinopatia

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...