Estos trabajos fueron apoyados a través de becas de Ciencias Naturales e Ingeniería de Investigación de Canadá (RGPIN 238382 a 2.011) y los Institutos Canadienses de Investigación en Salud (MOP115076). Heather Paul recibió el apoyo de una Beca de Postgrado de Ciencias Naturales e Ingeniería de Investigación de Canadá y una beca Health Solutions Alberta innova. Megan Hallam fue apoyado por una de Ciencias Naturales e Ingeniería del Consejo de Investigación de Postgrado de Becas, una beca de Frederick Banting y Charles Best Graduate Canadá, y el Premio a la Infancia de Alberta Instituto de Investigación del Hospital de Capacitación en Genética, Desarrollo Infantil y Salud.

Este protocolo fue aprobado por la Universidad de Comité de Cuidado de Animales de Calgary y conforme a la Guía para el Cuidado y Uso de Animales de Laboratorio. 1. Separe la presa de Offspring <ol><li> Separar la presa de su descendencia por un mínimo de 5 minutos antes de ordeño 12. NOTA: La presa puede ser ordeñada hasta 5-6 horas después de la separación 1,6,13, sin embargo períodos de separación de más de 4 horas pueden alterar la composición de la leche 14. Mientras que el tiempo de separación no parece afectar el volumen de recogida de leche 12, se aconseja que un tiempo de separación consistente mantenerse durante todo el estudio. Composición de la leche pueden cambiar durante la lactancia 15, por lo tanto, se debe hacer intentos para mantener el día de la recogida de leche consistente. Se sugiere la producción máxima de leche que se producen en días de lactancia 14 12. </li><li> Utilizando una cámara de calentamiento, asegúrese de que los cachorros son capaces de mantener temperatu de la carrocería adecuadare sin la presencia de su madre durante la duración del procedimiento de ordeño. NOTA: En el estudio se presenta a continuación, el ordeño se realizó en el destete, cuando las presas eran aproximadamente 22 semanas de edad y de las crías de 21 días de edad, se utilizó por lo tanto ninguna cámara de calentamiento. </li></ol> 2. Instalación y Preparación <ol><li> Recoger todos los materiales necesarios para el procedimiento de ordeño. NOTA: Todos los materiales se pueden encontrar en la tabla de materiales y equipos. </li><li> Coloque una almohadilla térmica en el banco donde ordeño se llevará a cabo y para abarcar la almohadilla con un banco absorbente bajo-pad. </li><li> Configurar el sistema anestésico. Asegúrese de que el sistema tiene oxígeno e isoflurano antes de comenzar suficiente. Coloque la máscara de anestesia que se utilizará para la inducción de la anestesia inicial a la máquina. Coloque la máscara que se utilizará para el mantenimiento de la anestesia cercana si es diferente de la máscara de anestesia inicial. </li><li> Adjunte una aguja de 25 G a una jeringa de 1 ml de oxitocinainyección utilizando una técnica aséptica. </li><li> Encienda la almohadilla térmica de modo que se mantiene la temperatura del cuerpo de la madre durante el procedimiento de ordeño. NOTA: Controle la temperatura de la almohadilla térmica para asegurar la almohadilla no se convierta en demasiado caliente y causar quemaduras. Alternativamente, utilizar una fuente de calor, tal como una mesa quirúrgica calentada, que se puede ajustar a una temperatura específica. </li></ol> 3. Anestesiar la presa de Uso isoflurano <ol><li> Abra el tanque de oxígeno y gire el flujo a 1 L (1000 cc) por min. Encienda el flujo de isoflurano y se puso a 5%. ATENCIÓN: Evitar la inhalación directa de anestesia y evitar la acumulación de vapores anestésicos. </li><li> Anestesiar la presa. </li><li> Cambiar a la máscara de mantenimiento, si es necesario, la colocación de la supina presa en la plataforma de banca absorbente. Confirme anestesia por la falta de reflejo de pedal. </li><li> Reducir el flujo de isoflurano al 2-3% para el mantenimiento de la anestesia. Monitorear continuamente presa durante todo el procedimiento