We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

Todos los protocolos experimentales con animales fueron aprobados por el comité de cuidado de los animales y el uso institucional en el Instituto de Ciencias de la Nutrición, Shanghai Institutos de Ciencias Biológicas, la Academia China de Ciencias (Shanghai, China). 1. Modelo de ratón DIO <ol><li> alimentación HFHS <ol><li> Alimentar a ratones C57BL / 6 macho de ocho semanas de edad, con un HFHS que contiene 40 kcal% de grasa y 40% de sacarosa kcal. Alojarlos en 12 h condiciones del ciclo de luz-oscuridad. </li><li> Mantener los ratones en HFHS condición de la dieta materna durante cuatro a dieciséis semanas. Compruebe semanalmente el peso corporal. </li></ol></li><li> Cuerpo análisis de la composición y análisis de imágenes pseudo-color de <ol><li> Se somete a los ratones a un análisis de la composición corporal analizador. <ol><li> Asegure ratones conscientes, no anestesiados en un tubo de plástico en el. Medir la masa grasa corporal, masa magra, y los fluidos que utilizan un sistema de RMN, como se ha descrito anteriormente 2. </li></ol></li><li> Escanear todo el cuerpo de cada ratón utilizando un sistema de resonancia magnética. <ol><li> Anestesiar a los ratones con 1% Avertin a través de inyección intraperitoneal (25 l / g) y llevar a cabo el análisis de imágenes en tubos de vidrio especializados. NOTA: El proceso de formación de imágenes tiene una duración de casi 0,5 horas para cada ratón. Cuando se completa la medición, volver a los ratones a su jaula. </li></ol></li><li> Análisis de todo el cuerpo de los ratones en dorsal, medio y capas ventrales; escanear el hígado en la dirección vertical. Utilice un imán 0,55 T y los siguientes parámetros instrumentales: el espacio K = 192 x 256 m, espesor de la sección = 3 mm, TE = 14,8 ms, TR = 400 ms, FOVRead = 100 mm, FOVPhase = 100 mm, y el número de exploraciones = 8. Scan usando la misma condición magnética para todos los grupos de ratones. </li><li> Mapear la imagen en escala de grises de una imagen de pseudo-color en función de la intensidad de la señal 9. </li></ol></li><li> la eutanasia del ratón <ol><li> esterilizar dissectiherramientas Ng, tales como pinzas y tijeras. </li><li> Vierta suficiente isoflurano en una toalla de papel y dejar que el isoflurano se volatiliza en un frasco de vidrio. Asegúrese de que los ratones son sacrificados por el isoflurano. </li><li> Fijar los ratones en una mesa de operaciones. Pulverizar cada abdomen con 75% de etanol, hacer una incisión en la dermis de la apófisis xifoides con unas tijeras, y rasgar la dermis longitudinalmente a través de la línea media con las manos para exponer el peritoneo. </li><li> Abrazadera de la apófisis xifoides con unas pinzas y cortar el peritoneo a lo ancho a través de la parte inferior de las costillas para exponer las vísceras. </li><li> Recoger la sangre en tubos con EDTA recubiertos por punción cardiaca. </li><li> Cortar el diafragma a lo largo del hígado y retirar con cuidado el hígado a partir de las enterocoelia utilizando unas tijeras finas. Cortar tres piezas de tejido hepático para las siguientes mediciones de lípidos: a) 6 x 3 x 3 mm para la tinción H &amp; E, b) 6 x 3 x 3 mm para Oil Red O tinción, y c) 20-40 mg de hígado de cloroformo / extracción con metanol. </li></ol></li><li> H &amp; E tinción <ol><li> Fijar el hígado en 4% de acetato de formalina tamponada con fosfato a 4 ° C durante la noche y luego incrustarlo en cera de parafina. </li><li> Los cortes de secciones de parafina (5 micras) y montarlos en portaobjetos de vidrio para tinción H &amp; E, como se ha descrito previamente 3, 10. </li></ol></li><li> Oil Red O tinción <ol><li> preparación Regent <ol><li> Preparar la solución de trabajo Oil Red O con 10% de formalina, 60% de isopropanol, hematoxilina, y gelatina de glicerol. Disolver 0,5 g de Oil Red O en polvo con 100 ml de isopropanol y calentar en un baño de 60 ° C para hacer una solución de 0,5%. </li><li> Diluir con agua doblemente destilada (ddH2O) en una proporción de 3: 2 (3 ml de solución de reserva y 2 ml de ddH2O). Se filtra la solución de trabajo y dejar reposar durante al menos 10 minutos a temperatura ambiente. </li></ol></li><li> Incrustar el hígado en el compuesto temperatura óptima de corte (OCT) en APLastic caja de empotrar. Congelar a -20 ° C durante 30 min. Cortarlo en secciones de 8 micras en un micrótomo de congelación, montar el tejido en una diapositiva, y colocarlo a temperatura ambiente durante 30 min. </li><li> Coloque una toalla de papel en el fondo de una bañera de tinción y se vierte una pequeña cantidad de formalina al 10% para mojar la toalla de papel. Colocar el portaobjetos en la bañera de tinción y fijar las secciones congeladas de vapores de formaldehído durante 5 min a 4 ° C. </li><li> Sumergir el portaobjetos en isopropanol al 60% en la bañera de tinción durante 3 s; repita una