Este trabajo fue apoyado por la Fundación Nacional de Ciencias Naturales de China (81573992). Damos las gracias a Emily K. Lo y Kathleen DiNapoli por su ayuda en la edición de idiomas.

El protocolo sigue las pautas del cuidado animal de la universidad de Shangai de la medicina china tradicional y es aprobado por el experimento animal Comité de Ética animal de Shangai. 1. Protocolo I: Preparación de EXD <ol><li> Cálculo <ol><li> Administrar el EXD a un grupo de tratamiento de 10 ratones hembra de seis meses de edad de Imprinting Control Region (ICR) (0,02 kg / ratón), 5 días a la semana durante 12 semanas. NOTA: Se necesitan un total de 1.702 EXDs. Debido a que los EXDs se miden en incrementos discretos, en la práctica real, administrar 2 EXDs. </li><li> Calcular el volumen de EXD aplicado por ratón ( es decir, V = (0,1418 EXD / kg • día) * (0,02 kg / ratón) * 50 ml / EXD = 0,14 ml / ratón • día). </li></ol></li><li> Remojo <ol><li> Colocar todas las materias primas de dos EXD ( es decir, Curculigo orchioides Gaertn (18 g), Herbaa Epimedii (18 g), Radix Morindae Officinalis (18 g), RAdix Angelicae Sinensis (18 g), Cortex Phellodendri (12 g) y Rhizoma Anemarrhenae (12 g), 96 g en total) en un recipiente con tapa. NOTA: Se recomienda un recipiente de cerámica. </li><li> Macerar las hierbas crudas con 500 mL de agua destilada durante 1 h; El agua debe cubrir las hierbas por cerca de una pulgada. Deja que todas las hierbas se empapen completamente. </li></ol></li><li> Primera decocción acuosa <ol><li> Calentar las hierbas utilizando una cocina de gas o una cocina de inducción de alta potencia hasta que el agua hierve (alrededor de 5 - 10 minutos, el tiempo depende de la potencia de calefacción, el recipiente y la cantidad de hierbas y agua). Baja la potencia a fuego lento durante 2 h. </li></ol></li><li> Primera filtración <ol><li> Cubra un vaso de precipitados de 500 ml con papel de filtro regular o con gasa y algodón. Vierta cuidadosamente la decocción en el vaso de precipitados a través del filtro. Deje las hierbas en el recipiente. </li><li> Devolver el residuo de hierbas restanteN el papel de filtro al recipiente. </li></ol></li><li> Segunda decocción acuosa <ol><li> Añadir agua destilada al recipiente con las hierbas; Deje cubrir las hierbas por cerca de una pulgada. </li><li> Repita el paso 1.3. </li></ol></li><li> Segunda filtración <ol><li> Repita los pasos 1.4.1 y 1.4.2. </li><li> Vierta la segunda decocción en el mismo vaso. Mezclar la primera y la segunda decocciones juntas. </li></ol></li><li> Concentración <ol><li> Colocar el vaso en una cocina de gas con gasa de alambre libre de amianto entre ellos, calentar la decocción con un bajo cocción a fuego lento y revolver lentamente y continuamente con una barra de vidrio. </li><li> Cuando la decocción se reduce a 200 ml, se transfiere a un vaso de precipitados de 500 ml. </li><li> Repita el paso 1.7.1 hasta que la decocción se reduzca a 100 mL. </li></ol></li><li> Almacenamiento y administración <ol><li> Transferir la decocción concentrada a una botella de vidrio estéril. Dejar enfriar a temperatura ambienteRe (RT). Almacenar a 4 ° C si se utiliza dentro de una semana oa -70 ° C para el almacenamiento a largo plazo. </li><li> Usando una aguja de sonda, administre 0,14 mL / ratón de la decocción a ratones ovariectomizados (OVX) una vez al día, 5 días a la semana durante 12 semanas. </li></ol></li></ol> 2. Protocolo II: Preparación de suero que contiene EXD <ol><li> Cálculo <ol><li> Calcular la dosis equivalente de rata (RED) usando 0,15 kg como el peso corporal de cada rata (1 mes de edad): dB = (1/60) * (90/100) * (60 / 0.15) 1/3 = 0.111 EXD / Kg / día. NOTA: Se necesita un total de 10 ml de suero que contenga EXD para preparar 100 ml de medio de cultivo (10% de suero que contenga EXD). Se espera que cada rata proporcione 2 ml de suero. Por lo tanto, 6 ratas (una pérdida del 20% se tienen en cuenta) son necesarios para la administración de EXD una vez al día durante 3 días. El número de EXD es: (0.111 EXD / kg • día) * (0.15 kg / rata) * (6 ratas) * (3 días) = ​​0.300 EXD. De nuevo, debido a que los EXDs se miden en incrementos discretos,Utilice 1 EXD. </li><li> Calcular el volumen de EXD aplicado por rata ( es decir, V = (0.111 EXD / kg • día) * (0.15 kg / rata) * (50 mL / EXD) = 0.83 mL / rata • día). </li></ol></li><li> Remojo <ol><li> Ponga las materias primas para 1 EXD en un recipiente con una tapa. Macerar las hierbas crudas en agua destilada durante 1 h; El agua debe cubrir las hierbas por cerca de una pulgada. Deja que todas las hierbas se empapen completamente. </li></ol></li><li> Primera decocción acuosa <ol><li> Calentar las hierbas utilizando una cocina de inducción de alta potencia hasta que el agua hierve (alrededor de 5 - 10 min, el tiempo depende de la potencia calorífica, el recipiente y la cantidad de hierbas y agua). Apague la alimentación a fuego lento durante 2 h. </li></ol></li><li> Primera filtración <ol><li> Cubra un vaso de precipitados de 500 ml con papel de filtro regular o con gasa y algodón. Vierta cuidadosamente la decocción en el vaso de precipitados a través del filtro. Deje las hierbas en el recipiente. </li><li&gt; Devuelva el residuo de hierbas en el papel de filtro al recipiente. </li></ol></li><li> Segunda decocción acuosa <ol><li> Añadir agua destilada al recipiente con las hierbas; Deje cubrir las hierbas por cerca de una pulgada. </li><li> Repita el paso 2.4. </li></ol></li><li> Segunda filtración <ol><li> Repita los pasos 2.5.1 y 2.5.2. </li><li> Vierta la segunda decocción en el mismo vaso de precipitados que en el paso 2.4.1 y mezcle la primera y la segunda decocciones juntas. </li></ol></li><li> Concentración <ol><li> Colocar el vaso en una cocina de gas con gasa de alambre libre de amianto entre ellos, calentar la decocción con un bajo a fuego lento y revolver lentamente y continuamente con una varilla de vidrio. </li><li> Cuando la decocción se reduce a 100 ml, se transfiere a un vaso de precipitados de 100 ml. </li><li> Repita el paso 2.7.1 hasta que la decocción se reduzca a 50 ml. </li></ol></li><li> Almacenamiento y administración <ol><li> Transferir la decocción concentrada en unaBotella de vidrio. Dejar enfriar a RT. Almacenar a 4 ° C si se utiliza dentro de una semana oa -70 ° C para el almacenamiento a largo plazo. </li><li> Administrar intragástralmente 0,83 mL / rata una vez al día durante 3 días. </li></ol></li><li> Preparación de suero que contiene EXD <ol><li> Anestesiar las ratas por inyección intraperitoneal de 300 ml / 100 g de 80 mg / kg de ketamina y 10 mg / kg de xilazina a 1 h después de la última administración de EXD. Confirme la adecuada anestesia por pellizco del dedo del pie. </li><li> Inciso de la piel y el peritoneo de la rata desde el abdomen hasta el fondo del tórax con un bisturí (o tijeras de operación recta). Crear una incisión de longitud y profundidad de unos 5 cm y 0,5 cm, respectivamente. Mueva las vísceras abdominales a la izquierda utilizando papel de seda. </li><li> Usando papel tisú, retire el tejido conectivo de la aorta abdominal para exponer claramente el vaso. </li><li> Saque la sangre lentamente de la aorta abdominal usando una jeringa calibre 22 de 10 ml. Transferirlo aUn tubo estéril de 15 ml después de retirar la aguja; Una rata puede producir 8-10 ml de sangre. NOTA: La rata debe estar viva ( es decir, con la aorta abdominal pulsando) al comenzar el dibujo de sangre y muerta después de que el dibujo se complete. </li><li> Coagular la sangre en posición vertical durante 30 - 60 min a la habitación RT. Centrifugar a 500-600 xg durante 20 min. Colocar cuidadosamente todo el sobrenadante (suero) en un tubo estéril de 50 ml y mezclar todo el suero (de diferentes animales). </li><li> Realizar la inactivación por calor mediante la incubación del suero en un baño de agua a 56 ° C durante 30 min. Filtrar el suero utilizando un filtro de jeringa con una membrana de polietersulfona hidrófila de 0,22 μm de tamaño de poro. Utilice el suero fresco o guárdelo a -20 ° C. </li></ol></li><li> La preparación de suero que contiene un fármaco de control (utilizada en el grupo de control) <ol><li> Administrar cada rata con el mismo volumen de solución salina (0,83 mL) una vez al día durante 3 días. Los otros pasos son los mismos que en el paso 2.9.</li></ol></li><li> Solicitud <ol><li> Añadir 10 ml de suero que contenga EXD o suero que contenga fármaco a cada 100 ml de medio, más 1% de penicilina-estreptomicina (PS) [ 23] . </li></ol></li></ol>

