Este trabajo fue apoyado por el Instituto Karolinska KID-financiación, la Autoridad Sueca de Protección Radiológica, Instituto Karolinska Eye Research Foundation, Gun och Bertil Stohnes Stiftelse, Eye St. Erik el Hospital Research Foundation, Ögonfonden, Konung Gustav V: s och Drottning Viktorias Frimurarstiftelse.

1. La exposición a la radiación ultravioleta <ol><li> 15 min antes de la exposición, anestesiar una hembra Sprague-Dawley con una mezcla de 90 mg / kg Ketalar (ketamina) y 10 mg / kg Rompun (xilazina) por inyección intraperitoneal. </li><li> Colocar el animal en un limitador de rata y apretar las correas hasta la inmovilización de la rata sin causar una compresión de tronco 13. </li><li> Infundir Mydriacyl (tropicamida), 10 mg / ml, en ambos ojos de la rata para inducir midriasis. </li><li> Colocar el animal de modo que un ojo se coloca contra un haz estrecho de radiación UV a 300 nm con un ancho de 14 nm 10 a media altura y proteger el ojo contralateral con cinta de color negro. </li><li> Exponer la rata unilateralmente por debajo del umbral de dosis 1 kJ / m 2 RUV-B a 300 nm durante 15 minutos 15. </li></ol> 2. Disección <ol><li> Después de la pre-determinado intervalo de post-exposición (1, 5, 24 y HR 120), sacrificar la rata de seis semanas de edad (peso &lt;200 g) por carbon asfixia dióxido de carbono y la dislocación de las vértebras cervicales. </li><li> Ojos enucleación. A continuación, coloque la córnea del ojo hacia abajo, lado posterior hacia arriba. Entonces, perfore un agujero mínimo mediante la aplicación de una cánula de calibre 27 y empuje tangencial a la esclerótica para evitar dañar la lente que está muy cerca de la esclerótica. A continuación, utilice un par de tijeras de cirugía oftálmica para cortar circunferencialmente justo detrás del limbo hasta que la parte posterior de la esclerótica se puede quitar. A continuación, levante la lente con unas pinzas curvas romas. </li><li> Eliminar los restos del cuerpo ciliar desde el ecuador del cristalino bajo un microscopio, manteniendo la lente en solución salina equilibrada de no más de 5-10 min. </li></ol> 3. RT-PCR cuantitativa <ol><li> Colocar una lente en una mezcla de 350 l tampón de lisis RA1 (NucleoSpin RNA II Kit de aislamiento de ARN total) y 3,5 l β-mercaptoetanol en un tubo Eppendorf de 2 ml. </li><li> Mantener la lente en esta mezcla durante 30 min a temperatura ambiente. </li><li> Homogeneizar la lente conuna aguja hasta sólo núcleo duro del cristalino que queda de la lente (la corteza y la cápsula de la lente se lisaron en tampón de lisis). Retire el núcleo de la mezcla. </li><li> Almacenar las muestras inmediatamente a -70 ° C. </li><li> Descongele las muestras y seguir el protocolo &quot;Total purificación de ARN a partir de células cultivadas y tejidos&quot; (NucleoSpin RNA II Kit de aislamiento de ARN total). </li><li> Tienda de muestras de ARN a -70 ° C. </li><li> Para verificar la eliminación suficiente de ADN de cada muestra, ejecutar una PCR en las condiciones siguientes (paso 1: 95 º C durante 2 min; paso 2 durante 40 ciclos: 95 ° C durante 20 s, 55 ° C durante 20 s, 72 ° C durante 20 seg; paso 3: 72 º C durante 7 min) con cebadores específicos de ADN de p53, adelante 5&#39;-ACCCTCTGACCTTTTTCCCA-3 &#39;y revertir 5&#39;-TGCTGGGATCTTAGGCACTC-3&#39; y Taq ADN polimerasa (dNTPack), de acuerdo con la fabricación protocolo. El producto de PCR esperada es 243 pares de bases. </li><li> Ejecutar un 1,5% electroforesis en gel de agarosa para detectar el producto de PCR específico de ADN de 243 pares de bases. Para preparar1,5% de gel de agarosa en tampón TBE tomar 3,75 g de agarosa y resolverlo en 250 ml de tampón TBE. Añadir bromuro de etidio (0,5 mg / ml) 500 l en 500 ml de solución de gel de agarosa al 1,5%. Calentar la mezcla en un horno de microondas y se agita para resolver la agarosa. </li></ol> Ninguna de las muestras tiene que revelar cualquier producto de PCR específicas de ADN en electroforesis en gel de agarosa al 1,5%. <ol start="9"><li> Medir la concentración de las muestras de ARN (1 l) y la relación de absorbancia de la muestra a 260 nm (RNA) y 280 nm (proteína) en un NanoDrop ND-1000 espectrofotómetro. Si el ARN a la proteína de la relación de absorbancia de la muestra de ARN es 2,0 o más, entonces la muestra de ARN es puro. Si la relación de absorbancia de la muestra de ARN es menor que 2,0, rehacer etapa de purificación de ARN 3,5. </li><li> Tomar un volumen de muestra de RNA correspondiente a 1 mg y sintetizar cDNA siguiendo el protocolo 1 Kit st Strand cDNA de síntesis (1 kit st Strand cDNA de síntesis para la RT-PCR). </li><li> Almacenar las muestras de ADNc a -20 º C(Por un período de 1 y más años, almacenan a -70 ° C). </li><li> Ejecutar cuantitativa en tiempo real PCR en iCycler MyiQ único sistema de detección de color real PCR en tiempo. Carga triplicados en placas de 96 pocillos con 1 l de muestra cDNA, TaqMan Gene Expression