para asegurarse depression de la respiración no se produce. NOTA: Una vez bajo anestesia, los ojos de la presa deben protegerse utilizando un lubricante ocular estéril para evitar que los ojos se sequen o se raye. </li></ol> 4. La oxitocina Inyección <ol><li> Asegúrese de que la oxitocina (20 unidades USP / ml) no ha pasado su fecha de caducidad. Desinfectar el vial de oxitocina con un alcohol estéril limpie / almohadilla de limpieza de alcohol. </li><li> Utilizando una técnica aséptica, elaborar 2 UI (0,1 ml) de la oxitocina en la jeringa. Use una aguja y una jeringa para cada presa que se ordeñaba. NOTA: La oxitocina dosis varían generalmente de inyecciones únicas de 1 a 5 UI 1,6,12,16. Alternativamente, una dosis de 4 UI / kg de peso corporal se puede utilizar 12. Una sola dosis de 2 UI puede repetirse una vez si se encuentra dificultad de ordeño. </li><li> Inyectar la oxitocina por vía intraperitoneal. Inserte la aguja en el cuadrante inferior derecho del abdomen con la aguja apuntando hacia la cabeza, en un ángulode 15-30 °, aproximadamente 0,5 cm de profundidad. </li><li> Tire hacia atrás el émbolo para asegurarse de presión negativa antes de la inyección. Si cualquier líquido (sangre, orina, contenido intestinal, etc.) se aspira con la jeringa, retire la aguja e intentar la inyección con una aguja y jeringa. Si hay líquido se aspira inyectar la oxitocina y desechar la aguja y jeringa inmediatamente en un contenedor de riesgo biológico. </li><li> Espere aproximadamente 5 a 15 minutos para la oxitocina para estimular la bajada de la leche. </li></ol> 5. Preparación de Sitios de ordeño <ol><li> Elija los sitios / los pezones de la que se obtuvo leche. La leche puede ser recogida desde cualquier tetina 12. </li><li> Retire suavemente la piel alrededor de los pezones para ser ordeñadas con los trimmers, como la piel puede causar dificultad en la recolección de la muestra debido a la absorción de la leche. Sea amable - la piel alrededor de los pezones es extremadamente sensible y puede estar seca y, como tal, es susceptible a los rasguños y las lágrimas. </li><li> La esterilización de la i tetinas no es necesario, pero opcionalmente, limpiar el pezón con agua tibia después de retirar la piel. Preparar al menos dos sitios como más de un sitio puede ser necesaria para la recogida de leche. NOTA: Si el análisis de la leche incluye perfiles microbiana, el área del pezón puede requerir esterilización con yodo 5. </li></ol> 6. Leche Colección <ol><li> Apriete suavemente la base del pezón, expulsar manualmente la leche para la colección. NOTA: Si el análisis de la leche incluye perfiles microbiana, las primeras gotas de leche deben ser desechados. </li><li> Recoger las gotas de leche en un tubo capilar, llenando el tubo capilar. Tubos capilares que se adaptan a volúmenes más grandes (por ejemplo, 50 l) facilitar el proceso. NOTA: Si se encuentra dificultad en la recogida de la leche, una segunda dosis de oxitocina puede ser administrado. Se recomienda que la dosis sea superior a 4 UI totales. </li><li> Prescindir de la leche desde el tubo capilar en un tubo de microcentrífuga estériltocando el extremo del tubo que se utilizó para extraer la leche de la teta a un lado del tubo de microcentrífuga - observar la leche que se extrae del tubo capilar a través de la acción capilar. NOTA: &#39;Blow out&#39; de la leche que no se introduce en el tubo usando una aguja de 18 G unida a una jeringa de 1 ml. </li><li> Monitorear continuamente la presa para detectar signos de dolor o depresión respiratoria y ajustar el flujo de isoflurano en consecuencia. </li><li> Continuar para recoger la leche como se describe en esta sección hasta suficiente leche se ha recogido para el análisis de la leche elegido. Para la determinación de la concentración creamatocrit y la proteína se describe a continuación, recoger 0,25 ml de leche. NOTA: Utilice un sitio de ordeño diferente si bajada