A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

La incubación 1. Huevo <ol><li> Obtener 8 días de edad huevos embrionados libres de patógenos específicos. </li><li> Coloque los huevos en rotación bandeja de huevos, extremos estampadas hacia arriba y coloque la bandeja giratoria dentro de una incubadora de huevos. Incubar los huevos durante 48 horas a 36 ° C y 50% de humedad. </li></ol> 2. Dejar caer la CAM y la apertura de los huevos <ol><li> Recoger los huevos de la incubadora. Coloque los huevos en la canasta de huevos, estampadas hacia arriba, y llevar ante la campana de flujo laminar. </li><li> Apague las luces de la sala y la capilla. </li><li> Mantenga el extremo sellado del huevo ligeramente a la candler huevo para exponer la vasculatura de la CAM, así como el saco de aire. NOTA: El candler huevo es una fuente de luz que se utiliza para visualizar el desarrollo del embrión y el saco de aire asociado y la vasculatura. </li><li> Coloque una marca de lápiz a entre dos grandes vasos sanguíneos. </li><li> Encienda las luces y usar un alfiler estéril para hacer un agujero en la punta del huevo por encima de la salida de aire (extremo sellado) y un agujero en el PEmarca NCIL. No empuje el pasador de empuje hasta el final; un agujero de unos 3 mm de profundidad suele ser suficiente. </li><li> Apagar las luces. Apriete la bombilla de seguridad, presionando firmemente el extremo abierto contra el agujero por encima de la bolsa de aire. Aplicar succión para extraer el aire en el agujero en la marca de lápiz, haciendo que la CAM caiga fuera de la concha en la marca de lápiz. </li><li> Compruebe que la bolsa de aire se ha movido desde el extremo sellado del huevo al agujero lápiz marcada con el Candler. Si no es así, utilice el alfiler para hacer dos agujeros un poco más profundo y luego volver a aplicar el vacío utilizando la bombilla seguridad. </li><li> Enciende las luces. Sostener el huevo en una mano, active la herramienta rotativa Dremel con un disco de corte 15/16 pulgadas conectado y crea dos cortes transversales lo suficiente profundo para cortar a través de la cáscara, pero no lo suficientemente profundo para cortar todo el camino a la depresión CAM. Haga los cortes alrededor de 2 cm de longitud y 1 cm de distancia entre sí. Haga los cortes lo suficientemente amplia como para que un anillo de silicona de 1 cm de diámetro puede pasar a través depero más delgado que la anchura de cinta adhesiva estándar. </li><li> A continuación, hacer 1 corte longitudinal entre y en los extremos de los dos cortes transversales tocando ligeramente la rueda de corte a la cáscara. </li><li> Deslice pinzas estériles debajo de la pieza de shell y paralelo a la cáscara. Coge la pieza de concha con las pinzas y quitar toda la pieza limpiamente. </li><li> Coloque cinta adhesiva sobre la nueva ventana abierta, por lo que asegúrese de doblar un extremo sobre sí misma para facilitar su extracción. </li><li> Coloque los huevos de vuelta en la incubadora en la bandeja de huevos (NO en los bastidores de huevo rotativas) hasta el momento de la inoculación. </li></ol> 3. Inoculación <ol><li> Mantenga sótano mezcla de proteínas de membrana, como matrigel, en hielo (para evitar la polimerización) y el lugar en el capó. </li><li> Utilice EDTA 2 mM para desprender las células (4 - 5 ml por matraz T75) y matraces de cultivo de tejidos en lugar incubadora durante 5 min. </li><li> Resuspender las células desprendidas en los medios de comunicación y contar utilizando un hemocitómetro y azul de tripano. Usar entre 500.000 to 2.000.000 de células de injerto sobre la CAM, dependiendo de la