La investigación en mi laboratorio es apoyado por el Departamento de los fondos de inicio Biología WKU, WKU Investigación Fundación RCCA-Concedo # 11-8032 y con una donación KBRIN-AREA financiado mediante una donación de padres, del Instituto Nacional de Ciencias Médicas Generales de los Institutos Nacionales de Salud con el número premio 5P20GM103436-13. También me gustaría agradecer al Dr. Tian Xu, en cuyo laboratorio se me presentaron a esta técnica y el Dr. Raymond Pagliarini que estableció por primera vez esta técnica en el laboratorio de Xu.

1. La inducción de tumores no invasivos benignos <ol><li> Utilice las poblaciones enumeradas en la tabla 2 para la inducción de tumores benignos. </li><li> Preparar un cultivo iniciador para el &quot;tester&quot; balance de la siguiente genotipo: y, w, ey-FLP1; Tub-GAL80, FRT40A; Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Preparar un cultivo iniciador para la población &quot;prueba&quot; del siguiente genotipo: w; FRT40A, UAS-Ras V12 / CyO </li><li> Recoge diez vírgenes femeninas de la acción &quot;tester&quot; y diez machos de la población &quot;probado&quot;. </li><li> Cruce los hombres y mujeres de la &quot;tester&quot; y la acción &quot;probado&quot; colocando a ambos en un vial con comida mosca. </li><li> Coloque la cruz en una incubadora a 25 ° C y deje que los machos y las hembras para aparearse y poner huevos durante 24-48 horas. </li><li> Compruebe los viales para la deposición de huevos y suficiente para la presencia de larvas de primer instar de asegurarse de que la cultura no se está secando. <ol><li> Añadir unas gotas de autoclavede agua destilada a la cultura para mantenerlo húmedo (si la cultura se está secando). </li><li> Seguir controlando la cultura y repita el paso 1.7.1, según sea necesario. </li></ol></li><li> Observe deambular larvas de tercer estadio bajo un microscopio estereoscópico de fluorescencia como se describe en la sección 3. </li></ol> 2. La inducción de tumores invasivos <ol><li> Utilice las poblaciones enumeradas en la tabla 2 para la inducción de tumores invasivos. </li><li> Utilice el cultivo iniciador desde el paso 1.2 anterior de la siguiente genotipo: y, w, ey-FLP1; Tub-GAL80, FRT40A; Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Preparar un cultivo iniciador para la población &quot;prueba&quot; del siguiente genotipo: w; LGL 4, FRT40A, UAS-Ras V12 / CyO </li><li> Recoge diez vírgenes femeninas de la acción &quot;tester&quot; y diez machos de la población &quot;probado&quot;. </li><li> Cruce los hombres y mujeres de la &quot;tester&quot; y la acción &quot;probado&quot; colocando a ambos en un vial con mosca fud. </li><li> Coloque la cruz en una incubadora a 25 ° C y deje que los machos y las hembras para aparearse y poner huevos durante 24-48 horas. </li><li> Compruebe los viales para la deposición de huevos y suficiente para la presencia de larvas de primer instar de asegurarse de que la cultura no se está secando. <ol><li> Añadir unas gotas de agua destilada en autoclave a la cultura para mantenerlo húmedo (si la cultura se está secando). </li><li> Seguir controlando la cultura para la sequedad y repita el paso 2.7.1, según sea necesario. </li></ol></li><li> Observar las larvas de tercer estadio bajo un microscopio estereoscópico de fluorescencia como se describe en la sección 3. </li></ol> 3. La observación de los tumores benignos y invasoras <ol><li> Utilice larvas de tercer estadio para el aislamiento tumor benigno y visualización. <ol><li> Moje un cepillo de pintura en agua destilada, y utilizar este pincel raspar unas pocas larvas de tercer estadio de la pared del vial de cultivo. </li><li> Coloque las larvas en una diapositiva de la depresión con 1X PBS y luego con un cepillo de pintura húmeda chatarrae de descuento en cualquier producto alimenticio de la epidermis de las larvas. </li><li> Coloque las larvas en una placa de Petri y dejar a -20 ° C congelador durante 30 min para inmovilizar la larva. <ol><li> Método alternativo para inmovilizar larvas utilizando FLYNAP. </li><li> Coloque una varita FLYNAP sumergido en FLYNAP al vial de 3.1.3.2 y después de enchufar el let vial vial tapado con larvas reposar durante 30 a 40 min. </li></ol></li><li> Coloque la larva inmovilizada sobre un portaobjetos de vidrio, agregar una gota de aceite de halocarbonos luz, cubrir con una cubierta de vidrio y observar bajo