de la leche disminuye o el sitio elegido no expulsa la leche suficiente. Un máximo de aproximadamente 2,5 ml de leche por animal puede ser recogida 17, o hasta 0,5 ml por pezón. Los autores recomiendan que el tiempo total bajo anestesia se limitará a aproximadamente 45 a 60 minutos, or 45 min de ordeño. </li><li> Recogida de la leche en un tubo microhematocrito para la medición creamatocrit partir de muestras de leche fresca, y sellar el extremo del tubo con sellador de arcilla. Etiquetar el tubo microhematocrito con el ID de presa y almacenar la muestra de leche en posición vertical. </li><li> Cuando el ordeño es completa, apague el flujo de isoflurano y oxígeno. Retire la máscara de la presa y continuar monitoreando la presa hasta que despierto. Se recomienda que si no es plenamente consciente, la presa se debe colocar en una almohadilla absorbente banco durante el período de recuperación, en lugar de hacerlo directamente sobre la ropa de cama de la jaula, para evitar que la ropa de cama de ser aspirados o rascarse los ojos de la presa durante la recuperación. NOTA: No deje desatendida la presa hasta que se haya recuperado el conocimiento suficiente para mantener recumbancy esternal. </li><li> Si no se requieren nuevos análisis, congelar la leche a -80 ° C. Otros han sugerido que la leche se puede almacenar durante un máximo de 3 horas a 4 ° C o 5 meses a -20 ° C 18. </li></ol> 7. Medición Creamatocrit <ol><li> Estimar el contenido de grasa en la leche mediante el cálculo del creamatocrit leche (el porcentaje de la crema en la muestra de leche) 19. NOTA: Las mediciones con la leche humana han demostrado que la leche ya sea fresco o congelado puede ser usado para una medición creamatocrit, sin embargo leche fresca es más altamente correlacionado (r = 0.92 r = 0,90 frente) con la concentración de lípidos 11. El uso de leche fresca o congelada para mediciones creamatocrit debe mantenerse coherente en el estudio, como la leche descongelada se asocia con una pequeña disminución en los valores creamatocrit 11. </li><li> Para la leche fresca, se recoge una muestra de leche de la teta en un tubo microhematocrito; rellenar al menos ¾ de su capacidad (aproximadamente 15-20 l). Alternativamente, dibujar leche fresca de la muestra recogida en el tubo capilar después de mezclar bien. Selle el extremo con sellador de arcilla. </li><li> Coloque el tubo capilar en el spinner hematocrito, con elextremo sellado apuntando hacia el exterior, asegurando la centrífuga es equilibrada. </li><li> Comience el spin hematocrito (120 segundos a 13.700 xg). NOTA: El tiempo de la vuelta o la velocidad pueden variar dependiendo del modelo de centrífuga utilizada. </li><li> Retire el tubo de la centrífuga después de la vuelta es completa y realizar las mediciones para calcular el creamatocrit. Observe la separación de la muestra en una capa de crema y una capa transparente. </li><li> Medir y registrar la longitud total de fluido en el tubo y la longitud de la grasa (crema) capa usando pinzas o una regla. NOTA: El creamatocrit se expresa como el porcentaje de la capa de crema dentro de la muestra de la leche 19, calculado como (longitud de capa de crema / longitud total de la columna de la leche) x 100 (Figura 1A). Calcular los valores de concentración y la energía de grasa de la medición creamatocrit como sigue: concentración de grasa (g / L) = (creamatocrit (%) - 0.59) /0.146 (Figura 1B) 19; Valor energético (kcAl / L) = 290 + (66,8 * creamatocrit (%) (Figura 1C) 19. </li></ol> 8. Proteína Concentración Determinación <ol><li> El uso de albúmina de suero bovino como un estándar de proteína, determinar la concentración total de proteína de la leche utilizando un ensayo estándar de la proteína, tal como una proteína ensayo de Lowry 6. NOTA: La dilución de la leche puede ser necesario para las mediciones de proteína de leche que caigan dentro de la curva estándar del ensayo. </li></ol>