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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effect+of+Chinese+herbal+drug-containing+serum+for+activating+blood,+activating+blood+and+dispelling+toxin+on+TNF-alpha-induced+adherence+between+endothelial+cells+and+neutrophils+and+the+expression+of+MAPK+pathway].">[Effect of Chinese herbal drug-containing serum for activating blood, activating blood and dispelling toxin on TNF-alpha-induced adherence between endothelial cells and neutrophils and the expression of MAPK pathway].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 35 (2), 204-209 (2015).</li><li>Li, Y., Xia, J. Y., Chen, W., Deng, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ling+Qi+Juan+Gan+capsule+drug-containing+serum+on+PDGF-induced+proliferation+and+JAK/STAT+signaling+of+HSC-T6+cells].">Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].</a> Zhonghua Gan Zang Bing Za Zhi. 21 (9), 663-667 (2013).</li><li>Li, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Some+issues+in+methology+of+Chinese+herbs+serum+pharmcology].">[Some issues in methology of Chinese herbs serum pharmcology].</a> Zhong Yao Xin Yao Yu Lin Chuang Yao Li. 10 (5), 263 (1999).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[A+review+of+Chinese+herbs+serum+pharmcology+methodological+study].">[A review of Chinese herbs serum pharmcology methodological study].</a> Nan Jing Zhong Yi Yao Da Xue Xue Bao. 18 (4), 254 (2002).</li><li>Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Issues+and+strategies+for+study+of+serum+pharmcology+in+oncology].">[Issues and strategies for study of serum pharmcology in oncology].</a> Zhong Yi Yan Jiu. 17 (5), 13-14 (2004).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

En los últimos años, se ha prestado más atención a las hierbas medicinales, un tipo de medicina alternativa que se ha aplicado en el entorno clínico en el mundo oriental durante miles de años. Diferente del patrón de &quot;banco a la cama&quot; de la medicina moderna, la medicina herbal tradicional primero requiere la &quot;cama al banco&quot; patrón para explicar sus mecanismos. Esto puede ser seguido por la validación realizada en la etapa de banca y el procesamiento para el desarrollo de fármacos recién op...