un microscopio estereoscópico de fluorescencia con la capacidad de visualizar la proteína verde fluorescente (GFP). </li><li> Las larvas que tiene un tumor benigno será fluorescente verde en la región anterior donde el complejo cefálica está presente. </li></ol></li><li> Seleccione el día 10 larvas (después de la inducción) de la cultura definida en el paso 2 anterior para la observación de los tumores invasivos. Día 10 larvas son seleccionadas porque las larvas portador de un tumor invasivo tiene un l extendidaetapa arval y generalmente no logran pupariate. </li><li> Siga los pasos 3.1.1 al 3.1.4 excepto que en lugar de tercero día de uso en estadio las larvas 10 larvas. <ol><li> Las larvas día 10 portador de un tumor invasivo será fluorescente verde en la región anterior donde el complejo cefálica está presente. </li></ol></li><li> Si el microscopio de fluorescencia está equipado con una cámara digital y luego tomar imágenes de las larvas que tiene un tumor benigno e invasiva fluorescente. </li></ol> 4. La disección de la Observación Complex y más cefálica de tumores benignos y invasoras La epidermis translúcidas de la larva hace que sea difícil de observar el grado de invasión de los tumores. Así, la extensión de la invasión es mejor visualizado mediante la disección del complejo cefálica de la larva. Los siguientes pasos se deben utilizar para diseccionar el complejo cefálica. <ol><li> Usando un pincel mojado seleccionar larvas de tercer estadio (para los tumores benignos) y el día 10 larvas (para los tumores invasivos) fesde los viales de cultivo respectivos. </li><li> Coloque las larvas en un pocillo de una placa que contiene la disección en frío PBS 1X. Utilice el pincel para raspar las partículas de comida de la epidermis de las larvas. <ol><li> Traslado larvas limpia para un pozo fresco del plato que contiene la disección en frío PBS 1X. </li><li> Comprobación de la existencia de la fluorescencia de GFP usando un microscopio estereoscópico capaz de detectar la fluorescencia. Deseche las larvas que no devenga GFP. </li></ol></li><li> Añadir 1,0 ml de 1X PBS frío a un pozo fresco en el plato de disección y el uso de un par de fórceps transferir una larva de cojinete o bien los tumores benignos o invasivos (el protocolo de disección sigue siendo el mismo para estos dos tipos de tumores). </li><li> Sostenga la larva hacia abajo con un par de pinzas alrededor de 2/3rds desde el extremo anterior. <ol><li> Utilice el otro par de pinzas y elegantemente separar el 1/3o posterior de la larva y deséchelo. </li><li> Suelte el anterior 2/3rds de la larva. A medida que se libera la presión en el interior de la larva el contenido dellarva rezumar de la cavidad del cuerpo. </li><li> Use un par de pinzas para eliminar el intestino, grasa corporal y otros contenidos interiores de la larva que salir al exterior de la cavidad del cuerpo. </li><li> Use un par de pinzas para sujetar el gancho de la boca de la larva y empujarla hacia la epidermis de larva y usando el otro par de pinzas invertir la larva completamente. </li><li> Utilice las pinzas para quitar suavemente y con cuidado la grasa corporal, las glándulas salivares, el intestino, el complejo ala disco y cualquier tubo traqueal. El complejo cefálico debe ser visible en el gancho de la boca de la larva invertida. Los hemisferios cerebrales y el cordón nervioso ventral (VNC) todavía estaría conectado a la epidermis de larvas a través de los nervios que emanan de la VNC. </li><li> Use un par de pinzas para romper suavemente las conexiones entre los nervios cefálicas y la epidermis de larvas. </li><li> A medida que las conexiones nerviosas entre el VNC y la epidermis de larvas se rompen y el exceso de grasa corporal y otros tejidos removidos, el co cefálicamplex se convertiría claramente visible y se adjunta a la epidermis de larvas sólo en los ganchos de la boca. </li><li> El complejo cefálica puede ser dejada en la epidermis de las larvas si el complejo tiene que ser fijo para aplicaciones posteriores, como la tinción de anticuerpos o para la visualización posterior. </li><li> Si no hay más aplicaciones posteriores se realizaron a continuación, el complejo cefálica se puede extraer de la epidermis de las larvas y se coloca en una gota de un glicerol medios de montaje basado en observación y análisis. Utilice VECTASHIELD como los medios de montaje. </li></ol></li></ol>