línea celular. Optimizar el número apropiado en experimentos preliminares. </li><li> Medir el volumen requerido para el número de células apropiadas y centrifugar a 500 xg durante 5 min. </li><li> Desechar el sobrenadante y resuspender a continuación, sedimento celular en 40 μ l de PBS ++ y 20 μ l de mezcla de membrana basal. NOTA: En este punto, las células puede ser tratada con fármacos u otros agentes de prueba según sea necesario para los experimentos mediante la resuspensión en la célula / sótano suspensión mezcla de membrana. Alternativamente, los agentes se pueden pipeta en la célula / sótano suspensión mezcla de membrana después del injerto. </li><li> Recuperar estante huevo con huevos de la incubadora y lugar en la campana de flujo laminar. </li><li> Cinta Despegue de la ventana, recuperar un anillo de silicona 1 mm de la tapa de un vial criogénico utilizando pinzas esterilizadas, y colocar el anillo a través de la ventana. </li><li> Pipetear la solución l celular 60 μ de PBS ++ y membrana basal Mixture directamente en el centro del anillo de silicona que descansa sobre la CAM. </li><li> Re-cinta de la ventana y mover estante huevo de nuevo a la incubadora. En este punto, tenga cuidado al mover los huevos como el anillo y la suspensión celular fácilmente pueden quedar desplazados. </li><li> Permitir que los tumores crezcan durante 5 a 7 días. </li></ol> 4. Recolección de Tumores <ol><li> Coloque bajo-pad en la capucha. Preparar 35 x 10 mm platos y recipientes paraformaldehído según sea necesario para la histología. Ponga 1 ml de PBS ++ en cada placa de 35 mm x 10. </li><li> Coloque la bolsa de riesgo biológico en el capó. Tome las tijeras de disección estéril e introduzca el extremo puntiagudo en el huevo en el agujero cerca del extremo sellado. </li><li> Corte alrededor de todo el huevo longitudinalmente. NOTA: la CAM y el tumor con el anillo deben atenerse a la mitad superior de la carcasa con la ventana. </li><li> Con unas tijeras de disección y pinzas, diseccionar el tumor fuera de la CAM y el lugar del tumor en la placa de 35 mm x 10. Medir y pesar tumores. Tumores fijos en paraformaldehyellos incrustar en parafina de y para su uso en histología e inmunohistoquímica experimentos. </li></ol> 5. (Opcional): Disociación Celular Tumoral <ol><li> Con unas tijeras de disección para picar el tumor en la placa de 35 mm x 10. Vierta tumor picada en un tubo de centrífuga de 15 ml y eliminar la mayor cantidad de PBS ++ como sea posible a través de la pipeta. </li><li> Añadir 2 ml de colagenasa (1 mg / ml en PBS ++), gire tubo varias veces, y se incuba el tubo a 37 ° C durante al menos 30 min (hasta varias horas). </li><li> Añadir 5 ml de medio a cada tubo y resuspender a fondo por pipeteando arriba y abajo 20 - 40x. Pipeteado a fondo es crítico para la disociación celular completa. </li><li> Deje que el sedimento se asiente, y transferir el sobrenadante a un tubo nuevo. Centrifugar a 500 xg durante 5 min. Aspirar el sobrenadante, dejando tras de sedimento celular disociada. NOTA: En este punto, los sedimentos celulares se pueden utilizar para diversos experimentos (citometría de flujo, la lisis para su uso en la transferencia Western, aislamiento de ARN, etc). Recuento total de células tumorales también se puede obtener usando un hemocitómetro y tinción con azul de tripano. Tenga en cuenta que, células de forma ovalada más pequeñas son las células de pollo de la CAM y no deben ser considerados como células tumorales. Debe haber un número mínimo de contaminación de las células no tumorales (CAM) si el tumor se diseca lejos de la CAM con cuidado. </li></ol>