El interés en el estudio y la aplicación de la medicina herbaria tradicional está creciendo actualmente. A diferencia de las drogas modernas, en las cuales los ingredientes químicos son definidos, las fórmulas herbarias tienen algunos ingredientes desconocidos y requieren procesos de extracción para permitir la entrega de sus compuestos activos. Aunque muchos estudios tratan de seleccionar un pequeño compuesto con una estructura bien conocida como representante de toda la hierba o fórmula herbaria, ni las eficacias farmacológicas ni los mecanismos pueden considerarse equivalentes 1 , 2 . Mientras que las huellas dactilares permiten el análisis de los constituyentes de fórmulas herbarias complejas, algunos constituyentes todavía no se analizan claramente, lo que conduce a un desafío cuando se combinan todos los extractos para el estudio 3 . Las interacciones de numerosos constituyentes / extractos median los efectos terapéuticos de las hierbas medicinales. Para conservar esta ventaja, la forma tradicional de extracto-decocciónIon-todavía es ampliamente utilizado en la clínica. Dado que el procedimiento de extracción tiene un gran impacto en la eficacia terapéutica, es necesario un protocolo estándar de decocción tradicional de agua, especialmente para estudios in vivo 4 , 5 . Por otro lado, al explorar mecanismos farmacológicos, la administración de decocciones a base de hierbas durante estudios in vitro o ex vivo es también un desafío. El concepto de un suero que contenía fármacos fue propuesto por primera vez por Tashino en 1988 6 . Desde entonces, un número creciente de investigadores lo han aplicado a la medicina herbal 7 , 8 , 9 . Aunque el método del suero que contiene fármaco tiene algunas limitaciones, tales como la influencia de ciertos componentes del suero, todavía se considera que es un método que imita de cerca las condiciones fisiológicas. 10 , 11 , 12 , 13 . También se ha aplicado al tratamiento de la anemia aplásica 14 , osteoporosis menopáusica 15 , 16 , 17 , fallo ovárico prematuro 18 , cáncer de mama 19 , cáncer de ovario 20 y pubertad tardía 21 . Aquí presentamos protocolos detallados para la preparación de la decocción er-xian (EXD) y su suero que contiene fármaco. Además, se describe la aplicación de EXD y EXD conteniendo suero a los modelos de osteoporosis menopáusicas murinos. Un EXD se compone de 9 g cada uno de Curculigo orchioides Gaertn , Herbaa Epimedii </eM, Radix Morindae Officinalis y Radix Angelicae Sinensis y 6 g de Cortex Phellodendri y Rhizoma Anemarrhenae por paciente adulto al día. La dosis equivalente para un ratón es de 0,1418 EXD / kg / día basándose en la siguiente ecuación 22 : dB = dA * RB / RA * (WA / WB) 1/3 . DA y dB se refieren a la dosis por peso corporal (mg / kg) del ser humano y del ratón, respectivamente. La dosis, en mg / kg, se sustituye por el número de EXD / kg. RA y RB representan el factor del cuerpo humano y el factor corporal del ratón respectivamente, que son proporcionales a la (superficie corporal (m 2 ) / peso corporal (kg)) 2/3 (ver Tabla 1 ). WA y WB indican el peso corporal (kg) del humano y del ratón, respectivamente.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	
	<tbody>
		<tr height="165">
			<td height="165" style="height:165px;width:216px;">Curculigo orchioides Gaertn (9&#8201;g), Herbaa Epimedii (9&#8201;g), Radix Morindae Officinalis (9&#8201;g), Radix Angelicae Sinensis (9&#8201;g), Cortex Phellodendri (6&#8201;g), and Rhizoma Anemarrhenae (6&#8201;g)</td>
			<td style="width:149px;">Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td>
			<td style="width:119px;">160922</td>
			<td style="width:167px;">EXD components</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Filter paper</td>
			<td style="width:149px;">GElifesciences</td>
			<td style="width:119px;">99-103-952</td>
			<td style="width:167px;">Filter EXD decoction before concentration</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Imprinting Control Region (ICR) mice</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">In vivo study</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprague Dawley rats</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">EXD-containing serum preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringe filter</td>
			<td style="width:149px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td style="width:167px;">0.22 &#181;m</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">gavage needles (10 ml)</td>
			<td style="width:149px;">Shanghai BO Ge trade sales department</td>
			<td align="right" style="width:119px;">59104274</td>
			<td style="width:167px;">Adminstration of EXD</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketamine (80 mg/kg)&#160;</td>
			<td style="width:149px;">Fujian Gutian Pharma Co. Ltd</td>
			<td style="width:119px;">H35020148</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Xylazine (10 mg/kg)</td>
			<td style="width:149px;">Sunway Pharma Co. Ltd</td>
			<td style="width:119px;">CB07591</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Dulbecco&#8217;s modification of Eagle&#8217;s medium Dulbecco (DMEM) culture medium</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">12800-116</td>
			<td style="width:167px;">DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">1277</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">4687</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
	</tbody>
</table>