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C., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Ras(V12)+and+scribbled+clones+induces+tumour+growth+and+invasion.">Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion.</a> Nature. 463, 545-548 (2010).</li></ol>

No tengo nada que revelar.

El cáncer es una enfermedad compleja con una comprensión mucho mejor hoy que en el pasado. Sin embargo, todavía queda mucho por aprender y explicado antes de que tengamos una imagen completa de los mecanismos subyacentes. El protocolo simple que aquí se presenta permite inducir genéticamente tumores benignos y invasivos en un organismo completo y, a continuación estudiar la biología asociada con la progresión de los tumores en este modelo. La mayoría de las técnicas existentes en Drosophila y otros or...

La progresión de los tumores de un benigna a un estado metastásico es un proceso sabia paso que se caracteriza por la evasión de los mecanismos protectores presentes en el cuerpo 8. Por ejemplo células tumorales en el cuerpo ha de ser capaz de evadir la apoptosis y el sistema inmune, avance la matriz extracelular especializada (ECM) denominada membrana basal, y superar los controles sociales impuestas por las células circundantes 8. Es a través de un paso de progresión prudente que las células cancerosas adquieren la capacidad de migrar y colonizar sitios distantes en un proceso denominado metástasis. Nuestra comprensión de cómo la célula tumoral supera las barreras impuestas por el cuerpo está todavía en su infancia, sin embargo, el cuadro que surge de las investigaciones realizadas hasta el momento los puntos a un uso repetido de los procesos normales de desarrollo y vías de señalización de las células de cáncer de 11 a 13. La mosca de la fruta Drosophila melanogaster, ha contribuido enormemente a nuestro understanding del desarrollo normal y la enfermedad a través del uso de técnicas genéticas sofisticadas desarrolladas a lo largo de las últimas décadas 14-17. El uso de mutagénesis y herramientas sobreexpresión hemos llegado a una mejor comprensión de diversos oncogenes y genes supresores de tumores 18-22. Sin embargo, la metástasis tumoral es el resultado de la cooperación entre varias lesiones genéticas que se ha estudiado principalmente en modelos de cultivo de células 23,24, así como diversos modelos de xenoinjerto 25-27. Estos modelos aunque poderosa tienen sus limitaciones, ya que no reproducir todas las condiciones que se encuentran en un organismo vivo. Además, los modelos transgénicos disponibles en ratones son engorrosos y no es propicio para el análisis genético de la conducta invasiva 28,29. Varios estudios han intentado entender invasión de células tumorales en Drosophila 30,31. Estas técnicas utilizan principalmente el trasplante de tumores primarios a anfitriones y luego se basan en el seguimiento de la transplanted tumores para la invasión de los tejidos vecinos 32,33. Una poderosa técnica llamada MARCM 10 fue adaptada por Pagliarini y Xu para modelar la invasión tumoral en Drosophila 9. Esta elegante modelado genética de la invasión tumoral explotado la cooperación entre un oncogén activado y la pérdida de la polaridad celular. El poder de este modelado se encuentra en el hecho de que los tumores invasivos se crean en un organismo intacto eludiendo así la necesidad de trasplante de tejidos. Para llevar a cabo la cooperación oncogénica, un oncogén activado como Ras V12 se expresa en los clones de células en el disco de ojos antenal de larvas. Como resultado de la técnica de MARCM estos clones también están marcadas con proteína fluorescente verde (GFP) para la visualización fácil y se hacen homocigotos para los mutantes de la polaridad celular como larvas letal gigante, íntima, y discos grandes. El resultado es GFP etiquetada tumores invasivos en el complejo cefálica. En este informe,demostrar cómo inducir, y visualizar estos tumores invasivos tanto en el contexto de una larvas intacto y en diseccionados complejo cefálica. La inducción de tumores que aquí se presenta utiliza reactivos en el segundo cromosoma de Drosophila. En la Tabla 2, proporciono una lista de acciones en X y 3 ª cromosomas que pueden ser utilizados para el mismo fin. Creo que este protocolo simplificado hará que esta técnica de fácil acceso para los investigadores interesados ​​en la comprensión de la base molecular de la progresión del tumor.