Master Mix, ensayo TaqMan Gene Expression para la caspasa 3, de acuerdo con las instrucciones de fabricación. Carga triplica en otra placa de 96 pocillos con 1 l de muestra cDNA, TaqMan Gene Expression Master Mix, ensayo TaqMan Gene Expression para mayores de 18, de acuerdo con las instrucciones de fabricación (TaqMan Gene Expression Protocolo de ensayo). Diluir en serie una fracción de cDNA a partir de tres elegidas al azar y no expuestas lentes. Ejecutar diluciones en serie junto con el ADNc de las muestras en una placa de 96 pocillos. </li><li> Usar el método de la curva estándar para la cuantificación de los resultados. El número de ciclos en la fluorescencia umbral se utiliza como la medida en software MyiQ. Una curva estándar que expresa el número de ciclos en el umbral como función de la concentración relativa de calibrador es consultadatablished para las diluciones en serie en cada placa. ADNc a partir de tres muestras no tratadas de lentes se utiliza para la calibración. Relacionar el número de ciclos de umbral para cada muestra a la curva estándar para obtener la concentración relativa de la muestra de ADNc medido. Finalmente, obtener el nivel de expresión de los genes diana mediante la relación de la concentración relativa de ADNc diana a los 18 años de control de cDNA internos. </li></ol> 4. La tinción inmunohistoquímica <ol><li> Fijación <ol><li> Diseccionar el ojo en PBS (Phosphate Buffered Saline, pH 7,4). </li><li> Poner el ojo en un tubo lleno con 4% de paraformaldehído (PFA) en frío de hielo durante 20 min. Preparar PFA 4% el día antes. Almacenar a 4 ° C hasta su uso. PFA es fresco durante aproximadamente una semana. </li><li> Eliminar PFA con una micropipeta. A continuación, lavar con PBS enfriado en hielo durante 20 min. </li><li> Poner el ojo en sacarosa 30% a +4 º C durante la noche. </li><li> Poner el ojo en una copa llena con la temperatura óptima de corte (OCT)-medio (Tissue-Tek). Poner el ojo en una posición adecuada para el corte. </li><li> Congelar en octubre mediano con hielo seco. </li><li> Almacenar a -70 ° C hasta su uso. </li></ol></li><li> Seccionamiento <ol><li> Coloque el ojo para la crio-corte en un plano adecuado. </li><li> Cortar tres 5 m de espesor medio sagital secciones desde una parte central de cada lente. Descartar al menos 6 secciones entre las secciones secuenciales para evitar manchar los mismos núcleos en diferentes secciones. </li><li> Una vez que una sección ha sido cortada, coloque suavemente una diapositiva en la parte superior de la misma. La sección a continuación, debe apegarse a la diapositiva. </li><li> Agregar los portaobjetos se seque al aire antes de su uso. Diapositivas se pueden almacenar a -20 ° C hasta su utilización. </li></ol></li><li> Fluorescencia inmunohistoquímica <ol><li> Deje que las muestras de alcanzar la temperatura ambiente (5-10 min). </li><li> Dibujar bordes alrededor de las muestras con un PAP-pen (Invitrogen). </li><li> Deje que la frontera seca durante un tiempo (10 min). </li><li> Etiquetar los portaobjetos de microscopio. </li><li> Rehidratar en 1 × PBS durante 15 min. </li><li> Permeabilizar en solución estándar de bloque durante 30 min. </li><li> Añadir anticuerpo primario (policlonal de conejo caspasa-3, troceados anticuerpo Asp175 9661; Tecnología de Señalización Celular, Inc), diluido 1:3000 en solución de bloqueo estándar. </li><li> Almacenar en una cámara humidificada a 4 ° C durante la noche. </li><li> Lavar en PBS por pipeteado (3 x 5 min) o por inmersión en un baño de gran tamaño (&gt; 15 min, 1-2 cambios). </li><li> Añadir anticuerpo secundario (anticuerpo de conejo anti secundaria con una específica de absorción / emisión del espectro) diluido 1:300 en la solución estándar de bloque. </li><li> Lavar en PBS por pipeteado (3 x 5 min) o por inmersión en un baño de gran tamaño (&gt; 15 min, 1-2 cambios). </li><li> Limpie el pen-PAP frotis. </li><li> Almacenar en una cámara húmeda a temperatura ambiente durante 3 h. Evite la exposición a la luz. </li><li> Añadir una gota de Vectashield y colocar un cubreobjetos sobre el portaobjetos. Evite hacer burbujas. </li><li> Dejar cuajar Vectashield (~ 20 min). </li><li> Mantenga apartados in la oscuridad hasta su análisis. </li><li> Todas las secciones de control se procesan en ausencia de anticuerpo primario. </li><li> Busque los resultados dentro de unas pocas horas bajo un microscopio de fluorescencia. </li><li> Contar núcleos de células epiteliales desde un lado del arco nuclear a proa del otro lado nuclear de cada lente. Aplicar el filtro azul estándar para ver todos los núcleos epiteliales de la lente en azul y un filtro estándar que se ajuste a la emisión del anticuerpo secundario para ver los núcleos positivos caspasa-3 en verde. </li><li> Registrar el número de todos los núcleos epiteliales de la lente y el número de núcleos positivos. Contar las células tres veces para cada sección. </li></ol></li></ol>

