Estamos muy agradecidos con el laboratorio del Dr. Robert S. Kerbel (Universidad de Toronto, el Instituto de Investigación Sunnybrook, Toronto, Canadá) para la ayuda técnica y la experiencia en el desarrollo de este procedimiento. También nos gustaría dar las gracias a la Dra. Sandra Sexton y el Roswell Park Departamento de Recursos Animales de Laboratorio del Instituto del Cáncer. Este trabajo fue apoyado por un premio de la Fundación Alianza Roswell Park (a JMLE).

1. Ortotópico tumor de riñón Implantación <ol><li> Cultivo Celular <ol><li> Antes de la implantación ortotópica, crecer células de ratón RENCA LUC como una monocapa a 75% de confluencia. </li><li> Después de tripsinización y resuspensión en 5% de FBS que contiene los medios de comunicación, centrifugar las células a 1000 rpm, 4 º C durante 5 min, repetir 3 veces para lavar en PBS. A continuación, volver a suspender las células en medio libre de suero a una concentración de 5 x 10 4 RENCA LUC / 5 medios libres de suero l. Nota: En función de la variante o línea celular utilizada, las células pueden ser resuspendidas en una proporción de matrigel y medio libre de suero 1:01 para frenar el crecimiento de células de ratón altamente agresivo y difusión después de la implantación. </li></ol></li><li> La cirugía y la implantación de células Nota: Todos los estudios con animales descritos en el mismo, incluyendo el mantenimiento y la determinación de los criterios de valoración experimentales, se llevaron a cabo de acuerdo con un Comité de Cuidado de Animales y el empleo Institucional (IACUC) protocol aprobado por el Instituto del Cáncer Roswell Park. <ol><li> Anestesie un ratón Balb / c usando isoflurano (2-3%). Una pizca de pie y comprobar si el reflejo de asegurar una buena anestesia. Aplique un ungüento veterinario para los ojos para evitar que se sequen. Coloque el ratón en decúbito lateral derecho y afeitar lado izquierdo entre proa y de las extremidades posteriores. Aplicar el alcohol y yodo a lo largo de área lumbar dorsal para preparar asépticamente área para la incisión. </li><li> Utilice tijeras quirúrgicas para iniciar una incisión en la piel 1 cm en una dirección longitudinal entre la última costilla y la articulación de la cadera. Aflojar tejido conectivo debajo de la piel utilizando una tijera disección roma. Con unas tijeras quirúrgicas para hacer una incisión de 0,5 cm en una dirección longitudinal en la pared abdominal. Utilice pinzas curvas para empujar suavemente hacia abajo alrededor de la herida abierta - esto exteriorizar el riñón y permitir la inmovilización suave de órgano antes de la inyección. </li><li> Cargue la jeringa Hamilton con 5 l de la mezcla de células preparado. Para implan sub-capsularestaciones, dos métodos de implantación pueden ser empleados: <ol><li> Superficie: En paralelo a los riñones, la aguja de inserción longitudinalmente orientado bajo la cápsula renal (pero por encima del parénquima) con el borde biselado hacia arriba. Una vez que la aguja está en su lugar, se inyecta todas las células, hasta las formas pequeñas burbujas blancas. Quite lentamente la aguja de la cápsula y secar de inmediato sobre la inyección con un algodón con punta de aplicador estéril para absorber cualquier fuga y prevenir la difusión celular. </li><li> Interna: Con borde biselado y hasta comenzando desde el lado opuesto de riñón para sitio de implantación final, insertar la aguja a través del interior de riñón hasta que la aguja es visible (pero no la punción) el espacio subcapsular. Inyectar todas las células hasta que se forme burbujas blancas y lugar de la inyección hisopo para evitar cualquier fuga. </li></ol></li><li> Regresar suavemente el riñón a la cavidad del cuerpo. Cierre de la pared corporal abdominal utilizando sutura absorbible 5-0 Vicryl. Una vez terminado, tire de la capa de la piel juntos y colocar 2-3 wound clips. Asegurar clips son firmes y uniformemente espaciadas para asegurar que no la exposición de la herida. Antes de la recuperación de administrar 500 l de NaCl al 0,9% y 100 l de buprenorfina (0,01 mg / ml) por vía subcutánea utilizando una aguja de 25. Monitorear rutinariamente la colocación y la estabilidad clip, y quitar después de 10 días. </li></ol></li></ol> 2. Nefrectomía / Extirpación de Primaria <ol><li> Siga el protocolo idéntico para anestesia ratón, inmovilización, incisión, y la exposición de los riñones, como se describe en las secciones 1.2.1 y 1.2.2 (arriba). </li><li> Utilice un segundo par de pinzas para aislar el riñón mediante la eliminación suavemente el tejido graso de conexión desde el extremo caudal y la glándula suprarrenal desde el extremo craneal del riñón. Mientras sujeta suavemente el riñón, el uso de sutura absorbible 5-0 Vicryl para hacer un doble nudo alrededor del uréter, la arteria renal y la