vez. </li><li> Añadir unas gotas de solución de trabajo de Oil Red O a la diapositiva para cubrir todo el tejido y se incuba durante 15 min. Proteger el tejido de la luz a temperatura ambiente. </li><li> Enjuague el portaobjetos en la nueva isopropanol 60% en la bañera de tinción durante 3 s; repetir dos veces. </li><li> Enjuague suavemente el portaobjetos a través del agua destilada dos veces y eliminar el agua alrededor del tejido. Asegúrese de no secar el tejido. </li><li> Colocar el portaobjetos en hematoxilina durante 1 min y enjuagar suavemente con agua del grifo durante 5-10min. Enjuague suavemente el portaobjetos en agua destilada dos veces y mantener la con agua destilada hasta que esté listo para poner en el cubreobjetos. </li><li> Calentar la gelatina de glicerol en el microondas y colocar varias gotas en la parte superior del tejido. Coloque con cuidado el cubreobjetos en la parte superior y tener cuidado de no formar burbujas. </li><li> Observe la mancha con un microscopio y capturar imágenes usando característicos de 20X y 40X. </li></ol></li><li> Medición de lípidos del hígado <ol><li> Coloque el tejido del hígado diseccionado en un nuevo tubo de centrífuga de 2 ml de plástico. Homogeneizar en 1 ml de PBS con un homogeneizador y transferir el lisado a un nuevo tubo de vidrio de 15 mL. </li><li> Añadir 5 ml de cloroformo / metanol (2:, 1 v / v), vórtice la mezcla vigorosamente durante 1 min, y se deja reposar durante 2 horas en hielo. </li><li> Centrifugar a 1650 xg durante 10 min a 4 ° C; la mezcla debe separarse en 3 capas: la capa superior de metanol, el disco proteína media, y la parte inferior de cloroformo y lípidos. </li><li> tran<html> sfer la fase inferior en un nuevo tubo de vidrio usando una pipeta Pasteur de vidrio; no hay necesidad de conseguir cada gota de la fracción inferior. Ajuste el tubo a un lado en el hielo. </li><li> Añadir 600 l de 4 mM MgCl 2 y 1,5 ml de cloroformo a la fracción sobrenadante que queda en el tubo anterior y agitar vigorosamente. Incubar en hielo durante 30 min y se centrifuga a 1650 xg durante 10 min a 4 ° C. </li><li> Combinar la fase inferior con el primer tubo fase inferior mediante pipeta Pasteur. Desechar las capas altas y medias. </li><li> Evaporar el cloroformo bajo gas nitrógeno en un baño de 37 ° C. Lavar los lados de los tubos de vidrio a fondo con 200 l de 1% de Triton X-100 / cloroformo (v / v). Se evapora bajo gas nitrógeno. Disolver el lípido con 200 l de ddH2O y agitar bien para hacer la emulsión final. </li><li> Mida el total de triglicéridos (TG) y colesterol (TC) niveles mediante el uso de un kit de triglicéridos o colesterol de acuerdo con el protocolo del fabricantexref &quot;&gt; 3. NOTA: Los valores de lípidos se normalizan a los de las concentraciones de proteína en el homogeneizado inicial, según lo determinado por la cuantificación de proteínas 3. </li></ol></li></ol> 2. Modelo de hepatocitos de ratón primaria <ol><li> Aislamiento de hepatocitos primarios de ratón <ol><li> Anestesiar a cada ratón con un 6% de hidrato de cloral (10 l / g por vía intraperitoneal). </li><li> Aislar hepatocitos primarios, como se describió anteriormente 10. Se cultivan las células en cubreobjetos de vidrio recubiertos de colágeno en una placa de 6 pocillos de Oil Red O tinción. Para la extracción de los lípidos, sembrar las células en 10 cm placa de cultivo celular 10. </li></ol></li><li> El tratamiento con células y Oil Red O tinción <ol><li> Reemplazar el medio con 2 ml de medio de inanición y se incuba a 37 ° C durante 24 h. Tratarla con glucosa 30 mM y 100 nM de insulina. Incubar en una incubadora de cultivo de C 37 ° durante 24 h. </li><li> Aspirar el medio. Añadir 2 ml de PBS y agitar suavemente para enjuagar el medio; repita una vez. </li><li> Fijar el cubreobjetos al portaobjetos con una banda elástica. Exponer las células adherentes a la superficie exterior. Realizar la tinción Oil Red O como se describe anteriormente (paso 1.5). NOTA: Los niveles basales de la acumulación de lípidos son estimuladas con un alto nivel de glucosa más el tratamiento con insulina después de la privación de suero. Las gotas de lípidos se visualizaron por tinción con Oil Red O. </li></ol></li><li> Medición de lípidos <ol><li> El tratamiento de los hepatocitos con alto contenido de glucosa e insulina en la misma condición (paso 2.2.1). Añadir 1 ml de PBS a cada 10 cm de placa de cultivo celular. Raspar las células y los cosechan en nuevos tubos de vidrio de 15 ml, de la que alícuotas de 100 l se transfieren a tubos frescos de 1,5 ml para la cuantificación de proteínas. </li><li> Añadir 4 ml de cloroformo / metanol (2: 1, v / v) a los restantes 900 l de células. Someter a ultrasonidos para aproximadas 20 pitidos y luego agitar vigorosamente para liberarel lípido en las células. Incubar la mezcla en hielo durante 2 h. Realizar la extracción de lípidos (paso 1.6). </li></ol></li></ol>

