Este trabajo es apoyado por The Jackson Laboratory Cancer Center Pilot Grant y La Fundación del Cáncer de Maine.

1. Protocolo <ol><li> Se anestesia el ratón con ketamina y xilazina a dosis de ketamina 0,1 mg y 0,01 mg de xilazina por 1 g de peso corporal. Carprofeno (0,005 mg por 1 g de peso corporal) se utiliza para la analgesia y se administra antes de la cirugía. </li><li> Todas las herramientas quirúrgicas, como la broca dental, son esterilizados con vapor en un autoclave. Si cirugías lotes se van a realizar, las puntas de los instrumentos quirúrgicos deben ser re-esterilizado con un esterilizador de cuentas de vidrio (Fine Ciencia Herramientas FST 250) antes de cada cirugía posterior. </li><li> Una vez que el ratón se ha alcanzado un plano quirúrgico de anestesia, evaluada por la ausencia de una respuesta a una pizca dedo del pie, que se coloca sobre una almohadilla de algodón que se coloca a continuación en una almohadilla de calefacción para mantener una temperatura corporal de ~ 37 ° C. La profundidad de la anestesia se monitoriza con frecuencia durante la cirugía. </li><li> Fur se retira de la cabeza de dorsal en un área aproximadamente triangular ~ 3 mm caudal a los orificios nasales y se extiende caudalmente a la cervical vértebras. Pomada oftálmica se aplica a los ojos para protegerlos de la deshidratación y la irritación. </li><li> El ratón se coloca en recumbancy ventral y la piel se desinfecta con yodo quirúrgica, 70% de etanol, y torundas estériles. D 0,8 x 1,0 cm colgajo de piel que cubre la cara dorsal de los huesos frontal y parietal del cráneo se retira con unas tijeras finas. </li><li> Un analgésico tópico, 0,5% de lidocaína, se aplica localmente en el periostio y los tejidos expuestos en la periferia de la herida. El periostio se quita raspando suavemente el cráneo con una hoja de bisturí, esto ayudará a que el pegamento de cianoacrilato para adherirse al hueso. </li><li> El área cortical de interés se resume en el cráneo con un marcador. El área de interés no deberían estar situados sobre las líneas de sutura del cráneo, para evitar daños a los buques grandes subyacentes. </li><li> Una capa delgada de adhesivo de cianoacrilato se aplica a los bordes de la herida, para evitar la filtración del fluido serosanguinolento, y en el cráneo, excepto enel área de interés. </li><li> Activado por la luz cemento dental se utiliza en el protocolo para el sellado de la calavera. Antes de la aplicación de cemento dental, todo el cráneo expuesto a excepción de la zona prevista para el adelgazamiento se trata con los cebadores y posteriormente con el agente de unión del kit de cemento dental activado por luz. Una vez que el agente de unión se secó, el cemento dental activado por luz se aplica para cubrir el cráneo. Para estabilizar la cabeza del ratón para la cirugía posterior y formación de imágenes, un estéril # 00-90 tuerca hexagonal está incrustado en el cemento dental a una distancia de la zona a ser adelgazada. El uso de luz activado por cemento dental nos permite preparar el cráneo sin tener que apresurarse para terminar el procedimiento. Una vez que la preparación del cemento dental en el cráneo se ha completado, el cemento dental se cura con luz visible. </li><li> Después de que el cemento dental se cura, un soporte de pieza de cabeza desde el dispositivo estereotáctico está montado en la tuerca hexagonal en la cabeza del ratón utilizando un tornillo # 00-90. </li><li> A drop de la temperatura ambiente, 0,9% de solución salina estéril se aplica a la zona del cráneo a ser adelgazada. Con la ayuda de un estereomicroscopio, una alta velocidad de micro-perforación se utiliza para adelgazar un área circular del cráneo (normalmente ~ 4-5 mm de diámetro) sobre la región de interés. Perforación y la aplicación de la solución salina al área perforada se realizan de forma intermitente durante el procedimiento de adelgazamiento para evitar la lesión térmica al tejido cerebral subyacente. Saline absorbe el calor y también ayuda a suavizar el hueso. </li><li> El cráneo de ratón comprende dos capas delgadas de hueso compacto, intercalando una capa gruesa de hueso esponjoso. La capa externa de hueso compacto y la mayoría del hueso esponjoso se eliminan utilizando el taladro. </li><li> Después de que la mayoría del hueso esponjoso se retira, las cavidades que quedan dentro del hueso esponjoso puede ser visto bajo un microscopio de disección, lo que indica que la perforación se acerca a la capa interna de hueso compacto. En esta etapa, el espesor del cráneo todavía debe ser más de 50 micras. Cráneo adelgazamiento debe sersiguió cuidadosamente para obtener una preparación