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

La medicina herbal tradicional, una medicina alternativa en el ambiente clínico, ha recibido una atención creciente en los últimos años. Antes de la entrega al cuerpo, se requiere comúnmente un procedimiento de extracción adicional para liberar los componentes activos de las hierbas crudas. La decocción acuosa es un procedimiento de extracción clásico que todavía se utiliza ampliamente en los entornos clínicos. Aquí, proponemos un protocolo detallado para la decocción er-xian (EXD) con el fin de aplicar las decocciones a base de hierbas a los estudios experimentales. Se describe el cálculo de una dosis apropiada para animales, así como los cuatro pasos principales de EXD: remojo, decocción de agua, filtración y concentración. Además, el suero que contiene EXD se introduce a las ratas como un medio de validación in vitro . En este caso, las ratas fueron administradas por vía oral EXD durante tres días. Las muestras de sangre se recogieron entonces, se inactivaron, se centrifugaron y se filtraron. El suero, diluido con el medio de cultivo, puede utilizarse para tratar células o tejidos en vItro Por ejemplo, EXD se aplicó tanto a estudios in vivo e in vitro y demostró que EXD mejora la osteogénesis. Este protocolo puede utilizarse como referencia para la preparación y aplicación de medicamentos herbales.

El efecto de EXD sobre la densidad ósea de ratones OVX La tinción con hematoxilina y eosina (H &amp; E) de la sección de la vértebra lumbar muestra un aumento de las trabéculas óseas después del tratamiento EXD in vivo ( Figura 1A , panel derecho) en comparación con las del grupo OVX ( Figura 1A , panel izquierdo). La Figura 1B muestra las imágenes representativ...

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Aquí, se presenta la preparación de una decocción er-xian (EXD) en cuatro pasos-empapamiento, decocción, filtración y concentración- y demuestran la administración de un suero preparado para EXD a ratas. Estos métodos son aplicables al estudio in vivo e in vitro de las decocciones a base de hierbas como las medicinas tradicionales chinas.

Preparación de Herbal Medicine: Decocción de Er-Xian y suero que contiene Er-Xian para<em&gt; In Vivo</em&gt; Y<em&gt; In Vitro</em&gt; Experimentos

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

Research

JoVE Journal

Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

10.9K vistas.  Shanghai University of Traditional Chinese Medicine.  Aquí, se presenta la preparación de una decocción er-xian (EXD) en cuatro pasos-empapamiento, decocción, filtración y concentración- y demuestran la administración de un suero preparado para EXD a ratas. Estos métodos son aplicables al estudio in vivo e in vitro de las decocciones a base de hierbas como las medicinas tradicionales chinas. 

Video: Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

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. ...

Investigación

Medicina

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 ...

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 ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validación de nanocuerpo y basados ​​en anticuerpos<em&gt; En Vivo</em&gt; Experimentos tumor de xenoinjerto NIRF-de formación de imágenes en ratones utilizando<em&gt; Ex Vivo</em&gt; Citometría de flujo y microscopía

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

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 ...

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.

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 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Preparación y<em&gt; In Vitro</em&gt; Caracterización de Magnetized miR-modificado células endoteliales

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

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 ...

Preparación de Herbal Medicine: Decocción de Er-Xian y suero que contiene Er-Xian para

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

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

Preparación de Herbal Medicine: Decocción de Er-Xian y suero que contiene Er-Xian para

Resumen

Explorar más videos

Capítulos en este video

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

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

Evaluación de la efectividad del medicamento para el cáncer Sensibilización

Validación de nanocuerpo y basados ​​en anticuerpos

Telemetría intrauterina para medir la presión del ratón contráctil

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

Análisis optimizada de

Preparación y

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

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

In vivo MRI ^{de 19F} para el seguimiento de la célula

Validación de nanocuerpo y basados en anticuerpos