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Los autores declaran que no tienen intereses financieros en competencia. Financiación: Este trabajo fue apoyado por becas de la Fundación Nacional de Ciencias Naturales de China (Nº 81270930, 31471129, 31671224 y) y los cien talentos del programa de la Academia de Ciencias de China (2013OHTP04) a YL

Hígado graso no alcohólico es una serie de enfermedades hepáticas progresivas que se asocia con el síndrome metabólico, la obesidad, la resistencia a la insulina, o diabetes mellitus tipo 2 (DM2) 11. El sello distintivo de hígado graso no alcohólico es la esteatosis, la acumulación de lípidos en los hepatocitos. Aquí, un espectro de métodos se presenta para caracterizar los fenotipos y los parámetros de la esteatosis hepática utilizando ratones DIO y hepatocitos primarios de ratón. ...

La obesidad es un problema de salud creciente en los países desarrollados y en desarrollo. Se ha informado de que una de las condiciones coexistentes frecuentemente asociados con la enfermedad de hígado graso no alcohólico (NAFLD), con una prevalencia que oscila entre 30 y 100 por ciento en pacientes con NAFLD 1. Debido a la fuerte correlación entre el hígado graso y la obesidad, obesidad (DIO) modelos de ratón inducidos por la dieta son ampliamente utilizados para estudiar los mecanismos moleculares complejos asociados con el desarrollo de hígado graso no alcohólico 2, 3, 4, 5, 6. La esteatosis hepática es la etapa más temprana de hígado graso no alcohólico, y puede progresar a la esteatohepatitis no alcohólica (EHNA), cirrosis, y en última instancia, el cáncer de hígado 7. Por lo tanto, el objetivo general de este método es generar modelos animales y celulares de las condiciones esteatósicos hepáticas y para prOvide protocolos detallados para un análisis de lípidos eficiente y precisa. Estos modelos y las mediciones son también útiles para la investigación de otros trastornos metabólicos, tales como resistencia a la insulina y la diabetes tipo 2. Como se identifica que la obesidad es uno de los factores de riesgo de hígado graso no alcohólico, un alto contenido de grasa, dieta alta en sacarosa (HFHS) que imita la dieta alta en grasas de tipo occidental se utiliza para inducir la obesidad en ratones. Posteriormente, el grado de esteatosis hepática puede ser evaluada utilizando diferentes métodos. En primer lugar, el peso corporal y el análisis de la composición corporal con la resonancia magnética nuclear (RMN) muestran la acumulación de lípidos durante la hora de comer. La masa grasa y masa magra se pueden cuantificar de una manera no invasiva y en tiempo real. Además, la resonancia magnética (MRI) se utiliza para mostrar tanto la de todo el cuerpo y la distribución de hígado de grasa. La señal de escala de grises del análisis de MRI se puede convertir una imagen de color pseudo-legible en, y la intensidad dela escala de grises y el color es hemi-cuantificable. Esta tecnología proporciona ventajas únicas para la medición de la acumulación de lípidos en animales vivos. En segundo lugar, el análisis histológico del hígado es el método más comúnmente utilizado para determinar la esteatosis hepática. Hematoxilina y eosina (H &amp; E) tinción histológica proporciona información, tales como la morfología de los hepatocitos y la infiltración de macrófagos, mientras que Oil Red O tinción muestra el tamaño y la posición de las gotas de lípidos en los hepatocitos. En tercer lugar, el análisis del contenido de lípidos mediante la extracción de cloroformo / metanol es una medida precisa y cuantitativa de los lípidos hepáticos. Los niveles totales de colesterol y triglicéridos se pueden medir con métodos bioquímicos. Es importante destacar que, el análisis de extracción de lípidos y Oil Red O tinción también se pueden utilizar en los hepatocitos tratados farmacéuticamente manipulado genéticamente o. La ventaja del presente método es su utilización de múltiples enfoques optimizados para generar modelos esteatósicos hepáticasy caracterizar exhaustivamente los fenotipos tanto in vivo como in vitro. Los modelos de ratones DIO pueden recapitular la patología y metabólicas fenotipos de la enfermedad de hígado graso humano. Otros parámetros metabólicos en humanos pueden ser replicados en este modelo, así 8. La generación del modelo de hepatocitos grasos sometidos en respuesta a los altos de glucosa e insulina es eficiente, útil y supera la limitación del trabajo costoso y lento ratón. Tomados en conjunto, estos métodos son suficientes y esencial para el estudio de la disfunción hepática de lípidos y resistencia a la insulina durante la sobrecarga de nutrientes.