muy delgada (~ 20 m) y suave. </li><li> Una vez que el grosor deseado de cráneo se consigue (~ 20 m), el cráneo es primero pule con un látigo de silicona a medida con pasta de diamante (6-15 micras) durante aproximadamente 10 min. El pulido de diamantes pasta sirve para dos propósitos. En primer lugar, además adelgaza el cráneo sin la aplicación de presión en el cráneo adelgazada, evitando así el daño accidental al tejido cerebral subyacente. En segundo lugar, sirve como un paso de pulido inicial para el cráneo antes de diluir el óxido de estaño de grano fino se utiliza para el pulido cráneo adicional. </li><li> Tras el pulido de diamantes de pasta, el cráneo adelgazada se pule con óxido de estaño de aproximadamente 10 min. Una vez que el cráneo es pulido, el óxido de estaño es arrastrada con solución salina hasta que el cráneo adelgazado vea limpia. </li><li> En este paso, si la ventana craneal se va a utilizar para longitudinal de imágenes in vivo, el área limpiada cráneo delgado se seca. Una pequeña gota de claropegamento de cianoacrilato se aplica para sellar la ventana craneal. La capa de pegamento de cianoacrilato entre el cráneo adelgazada y el cubreobjetos debe ser tan delgado como sea posible. </li><li> Si la inyección se debe realizar después de la creación de una ventana craneal puertos, en este paso, una aguja de jeringa de calibre 26-se utiliza para crear una pequeña abertura en un lado de la pulida delgada cráneo ventana. Esta abertura se utiliza para la inyección de células de glioblastoma multiforme. </li><li> Una pipeta de vidrio estéril con punta de abrir unas 50 micras de diámetro se tira con un extractor pipeta. Esta pipeta se utilizan para la inyección de células GBM, y se carga en un micromanipulador. La pipeta se carga en un ángulo de 30-grados para que el sitio de la célula de inyección GBM estará lejos del sitio de formación de imágenes final. La punta de la pipeta se retira bajo un microscopio de disección. La suspensión de células GBM es de carga frontal en la pipeta por succión con una bomba de jeringa. </li><li> Una vez que la pipeta de inyección se carga con células GBM, el ratón se mueve de nuevo bajo the microscopio. La pipeta de inyección se baja hacia la pequeña abertura en la ventana craneal y, finalmente, se inserta en el cerebro a través de la abertura utilizando un micromanipulador. 1 l de una suspensión de células GBM se inyecta en el cerebro 100-200 micras desde la superficie del cerebro (velocidad: 0,1 l / min, duración: 10 min). </li><li> El cráneo se seca, una pequeña gota de pegamento de cianoacrilato transparente se aplica al cráneo seco, y una pieza de 3-mm de tamaño # 1 cubreobjetos se adhiere a la superficie del cráneo delgado y pulido. El espacio entre el cubreobjetos y cemento dental adyacente está sellado con pegamento de cianoacrilato. </li><li> Después de la cirugía, el ratón se inyecta por vía subcutánea con 1 ml de solución salina estéril caliente y provisto de calor suplementario para mantener la temperatura del cuerpo hasta que esté completamente recuperado de la anestesia. </li><li> Para las imágenes, los ratones son anestesiados con ketamina y xilazina a dosis de ketamina 0,1 mg y 0,01 mg de xilazina por 1 g de peso corporal. Los ratones se inmovilizan utilizando un marco estereotáctico costumbrebajo el microscopio. Si repetitiva de imágenes in vivo se realiza en forma diaria, isofluorano será una mejor opción, ya que los ratones se muestran signos de dependencia con el uso repetitivo de la ketamina. </li></ol> 2. Los resultados representativos Una cirugía exitosa puertos ventana craneal permite que la ventana craneal a quedar claro por semanas o meses. Figura 1 muestra la vasculatura debajo de una ventana Puertos craneal como visualizó con luz retrodispersada. Estas imágenes de la vasculatura fueron utilizados como puntos de referencia para la localización de las mismas áreas del cerebro para imágenes repetitivas. Para visualizar tanto las células endoteliales y las células de la vasculatura GBM, cruzamos el B6.Cg-Tg (Tek-cre) 1Ywa / J ratones, que las etiquetas de las células endoteliales vasculares con el B6.Cg-Gt (ROSA) 26Sor TM9 (CAG-tdTomato ) Hze / J del ratón, y los ratones generados con las células endoteliales vasculares marcadas con el tdTomato proteína fluorescente roja. A continuación, realiza el procedimiento de craneotomía en estos puertos millasce, y se inyectaron células marcadas con GFP GBM en sus cerebros a través de una pequeña abertura en el lado de una ventana craneal. A continuación, realiza in vivo de dos fotones de imágenes longitudinalmente en los ratones inyectados. La longitud