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Los autores no tienen conflictos de intereses a revelar. Todos los experimentos con animales se realizaron de acuerdo con protocolos CCAC aprobado.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

La leche es la única fuente de nutrición para los mamíferos recién nacidos, el suministro de energía y nutrientes para el crecimiento infantil y desarrollo 1,2. Mientras que la leche se compone principalmente de células, lípidos, y proteínas 1, también contiene una gran cantidad de compuestos bioactivos que modulan el desarrollo primeros años de vida de la descendencia incluyendo enzimas, carbohidratos, hormonas, anticuerpos, factores de crecimiento, citoquinas, exosomas, microvesículas, y pequeños RNAs tales como microARN 1,2. El papel fundamental de la leche materna en el establecimiento de inmune y la salud intestinal 3 crías, junto con la evidencia de que los lactantes amamantados son menos susceptibles a la enfermedad 2, pone de relieve la importancia de identificar los componentes de la leche asociados a los procesos de enfermedad en la vida temprana y los mecanismos moleculares implicados en sus acciones. La rata en desarrollo es un modelo popular para investigar el efecto de diferentes nutricional, fisiológica, y las intervenciones químicas en principiosdesarrollo -life 4. El análisis de la leche de rata puede, por tanto, proporcionar nuevos visión de la salud materna y la descendencia. Los avances científicos actuales ofrecen ahora el aumento de oportunidades para investigaciones en profundidad de los efectos de los componentes de la leche específicos sobre la salud y la enfermedad. Por ejemplo, la secuencia de los perfiles de bacterias de la leche ha dilucidado su papel en la colonización intestinal temprana del intestino del bebé 5, el análisis de espectrometría de masas de los oligosacáridos de la leche han proporcionado información sobre la alteración de los perfiles de oligosacáridos de la leche a través de la dieta materna 6, y profundo secuenciación de microARN secretada en los glóbulos de grasa de la leche materna pone de manifiesto posibles funciones en la transcripción de genes, el metabolismo y la función inmune 7. Modelos de ratas representan uno de los organismos modelo más populares utilizados en los estudios maternos 8,9. Una ventaja es sus períodos de gestación y lactancia cortas, solamente approximat duraderosely 21 días cada uno; Por lo tanto, el tiempo total desde el comienzo del embarazo para la lactancia representa un corto período de tiempo en el que los datos valiosos pueden ser generados. El mayor tamaño de las ratas en comparación con ratones, en el contexto de recogida de leche, puede proporcionar una ventaja significativa con respecto al volumen de leche y la facilidad de recolección de la leche; la producción de leche en el ratón, por ejemplo, parece ser depende del peso corporal total con los ratones más pesados ​​que producen más leche 10. Aquí, se proporciona una descripción general para la recolección manual de la leche de ratas lactantes. Este protocolo requiere un mínimo de equipo, es no invasivo, de bajo costo, y se puede utilizar para recoger volúmenes adecuados de la leche para análisis posteriores aguas abajo. En resumen, la presa se anestesia con isoflurano, la bajada de leche es estimulada por la oxitocina, y la leche se recoge en tubos capilares a través de la extracción manual de la leche. Por último, como dos componentes principales de la leche son grasas y proteínas, una breve descriptiose presenta n de estimar el contenido de grasa de la leche mediante mediciones creamatocrit 11 y cuantificación de concentración de proteína total utilizando un ensayo de proteína estándar.

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			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
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			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
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			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
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			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
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			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
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milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

La leche se recogió como se describe en el destete de las presas Wistar (aproximadamente 22 semanas de edad, pesando 350 a 400 g) que consumió un control (AIN-93G, n = 5), alta en proteínas (40% de caseína p / p, n = 5) o alto contenido de fibra prebiótica (21,6% peso / peso, 1: 1 relación de oligofructosa e inulina, n = 4) dieta a lo largo embarazo y la lactancia. La dosis de oxitocina fue 2 IU. La leche se recogió usando tubos capilares, y un tubo se centrifugó utilizando una centrifugadora de hematocrito para...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Leche Colección de la rata utilizando tubos capilares y Estimación del contenido de grasa de la leche por Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K vistas.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Video: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Investigación

Biología

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

El aislamiento de fibroblastos portales rata por<em&gt; In situ</em&gt; Hígado de perfusión

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagen funcional de grasa marrón en ratones con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Métodos de recolección de leche Ratones y Reeves Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Recopilación y análisis de<em&gt; Arabidopsis</em&gt; Floema exudados mediante el método de EDTA-facilitado

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Perfilado Redox Celular Utilizando Microscopía de Alto Contenido

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinación de ácidos siálicos en hígado y muestras de leche de tipo salvaje y<em&gt; CMAH</em&gt; Ratones knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Análisis de la autofagia inducida por inanición en el cuerpo graso larvario de Drosophila melanogaster

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Cuantificación de la infiltración grasa del músculo esquelético mediante descelularización

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Autor destacado: Cuantificación basada en la descelularización de la infiltración grasa del músculo esquelético  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.