vena. Poco a poco seguro y luego se corta por encima del nudo para extraer el riñón. Si cualquier señal de inmediato la pérdida de sangre, o se corre el riesgo de retención insuficientede sutura en el uréter, la arteria y la vena, a continuación, utilizar cauterizador en lugar de tijeras para cortar por encima de nudo. </li><li> Una vez que se elimina el riñón, revise cuidadosamente cualquier sangrado de la arteria atada y use cauterizador si es necesario. Cierre de la pared abdominal del cuerpo usando Vicryl 5-0. Tire de la capa de la piel juntos y colocar 2-3 grapas para heridas tras mismas precauciones como se describe en 1.2.4. Seguir de cerca los animales todos los días después de la cirugía para detectar signos de malestar, sangrado o movilidad reducida. Siga las directrices del centro para animales de manera segura. </li></ol> 3. Monitoreo metastásico Progresión y localización en el punto final <ol><li> Cuantificación de la enfermedad metastásica: monitoreo bioluminiscente <ol><li> Ver metodología descrita previamente en JOVE para el seguimiento de metástasis utilizando células transfectadas con luciferasa y se cuantifica utilizando el sistema de imágenes de Xenogen IVIS 11,12. </li></ol></li><li> Guía visual necropsia para la evaluación comparativa de la distribución metastásicación <ol><li> Sacrificar animales de acuerdo a las directrices institucionales que utilizan criterios de valoración que incluyen signos de sufrimiento, dificultad para respirar, pérdida de peso, etc Nota: estrecha vigilancia de los animales es fundamental, ya la progresión metastásica espontáneo puede ser muy variable y rápida. </li><li> Utilice tijeras quirúrgicas para iniciar una incisión por encima de la uretra, que continúa hasta la cavidad torácica a la mandíbula inferior. Con unas tijeras de extremos romos para separar la piel del músculo de la pared abdominal. </li><li> Iniciar incisiones a lo largo de los brazos y las piernas. Pelar la piel de distancia. Examine de cerca la fascia de la piel y la pared abdominal de los nódulos, entonces los ganglios linfáticos superficiales: inguinal, braquial, axilar y cervical superficial. </li><li> Hacer una incisión en la pared abdominal y cortar a lo largo de los lados laterales izquierdo y derecho hasta el diafragma. Con un corte horizontal quitar la parte frontal de la pared abdominal para desnudar las vísceras. </li><li> Examine la apariencia tranquila de las vísceras fo gravemente tumores notables. También tenga en cuenta el contenido del estómago y los intestinos; si están vacíos, el ratón puede no haber sido capaz de comer. Si el contenido es oscuro, esto puede indicar evidencia de hemorragia gastrointestinal superior / inferior. </li><li> Visualmente la puntuación de cada órgano de la presencia o ausencia de tumor, la asignación de una puntuación total de cada grupo de ratones (4 animales por grupo se utiliza como ejemplo en la Figura 1D-i). Notas: 1) La ampliación de los ganglios linfáticos no significa necesariamente que se trata de un crecimiento tumoral; si es muy grande, muy firme y un color blanquecino, es probable un nódulo metastásico. 2) lechosa excesivo o líquido turbio en el abdomen indica ascitis. La ascitis pueden incluir acumulaciones sólidas sobre los órganos abdominales, en particular entre los lóbulos del hígado y el estómago. 3) para los órganos como los pulmones, nódulos múltiples pueden estar presentes y contados para las comparaciones entre animales individuales (véase 13, por ejemplo). </li><li> Identificar los tumores brutos o anomalías de los órganos abdominales individuales: hígado, bazo, riñones y páncreas mediante la eliminación y el lavado con PBS cuidadosamente una botella con atomizador. </li><li> Después de la eliminación de los órganos abdominales, inspeccionar los ganglios linfáticos abdominales, incluyendo la zona lumbar, sacra, renal, y ganglio linfático mesentérico. </li><li> Evaluar diafragma para evidencias de diseminación metastásica. Nota: Amplia difusión en el diafragma puede causar que pierda su contractilidad, lo que lleva a la dificultosa o pérdida de la respiración. </li><li> Retire el diafragma y hacer dos incisiones laterales en la cavidad torácica, la eliminación de la porción frontal para exponer los pulmones y el corazón. </li><li> Eliminar pulmones y el corazón agarrando y levantando suavemente el esófago y el corte de la tráquea por encima del corazón. Evaluar los tejidos de nódulos. Apriete corazón. La falta de consistencia elástica puede indicar tumor presente. </li><li> Utilice tijeras quirúrgicas para cortar linealmente a través del cráneo. Retire con cuidado los pequeños trozos de cráneo hasta ttodo él cerebro está expuesto. Busque en todo el cerebro en busca de nódulos visibles o anormalidades de las decoloraciones. </li></ol></li></ol>