de onda de excitación del láser se fijó en 910 nm, lo que resultó en la excitación tanto de GFP y tdTomato. Dos fotones de imágenes se realizó en una costumbre-construido de dos fotones láser de barrido microscopio con dos detectores para la detección de capacidad simultánea verde / rojo de fluorescencia. Figura 2 muestra un ejemplo en vivo de iniciación glioma de células GBM inyecta cerca de la vasculatura. La ventana Puertos craneal es también una excelente opción para la crónica time-lapse de imágenes in vivo. Figura 3 muestra la tinción GFAP secciones de la corteza de un ratón sin necesidad de cirugía puertos, un ratón de 7 días después de la cirugía de los puertos, y un ratón de 7 días después de la cirugía y Puertos inyección de solución salina. Es evidente a partir de las imágenes que el ratón sin necesidad de cirugía y el ratón con PoRCirugía TS no tienen gliosis, mientras que tras la inyección de solución salina, gliosis se observa en el tejido cerebral del ratón, debido a pipetear la penetración y la inyección de solución salina en el cerebro. Este resultado demuestra que gliosis no se produce en la ventana de puertos craneal, proporcionando validación independiente de que los puertos ventana craneal descritos aquí no conduce a la gliosis que se asocia típicamente con un &quot;cráneo abierto&quot; crónica ventana craneal. Figura 4 muestra de alta resolución imágenes de las espinas dendríticas de ratón del mismo en el día de la cirugía (día 0) y 32 días después de la creación de la ventana Puertos (día 32), lo que demuestra que la ventana de puertos es también una excelente opción para la alta resolución de imágenes in vivo. <img alt="Figura 1" src="/files/ftp_upload/4201/4201fig1.jpg" /> Figura 1. Visualización de la vasculatura debajo de una ventana cráneo pulido y reforzado adelgazado craneal con luz retrodispersada. Días representar t<html> que el número de días después de la cirugía; imágenes fueron tomadas a partir del día de la cirugía inmediatamente después de la finalización de la inyección de células GBM. Barra de escala:. 0,5 mm <a href="http://www.jove.com/files/ftp_upload/4201/4201fig1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img alt="Figura 2" src="/files/ftp_upload/4201/4201fig2.jpg" /> Figura 2. Visualización de las células inyectadas GBM y la vasculatura in vivo. Los ratones que llevan tanto Tek-Cre y ROSA26 CAG-tdTomato fueron utilizados para la inyección de células de glioblastoma multiforme. Las células endoteliales vasculares se marcaron con tdTomato (rojo), y las células se marcaron con GBM GFP (verde). Las imágenes fueron adquiridas a partir de 24 horas después de la inyección de células GBM. Un total de 10-12 campos adyacentes de vista fueron tomadas y se montaron una junto a otra, con las células inyectadas GBM identificados en el centro del campo total, para producir las imágenes finales. Días representan el número de días después de la cirugía. Barra de escala: 100 m.href = &quot;http://www.jove.com/files/ftp_upload/4201/4201fig2large.jpg&quot; target = &quot;_blank&quot;&gt; Haga clic aquí para ampliar la figura. <img alt="Figura 3" src="/files/ftp_upload/4201/4201fig3.jpg" /> Figura 3. Gliosis no se produce después de la cirugía puertos ventana craneal. Paneles superiores: Sección coronal de cerebro de ratón bajo condiciones de baja magnificación. Panel izquierdo: un ratón sin ninguna cirugía ventana craneal; no activación GFAP se observa en el tejido cerebral. Medio panel: un ratón con una ventana craneal puertos generado en el lado derecho del cerebro; no activación GFAP se observa en el tejido cerebral. Panel derecho: un ratón con una ventana craneal puertos e inyección de solución salina desde el lado de la ventana Puertos. Activación GFAP se observa en el lado de la ventana de puertos, pero no en el lado del tejido del cerebro intacto. Barra de escala: 1 mm. Panel inferior: de mayor potencia imágenes de los tejidos del cerebro en las áreas que se indican en el cuadro blanco de las imágenes de pantalla superior. Barra de escala: 100 m. <ahref = &quot;http://www.jove.com/files/ftp_upload/4201/4201fig3large.jpg&quot; target = &quot;_blank&quot;&gt; Haga clic aquí para ampliar la figura. <img alt="Figura 4" src="/files/ftp_upload/4201/4201fig4.jpg" /> Figura 4. Alta resolución de imagen de las espinas dendríticas de ratones Thy-1 YFPH. Las imágenes fueron adquiridas en el día de la cirugía de los puertos, y 32 días después de la cirugía de los puertos. En efecto, de las imágenes que la mayoría de las espinas presentan en el día 0 son claramente visibles 32 días después de la cirugía puertos. Barra de escala: 2 micras.