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No hay conflictos de interés declarado.

<html> En este trabajo se describen los métodos que permiten estudiar los eventos moleculares que ocurren durante la RUV-B inducida por la catarata. Teniendo en cuenta, que la mayoría de la información disponible para UVR in vivo inducida por catarata se derivó de los experimentos con ratas albinas Sprague-Dawley 7, 16, 17, 18, ​​19, se decidió utilizar la albinas Sprague-Dawley en el estudio actual. Edad de las ratas fue de seis semanas de edad. El género fue es...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Ketalar </td><td> Pfizer </td><td> 150086 </td><td> 50 mg / ml </td></tr><tr><td> Rompun </td><td> Bayer </td><td> 022545 </td><td> 20 mg / ml </td></tr><tr><td> Oculentum Simplex </td><td> Apoteket, Suecia </td><td> 336164 </td><td> 5 g </td></tr><tr><td> Mydriacyl </td><td> Alcon </td><td> 00352 </td><td> 10 mg / ml </td></tr><tr><td> Solución salina equilibrada </td><td> Alcon </td><td> 0007950055 </td><td></td></tr><tr><td> 3,5 ul β-mercaptoetanol </td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II Kit de aislamiento de ARN total </td><td> Macherey-Nagel GmbH &amp; Co, Duren, Alemania </td><td> 740.955,50 </td><td></td></tr><tr><td> cebadores específicos de ADN de p53 </td><td> biomers.net GmbH </td><td> Una medida que se </td><td></td></tr><tr><td> Taq ADN polimerasa, dNTPack </td><td> Roche </td><td> 04 728 866 001 </td><td></td></tr><tr><td> Agarosa </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> El bromuro de etidio solución de 0,5 mg / ml </td><td> Sigma </td><td> E1385 </td><td></td></tr><tr><td> Nano-Drop ND-1000 espectrofotómetro </td><td> Productos NanoDrop </td><td></td><td></td></tr><tr><td> 1 ª Strand cDNA síntesis kit de RT-PCR (AMV) </td><td> Roche Diagnostics GmbH </td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ un solo color en tiempo real PCR sistema de detección </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr> &lt;tr&gt; <td> Placa de 96 pocillos </td><td> Sarstedt </td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan Gene Expression Master Mix </td><td> Applied Biosystems </td><td> 4369016 </td><td></td></tr><tr><td> TaqMan Gene ensayo de expresión de la caspasa 3 </td><td> Applied Biosystems </td><td> Rn00563902_m1 </td><td></td></tr><tr><td> TaqMan Gene ensayo de expresión para mayores de 18 </td><td> Applied Biosystems </td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ software </td><td> Bio-Rad Laboratories </td><td></td><td></td></tr><tr><td> Exfoliados caspasa-3 (Asp175) </td><td> Señalización Celular Tecnología </td><td> 9661 </td><td></td></tr><tr><td> Anti IgG de conejo </td><td> Abcam </td><td> Ab6798 </td><td></td></tr><tr><td> Sacarosa </td><td> Sigma Aldrich </td><td> 84097 </td><td></td></tr&gt; <tr><td> Vectashield </td><td> Vector Laboratories </td><td></td><td></td></tr><tr><td> Universal microscopio Axioplan 2 Radiología </td><td> Carl Zeiss </td><td></td><td></td></tr><tr><td> Paraformaldehído </td><td> Sigma Aldrich </td><td> 441244 </td><td></td></tr><tr><td> TBE 10x tampón </td><td> Promega </td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