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Los autores no tienen nada que revelar.

El propósito de este protocolo es para evaluar la enfermedad metastásica espontánea clínicamente relevante utilizando un modelo de ratón del tumor singénico para describir las técnicas de implantación / resección. Actualmente, la mayoría de los estudios preclínicos evaluación de nuevas terapias experimentales no incluyen el estudio de la enfermedad metastásica y sólo unos pocos recapitular las etapas de crecimiento del tumor primario, la resección quirúrgica, y la eventual diseminación metastásica espo...

La causa principal de mortalidad en pacientes con carcinoma de células renales (RCC) es la enfermedad metastásica sistémica que se produce normalmente después de la extracción quirúrgica de un tumor primario de crecimiento en el riñón. Sin embargo, muy pocos modelos tumorales preclínicos que evaluaron terapéutica experimental en ratones incluyen la enfermedad metastásica, y menos aún recapitulan fielmente las etapas clínicas de crecimiento localizado, la cirugía, y el inicio de micro-metástasis espontánea y progresión 1-3. Esta brecha en la prueba se ha convertido cada vez más importante en la evaluación de nuevas terapias como a veces sorprendentes efectos antitumorales observados en modelos animales no siempre se tradujo en un éxito similar tratamiento de los pacientes 4. Estas diferencias en los resultados pueden deberse a eficacias de drogas diferenciales entre modelos de tumores localizados ectópicos o ortotópico primarios y la fase tardía de la enfermedad metastásica 5-7. En el caso de RCC, sólo unos pocos estudios han empleado establecieron unNimal protocolos que incluyen la enfermedad recurrente espontánea que imita los pacientes que típicamente han tenido riñones con tumores en su totalidad o parcialmente removidos 2,3. Las razones de esta escasez de pruebas de modelo de ratón varían. En primer lugar, es el alto costo de los animales y la variabilidad inherente de la selección de las células tumorales y el potencial metastásico. Por ejemplo, las líneas celulares de riñón humano tienden a hacer metástasis raramente, y deben ser seleccionados a través de múltiples rondas de implantación primaria ortotópico metastásico y selección para derivar las variantes que difunden de manera consistente y forman lesiones a distancia (véase la descripción de una línea celular tal derivación humana 8-10) . Por el contrario, las células de ratón en modelos inmunocompetentes tienden a comportarse de manera agresiva, y los números bajos de células deben ser inyectados con matrigel para reducir la diseminación sistémica inmediata 3. En segundo lugar, las dificultades técnicas para la realización de la implantación adecuada, la resección quirúrgica (nefrectomía), y el seguimiento (y cuantificar) metastati espontáneac del crecimiento puede ser un reto y varias variables críticas que plantearse cuando se emplea esta técnica (véase la discusión para más detalles). El objetivo de este protocolo es describir los pasos esenciales (así como los peligros potenciales) de la implantación ortotópica, la resección (nefrectomía), y el seguimiento de la enfermedad CCR metastásico espontánea y ofrecer una guía para estandarizada (y más extendido) utilizar los laboratorios científicos que evalúan la eficacia de la terapéutica experimental.

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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">DMEM-high glucose with Pyr. And L-Glutamine</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">FBS</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.25% Trypsin EDTA</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1x DPBS without Calcium &#38; Magnesium</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Matrigel</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td>must be kept on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Artifical tears-lubricant opthalmic ointment</td>
			<td style="width:147px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Pocket pro pet trimmer</td>
			<td style="width:147px;">Braintree scientific</td>
			<td style="width:119px;">CLP9931B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Alcohol swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Betadine solution swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">67618-152-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MICRO DISSECTING sissors straight,blunt - 25mm blades - 4.5&#34;&#160;</td>
			<td style="width:147px;">Southpointe surgical</td>
			<td style="width:119px;">RS-5982</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Iris Forceps, serrated, curved, 10cm long</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>need two of these</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">10&#181;l Hamilton syringe</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7635-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">30G, 45 degree, RN needle</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7803-07</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Sterile cotton tipped appicator</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10805-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">High temperature cautery kit</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">5-0 coated Vicryl, conventional cutting needle&#160;</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:119px;">J834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clip applier for 7mm clips</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clips, 7mm, non-sterile</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Removing forceps, 12cm lone</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% Sodium Chloride</td>
			<td style="width:147px;">Baxter Healthcare</td>
			<td style="width:119px;">2B1322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Buprenorphine 0.01mg/mL</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">25G 5/8&#34; needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1mL syringe w/out needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">D-Luciferin</td>
			<td style="width:147px;">Gold Bio technology</td>
			<td style="width:119px;">LUCK-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy