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No hay conflictos de interés declarado.

La clave de una ventana craneal puertos exitoso es el adelgazamiento y pulido. Mientras que el adelgazamiento inicial puede ser realizada rápidamente, se debe tener cuidado para asegurar homogénea adelgazamiento del cráneo sobre un área grande. Nos típicamente aplicar una capa delgada de solución salina al cráneo, y luego delgada del cráneo una pasada a la vez, tal que la solución salina se evapora poco después de la microtaladro ha pasado sobre el cráneo una vez. Esto nos permite lenta pero más homogénea d...

<table border="1"><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> DMEM/F12 (1:1) con piruvato de sodio </td><td> Thermo Sci Hyclone </td><td> SH3026101 </td><td></td></tr><tr><td> B-27 Suplemento libre de suero (50x) </td><td> Invitrogen </td><td> 17504-044 </td><td></td></tr><tr><td> GlutaMax-1 Supplement </td><td> Invitrogen </td><td> 35050061 </td><td></td></tr><tr><td> Solución de penicilina-estreptomicina </td><td> Thermo Sci Hyclone </td><td> SV30010 </td><td></td></tr><tr><td> Accutase-enzima Tono Medio Desprendimiento </td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> T25 frascos de ventilación tapa verde </td><td> SARSTEDT </td><td> 83.1810.502 </td><td></td></tr><tr><td> 70% de alcohol </td><td> JAX LAHS </td><td></td><td></td></tr><tr><td> 10% solución de povidona yodada tópica </td><td> JAX LAHS </td><td></td><td></td></tr><tr><td> La ketamina HCl </td><td> Mayordomo Animal Health Supply </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> Carprofen </td><td> JAX LAHS </td><td></td><td></td></tr><tr><td> De pomada oftálmica </td><td> Dechra veterinarios </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> Pegamento de cianoacrilato </td><td> Henkel Corp. </td><td> 46551 </td><td></td></tr><tr><td> Los hisopos estériles </td><td> Fisher Scientific </td><td> 23-400-114 </td><td></td></tr><tr><td> Diamond pasta </td><td> Widget de suministro </td><td> BBE60 </td><td></td></tr><tr><td> Óxido de estaño </td><td> LORTONE, Inc. </td><td> 591-038 </td><td></td></tr><tr><td> Liner 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 LAHS </td><td></td><td></td></tr><tr><td> Cubierta de vidrio </td><td> Warner Instruments </td><td> 64-0720 </td><td></td></tr><tr><td> Tuerca hexagonal </td><td> Small Parts, Inc. </td><td> HNX-0090-C </td><td></td></tr><tr><td> Syringe Pump </td><td> Syringepump.com </td><td> NE-1000 </td><td></td></tr><tr><td> Two-Photon Imaging System </td><td> Hecha a la medida (Cualquier sistema comercial seríatrabajar) </td><td></td><td></td></tr></table>