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

Drosophila ha iluminado nuestra comprensión de las bases genéticas del desarrollo normal y la enfermedad de las últimas décadas y hoy en día sigue contribuyendo en gran medida a nuestra comprensión de las enfermedades complejas 1-7. La progresión de los tumores de comportamiento benigno a un estado metastásico es un evento complejo 8 y ha sido el modelo en Drosophila para ayudarnos a entender mejor la base genética de esta enfermedad 9. Aquí presento un protocolo sencillo para inducir genéticamente, observar y luego analizar la progresión de los tumores en las larvas de Drosophila. La técnica de inducción del tumor se basa en el sistema de MARCM 10 y explota la cooperación entre un oncogén activado, Ras V12 y la pérdida de genes de polaridad celular (íntima, discos grande y letal larvas gigante) para generar tumores invasivos 9. Demuestro cómo estos tumores pueden ser visualizados en las larvas intactas yentonces, ¿cómo estos pueden ser disecados para su posterior análisis. El protocolo simplificado presentado aquí debería hacer posible que esta técnica para ser utilizada por los investigadores interesados ​​en la comprensión de la función de un gen en la invasión tumoral.

Como resultado del protocolo presentado aquí el usuario será capaz de inducir tumores benignos mediante la sobreexpresión de un oncogén activado en el ojo antenal disco imaginal de larvas. El usuario también será capaz de inducir tumores invasivos en el disco antenal ojo mediante la sobreexpresión de un oncogén activado en clones de células también de mutantes en un gen de polaridad de la célula. Los tumores pueden ser fácilmente visualizados con la ayuda de un estereomicroscopio fluorescente como tejido &qu...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperación entre un oncogén activado como RAS V12 y las mutaciones en los genes de polaridad celular como garabateado, resultar en el crecimiento tumoral en Drosophila donde las células tumorales también muestran comportamientos invasivos. Aquí se presenta un protocolo sencillo para la inducción y la observación de los tumores benignos y invasivos.

Un protocolo para la inducción genética y visualización de tumores benignos y invasoras en cefálica Complejos de<em&gt; Drosophila melanogaster</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

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Research

JoVE Journal

Medicine

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

9.4K vistas.  Western Kentucky University.  Cooperación entre un oncogén activado como RAS V12 y las mutaciones en los genes de polaridad celular como garabateado, resultar en el crecimiento tumoral en Drosophila donde las células tumorales también muestran comportamientos invasivos. Aquí se presenta un protocolo sencillo para la inducción y la observación de los tumores benignos y invasivos. 

Video: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Establecimiento y propagación de los tumores del retinoblastoma humanos en ratones con deficiencia inmune

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Investigación

Medicina

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

No invasiva de medición óptica del metabolismo cerebral en lactantes y Hemodinámica

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Renal Cápsula xenotrasplante y subcutánea Pellet Implantación para la Evaluación de la próstata Carcinogénesis y la hiperplasia prostática benigna

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Microscopia intravital y trombos de inducción en el lóbulo de la oreja de un ratón sin pelo

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Técnica mínimamente invasiva constricción aórtica transversal en ratones para la inducción de la hipertrofia Ventricular izquierda

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Evaluación no invasiva de la función muscular de los dorsiflexores en ratones

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualización de complejos endógenos de mitophagy in Situ en células beta pancreáticas humanas que utilizan el ensayo de ligadura de proximidad

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Un modelo preclínico de rata para el estudio de la lesión por isquemia-reperfusión en microcirugía reconstructiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...