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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

Varios pasos clave en este protocolo muy probablemente explican la mejora de la supervivencia embrionaria, así como una mayor fiabilidad de crecimiento del tumor. Dejar caer la CAM alejado de la cáscara aplicando succión a la bolsa de aire es menos invasivo que otros métodos existentes (utilizando una aguja para extraer la albúmina del huevo, disección roma, etc.). El uso de un alfiler estéril para crear los dos pequeños agujeros necesarios para este método es la parte más difícil del protocolo. El uso de una...

El carcinoma hepatocelular (CHC) es la 3ª causa de mortalidad por cáncer en el mundo 1. Actualmente sólo el 30% de los pacientes con HCC son elegibles para los tratamientos quirúrgicos potencialmente curativos 2, y la quimioterapia sistémica no es eficaz 3. Por lo tanto, hay una necesidad apremiante insatisfecha clínico para nuevas terapias HCC, y el desarrollo de sistemas de modelos adecuados para la prueba eficiente de nuevos agentes. La membrana corioalantoidea (CAM) de ensayo polluelo proporciona un método reproducible y rentable, y rápido rendimiento medio de probar posibles fármacos anti-tumorales in vivo. El ensayo de CAM se ha utilizado ampliamente para estudiar la angiogénesis 4. También se ha desarrollado con éxito en un modelo de xenoinjerto de tumor de cánceres, incluyendo glioblastoma 5, el cáncer de páncreas 6, melanoma 7-9, y osteosarcoma 10-11. Tanto in ovo 12 y ex ovo13 técnicas se han utilizado en la literatura, con detalles que varían de un protocolo a otro. Un gran desafío para el modelo de xenoinjerto CAM es la relativamente alta incidencia de muerte embrionaria después de la manipulación del huevo, con tasas de mortalidad de embriones de pollo publicados que van desde 25 hasta 50 por ciento de 11 a 14. En este artículo se describe el desarrollo de un modelo de xenoinjerto in ovo de CHC que produce de forma fiable el crecimiento de tumores vascularizados tridimensionales que histológicamente se asemejan a HCC indiferenciado. Hemos adaptado un protocolo descrito por primera Ossowski et. Al 14 y han logrado de embriones de pollo tasas de supervivencia de hasta el 93% con extremadamente alta injerto tumor.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

La membrana corioalantoidea de pollo (CAM) comienza a desarrollarse el día 7 después de la fecundación y madura por día 12. El CAM es naturalmente inmunodeficientes y altamente vascularizado, por lo que es un sistema ideal para la implantación del tumor. Además, la CAM contiene proteínas de matriz extracelular tales como fibronectina, laminina, colágeno, la integrina alfa (v) beta3, y MMP-2, por lo que es un modelo atractivo para estudiar la invasión tumoral y la metástasis. Los científicos han tardado mucho provecho de la fisiología de la CAM utilizando como modelo de la angiogénesis. Más recientemente, el ensayo de CAM se ha modificado para que funcione como un sistema modelo de xenoinjerto in vivo para varios tipos de cáncer que sirve de puente entre el trabajo básico in vitro y modelos animales de cáncer más complejos. El ensayo de CAM permite el estudio del crecimiento del tumor, las terapias anti-tumorales, y pro-tumorales vías moleculares en un sistema biológicamente relevante que es a la vez en tiempo y costo efectiva. Aquí se describe el desarrollo de la CAM xenograft modelo de carcinoma hepatocelular (HCC) con tasas de supervivencia de embriones de hasta 93% y tumor fiable tomar conduce al crecimiento de los tumores en tres dimensiones, vascularizados.

Imágenes representativas de los pasos clave en el protocolo se muestran aquí. Figura 1A muestra el uso de la candler visualizar el embrión en desarrollo, el saco de aire, y la vasculatura de la CAM. Figura 1B-1C muestran el proceso de abandonar la CAM al hacer los dos orificios y luego aplicando presión negativa utilizando la bombilla de seguridad, y la Figura 1d muestra una eliminado satisfactoriamente CAM con una gran burbuja de aire bajo el orifi...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

los<em&gt; En Ovo</em&gt; Chick corioalantoidea de membrana (CAM) Ensayo como un modelo de xenoinjerto eficiente de carcinoma hepatocelular

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

34.9K vistas.  Icahn School of Medicine at Mount Sinai. The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Video: The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotón imágenes de invasión de células tumorales en un modelo de ratón ortotópico de carcinoma oral de células escamosas

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Investigación

Medicina

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modelo de ratón ortotópico de carcinoma de tiroides anaplásico

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

La<em&gt; En ovo</em&gt; CAM-ensayo como un Xenograftmodel para el sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Generación de subcutánea e intrahepática hepatocelular carcinoma humano xenoinjertos en ratones inmunodeficientes

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

Confocal Lapso de tiempo de imágenes como un método eficaz para la Evaluación Cytocompatibility de composites dentales

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Un &quot;paciente-Like&quot; ortotópico Singénico modelo de ratón de carcinoma hepatocelular Metástasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Diseño de guías quirúrgicas CAD/CAM para la reconstrucción maxilar mediante un enfoque interno

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Predicción de respuesta al tratamiento tratamientos guiados por imágenes mediante aprendizaje automático: un ejemplo para el Trans-Arterial tratamiento de Carcinoma Hepatocelular

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...