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

El glioma es el de las formas más letales de cáncer humano. La terapia del glioma más eficaz hasta la fecha de la cirugía seguida de radiación con el tratamiento ofrece al paciente modestos beneficios, como la mayoría de los pacientes no sobreviven más de cinco años después del diagnóstico debido a glioma 1,2 recaída. El descubrimiento de las células madre del cáncer en los tumores cerebrales humanos promete tener un impacto enorme en el desarrollo de nuevas estrategias terapéuticas para el glioma 3. Las células madre cancerosas se definen por su capacidad tanto de auto-renovación y diferenciar a, y se cree que son las únicas células en un tumor que tiene la capacidad de iniciar nuevos tumores 4. Glioma terapia de recaída después de la radiación se cree que surgen de la resistencia de las células madre de glioma (GSCS) a la terapia 5-10. In vivo, GSCS se muestran a residir en un nicho perivascular que es importante para mantener sus madre similares a células características 11-14 . Central a la organizaciónción del nicho GSC son las células endoteliales vasculares 12. La evidencia actual sugiere que GSCS y su interacción con las células endoteliales vasculares son importantes para el desarrollo del tumor, e identificar GSCS y su interacción con las células endoteliales como importantes dianas terapéuticas para el glioma. La presencia de GSCS se determina experimentalmente mediante su capacidad de iniciar nuevos tumores en trasplante ortotópico 15. Esto se consigue normalmente mediante la inyección de un número específico de células aisladas de tumores GBM humanos en los cerebros de severamente inmuno-ratones deficientes, o de células de ratón de GBM en los cerebros de los ratones receptores congénicos. Los ensayos para el crecimiento del tumor se realiza entonces después de un tiempo suficiente para permitir GSCS entre las células inyectadas GBM para dar lugar a nuevos tumores-típicamente de varias semanas o meses. Por lo tanto, los ensayos existentes no permiten examinar el importante proceso patológico de la iniciación del tumor de GSCS solteras en vivo. Por consiguiente, es esencial quensights en las funciones específicas de GSCS y su interacción con las células endoteliales vasculares en las primeras etapas de la iniciación del tumor se carece. Estas ideas son fundamentales para el desarrollo de nuevas estrategias terapéuticas para el glioma, y ​​tendrá grandes implicaciones para la prevención de recaídas en pacientes con glioma. Aquí se han adaptado los puertos procedimiento de ventana craneal 16 e in vivo de dos fotones microscopía para permitir la visualización de la iniciación del tumor a partir de células de GBM inyectados en el cerebro de un ratón vivo. Nuestra técnica allanará el camino para los futuros esfuerzos por esclarecer los mecanismos clave de señalización entre GSCS y células endoteliales vasculares durante el inicio del 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

Mediante la combinación de un pulido y reforzado delgada cráneo (puertos) craneal ventana y glioblastoma (GBM) la inyección de células, se puede observar la iniciación glioma y el crecimiento de las células de GBM inyectados en el cerebro de un ratón vivo longitudinalmente.

Imágenes Glioma Iniciación<em&gt; En Vivo</em&gt; A través de un pulido y reforzado fina calavera ventana craneal

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 vistas.  The Jackson Laboratory. Mediante la combinación de un pulido y reforzado delgada cráneo (puertos) craneal ventana y glioblastoma (GBM) la inyección de células, se puede observar la iniciación glioma y el crecimiento de las células de GBM inyectados en el cerebro de un ratón vivo longitudinalmente.

Video: 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.

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

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

Investigación

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.

En vivo doble sustrato bioluminiscentes imagen

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

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.

Heterotípicos tridimensional<em&gt; In Vitro</em&gt; Modelado de estromal-epitelial interacciones durante la iniciación y progresión del cáncer de ovario

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 una ventana craneal para visualizar la arteria cerebral media Durante la endotelina-1 inducida por oclusión de la arteria cerebral media

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'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-Neuronavegación<em&gt; En Vivo</em&gt; A través del cerebro de un paciente durante una espontánea migraña

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.

Secuencial<em&gt; En vivo</em&gt; Imagen de células madre / progenitoras osteogénicas durante la reparación de la fractura

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  &#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; En vivo</em&gt; Óptica y μCT imágenes para vigilar la infección, inflamación y Hueso anatomía en una infección de implantes ortopédicos en ratones

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 del Arte craneal imágenes por ultrasonido en neonatos

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.

Telemetría intrauterina para medir la presión del ratón contráctil<em&gt; En 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.

En vivo de imágenes y Seguimiento de tecnecio-99m Etiquetada células madre mesenquimales de médula ósea en Equinos tendinopatía

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