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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">O.C.T compound</td>
			<td style="width:183px;">SAKURA</td>
			<td style="width:163px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Oil Red O</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">O0625-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Triglycerides kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR22421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Cholesterol kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR13421</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Collagen type I, Rat tail</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:163px;">08-115</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">DMEM (low glucose)</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">11885-092</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Penicillin / Streptomycin</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">15140-122</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">FBS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">10099141</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PBS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">R21-040-CVR</td>
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			<td height="20" style="height:20px;width:216px;">HBSS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">20-021-CV</td>
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			<td height="20" style="height:20px;width:216px;">Insulin</td>
			<td style="width:183px;">TOCRIS Bioscience</td>
			<td style="width:163px;">3435</td>
			<td>dissolve in PBS, 1mM for stock</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">G8270-100G</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:163px;">BX53</td>
			<td></td>
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			<td style="width:183px;">Longerpump</td>
			<td style="width:163px;">BT100-2J</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">10cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">420167</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">6-well-plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">3516</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">BCA assay</td>
			<td style="width:183px;">Beyotime</td>
			<td style="width:163px;">P0010</td>
			<td></td>
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			<td height="40" style="height:40px;width:216px;">Nuclear Magnetic Resonance</td>
			<td style="width:183px;">Niumag technology</td>
			<td style="width:163px;">MiniQMR23-060H-I</td>
			<td></td>
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			<td height="40" style="height:40px;width:216px;">High fat high surcose diet(HFHS)</td>
			<td style="width:183px;">Research Diets</td>
			<td style="width:163px;">D12327</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized analysis of in vivo and in vitro hepatic steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Como se muestra en la Figura 1A, el peso corporal del ratón se aumentó a 45 ± 1,2 g después de 16 semanas de alimentación HFHS, que es aproximadamente 1,5 veces mayor que en el grupo de alimentación de dieta de pienso. Los análisis de la composición corporal de RMN que muestra la masa grasa y la masa magra de ratones se indican (Figura 1B). Las distribuciones de grasa de todo el cuerpo y del hígado se determinó por resonancia magnética, y las ...

Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análisis optimizada de<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; La esteatosis hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

optimized-analysis-of-in-vivo-and-in-vitro-hepatic-steatosis

Research

JoVE Journal

Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

15.8K vistas.  Chinese Academy of Sciences. Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described. 

Video: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualización y análisis de flujo de la sangre y del consumo de oxígeno en la microcirculación hepática: Aplicación a un Modelo de cuadro de hepatitis aguda

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Investigación

Medicina

In vivo 19F MRI for Cell Tracking

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

In vivo 19F MRI for Cell Tracking

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

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

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

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

Monitoreo de Parámetros sistémicos y hemodinámicos hepática en ratones

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Evaluación de la efectividad del medicamento para el cáncer Sensibilización<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

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

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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

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

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

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

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

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

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

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

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

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

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

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Preparación de gránulos Gushukang (GSK) para experimentos in Vivo e In Vitro

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Ensayo de migración de arañazos y cámara dorsal Skinfold para análisis In Vitro e In Vivo de sanación de heridas

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...