La catarata es la principal causa de ceguera en el mundo 1. La Organización Mundial de la Salud define catarata como una opacidad del cristalino del ojo que impide la transmisión de la luz. La catarata es una enfermedad multifactorial asociada con la diabetes, el tabaquismo, la radiación ultravioleta (UVR), alcohol, radiación ionizante, los esteroides y la hipertensión. Hay una fuerte evidencia experimental 2-4 y epidemiológica 5,6 UVR que provoca catarata. Hemos desarrollado un modelo animal para UVR B catarata inducida en ambos anestesiados 7 y los animales no anestesiados 8. La única cura para las cataratas es la cirugía, pero este tratamiento no es accesible a todos. Se ha estimado que un retraso de la aparición de cataratas durante 10 años podría reducir la necesidad de cirugía de cataratas en un 50% 9. Para retrasar la incidencia de cataratas, que es necesario para comprender los mecanismos de la formación de cataratas y encontrar estrategia eficaz de prevencióngias. Entre los mecanismos para el desarrollo de cataratas, la apoptosis desempeña un papel crucial en la iniciación de cataratas en los seres humanos y los animales 10. Nuestro enfoque ha sido recientemente apoptosis en la lente como el mecanismo para el desarrollo de cataratas 8,11,12. Se prevé que una mejor comprensión del efecto de la radiación UV en la vía de la apoptosis se proporcionan posibilidades para el descubrimiento de nuevos productos farmacéuticos para prevenir cataratas. En este artículo se describe cómo catarata puede ser inducida experimentalmente por la exposición en vivo a los rayos UV-B. Además RT-PCR e inmunohistoquímica se presentan como herramientas para estudiar los mecanismos moleculares de la RUV-B inducida por la catarata.

Las diversas fuentes de variación en las mediciones se calcula con un análisis de varianza y se encontró que la consideración de tres mediciones por animal de la varianza para mediciones fue del orden de 15% del de los animales. Así, considerando el análisis de toda la lente, no es posible aumentar la precisión. Pruebas ortogonal dilucidado un contraste estadísticamente significativo para la caspasa-3 mensajes entre 120 intervalo de latencia de horas frente a cortos intervalos de latencia. <p class="jove_con...

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Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

La catarata es la principal causa de ceguera en el mundo. Radiación solar ultravioleta (UVR) es el principal factor de riesgo para el desarrollo de cataratas. Un modelo animal de lejos catarata inducida por UVR-B fue desarrollado. En este artículo se describen los métodos para la investigación de la formación de cataratas: La exposición a los rayos UV, cuantitativos RT-PCR e inmunohistoquímica.

Caracterización de los mecanismos moleculares de la<em&gt; En vivo</em&gt; Catarata inducida por RUV

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

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Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K vistas.  Karolinska Institutet. La catarata es la principal causa de ceguera en el mundo. Radiación solar ultravioleta (UVR) es el principal factor de riesgo para el desarrollo de cataratas. Un modelo animal de lejos catarata inducida por UVR-B fue desarrollado. En este artículo se describen los métodos para la investigación de la formación de cataratas: La exposición a los rayos UV, cuantitativos RT-PCR e inmunohistoquímica.

Video: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

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

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Un In vivo Modelo de roedores de la contracción inducida por la lesión y la monitorización no invasiva de la recuperación

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo MRI de 19F para el seguimiento de la célula

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Molecular Profiling del microambiente tumoral invasiva en un modelo de 3 dimensiones de las células del cáncer colorrectal y<em&gt; Ex vivo</em&gt; Los fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Inducida por la quimioterapia Vascular Toxicidad - En tiempo real<em&gt; En vivo</em&gt; Imagen de Deterioro del buque

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Establecimiento y caracterización de IU y ITUAC en un modelo de ratón

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo la detección de la mitofagia en células humanas, C. Elegansy los ratones

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Un modelo murino inducido por doxorrubicina de miocardiopatía dilatada in vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Generación y caracterización del infarto de miocardio del ventrículo derecho inducido por ligadura permanente de la arteria coronaria derecha en ratones

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...