Uno de los retos clave para la mejora de las pruebas de nuevas terapias experimentales en el carcinoma de células renales (CCR) es el desarrollo de modelos que fielmente recapitula progresión de la enfermedad metastásica temprana y tardía etapa. Modelos típicos implantación del tumor utilizan la implantación del tumor primario ectópico o ortotópico, pero pocos incluyen la enfermedad metastásica espontánea sistémica que imita el entorno clínico. Este protocolo describe los pasos clave para el desarrollo de la progresión de la enfermedad RCC en escena similar a los pacientes. En primer lugar, se utiliza un ratón línea celular de tumor altamente metastásica en un modelo singénico para mostrar la implantación de células del tumor ortotópico. Los métodos incluyen la implantación superficial e interna en el espacio subcapsular con células combinadas con Matrigel para evitar fugas y la propagación temprana. Siguiente que describe los procedimientos para la escisión de portador de un tumor de riñón (nefrectomía), con cuidado de ratón pre-y post-quirúrgico crítico. Por último, se describen los pasos necesarios para controlar y evaluarmicro y macro-evolución de la enfermedad metastásica, incluyendo imágenes bioluminiscentes, así proporciona una guía visual necropsia detallada para anotar distribución de la enfermedad sistémica. El objetivo de esta descripción del protocolo es facilitar el uso generalizado de los modelos CCR metastásico clínicamente relevantes para mejorar el valor predictivo de la futura prueba terapéutica.

Figura 1A muestra un esquema delineando los procedimientos detallados en este resumen de protocolo. Varios factores importantes deben ser considerados para cada paso. Por ejemplo, en el paso 1 se muestran dos métodos para la sub-capsular implantación de células tumorales en el riñón. Las células tumorales se pueden implantar en el espacio subcapsular con un pequeño blanco-burbuja de confirmar la colocación localizada de células con fugas impedido por la eliminación cuidadosa de la aguja y limp...

Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Modelos de progresión de la enfermedad espontánea carcinoma metastásico de células renales (CCR) pueden ser utilizados para la evaluación de los tratamientos en un entorno clínicamente relevante. Este protocolo demuestra diferentes procedimientos para la implantación de células tumorales de riñón ortotópico, nefrectomía adecuado, y finalmente describe una guía para la necropsia de puntuación visual y bioluminiscente de carga metastásica y la localización.

Modelado espontánea metastásico Carcinoma de células renales (CCR metastásico) en ratones después de la nefrectomía

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

modeling-spontaneous-metastatic-renal-cell-carcinoma-mrcc-mice

Research

JoVE Journal

Medicine

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

16.4K vistas.  Roswell Park Cancer Institute.  Modelos de progresión de la enfermedad espontánea carcinoma metastásico de células renales (CCR) pueden ser utilizados para la evaluación de los tratamientos en un entorno clínicamente relevante. Este protocolo demuestra diferentes procedimientos para la implantación de células tumorales de riñón ortotópico, nefrectomía adecuado, y finalmente describe una guía para la necropsia de puntuación visual y bioluminiscente de carga metastásica y la localización....

Video: Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

De un puerto nefrectomía del donante

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

Investigación

Medicina

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

El trasplante de células del estroma mamario y células de carcinoma en los ratones C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

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 ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Modelado Hemorragia intracerebral en ratones: La inyección de sangre autóloga o colagenasa bacteriana

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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  ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

La inducción de la transición de vejiga invasivo Carcinoma en inmune intacto humanos MUC1 Ratones Transgénicos: Un Modelo para el Desarrollo Inmunoterapia

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Inicio de la metástasis de carcinoma de mama mediante la Focalización del epitelio ductal con adenovirus Cre: Un modelo de ratón transgénico de Novela del Cáncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Carrera Modelado en ratones: Coagulación permanente de la arteria cerebral media Distal

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.