Los autores desean agradecer a Michael Goldberg para la preparación de solución siRNA estabilizado y Heather Tobin por su ayuda con los animales. Este estudio fue financiado por el Departamento de Defensa Premio Innovador W81XWH-08-1-0659 de DEI. La formación postdoctoral de SK fue apoyado por la fundación Susan G Komen (KG101329). Los autores desean agradecer a Kristin Johnson para la preparación del esquema.

1. Preparación de la solución de inyección <ol><li> Preparar 0,2% colorante azul de Evans en solución salina tamponada de fosfato (PBS) u otra solución acuosa de interés. Este colorante vital es útil para evaluar el éxito de las inyecciones intraductal y se recomienda como una ayuda para la visualización en el desarrollo de conocimientos especializados en la técnica. </li></ol> El volumen requerido dependerá del número y ubicación de las glándulas para ser inyectado. Todos los 10 glándulas mamarias de ratones hembras pueden ser inyectados, pero debido al tamaño más pequeño de la glándula, pares de glándulas 1 y 5 son típicamente inyectados con 10 l de solución. Todos los otros pares mamarias son inyectados con 20 l. Estos volúmenes son suficientes para cubrir todo el árbol ductal. 2. Preparación preoperatoria <ol><li> Anotar el peso corporal de cada ratón. Al igual que con todos los estudios preclínicos, los pesos de los animales deben ser controlados regularmente (dos veces por semana o más) para evaluar la toxicidad potencial. </li><li> Se anestesia la mOuse usando una cámara de isoflurano y aplicar lubricante ocular. Durante el procedimiento de ratones continuarán siendo anestesiado utilizando inhalado concentración de 2-4% de isoflurano en oxígeno a través de un cono de nariz. Es importante vigilar cuidadosamente el ratón para los cambios en la frecuencia respiratoria, el ajuste del nivel de isoflurano en consecuencia. </li><li> Inyectar el meloxicam (5-10 mg / kg) por vía subcutánea antes del procedimiento como la analgesia. </li><li> Aplique una crema de depilación over-the-counter de la zona del pezón. Espere 5 minutos y retire suavemente el cabello suelto con un aplicador de punta de algodón con un movimiento circular. Retire la crema usando toallas de papel húmedas mojadas con agua tibia. El afeitado no se recomienda debido al riesgo de dañar los pezones. </li><li> Asegure el ratón bajo el estereoscopio pegando suavemente las extremidades. </li><li> Lugares de inyección limpias con toallitas de alcohol. </li></ol> 3. Inyección intraductal <ol><li> Localice pezones adecuados para ser inyectado bajo el estereoscopio. Utilice fina micro-disección tweezers para eliminar la piel muerta que cubre la abertura del pezón. </li><li> Cargar 10-20 l de solución de inyección en una jeringa de 50 l con un G cubo de la aguja de metal 33 fijada. A veces, después de la inyección, pequeñas cantidades (1 mu l o menos) pueden filtrarse del pezón al tirar la aguja. Por lo tanto, se recomienda para inyectar 11 o 21 l, respectivamente, para tener en cuenta la pérdida potencial. </li><li> Sostenga el pezón suavemente con las pinzas finas y levántelo ligeramente para posicionarla para inyección. No es necesario cortar el pezón. </li><li> Se inyecta la solución lentamente para minimizar el daño potencial causado por el fluido que se mueve rápidamente dentro de los lúmenes ductales. La velocidad de inyección debe mantenerse a aproximadamente 40 l / min. </li></ol> 4. Cuidado Postoperatorio <ol><li> Observe el lugar de la inyección. No debe haber signos de trauma a la región del pezón o el tejido circundante. Hinchazón en el área que rodea el pezón probablemente indica una almohadilla de grasa mamaria Rathe inyecciónR que una inyección intraductal éxito. </li><li> Quite el animal del cono de la nariz y pasar a una jaula separada para la recuperación. Coloque la jaula bajo una lámpara de calor para prevenir la hipotermia y ayudar en la recuperación. Los ratones son alojados individualmente y serán monitoreados de cerca hasta que recobren el conocimiento y la movilidad. </li></ol> 5. Análisis de glándula mamaria de tejidos <ol><li> Al finalizar el estudio, los ratones se sacrifican por dislocación cervical después de CO 2 de gas comprimido en una cámara de aislamiento. Las glándulas mamarias se extirpan y se pueden usar para la preparación de todo el montaje 11, la histología, o ARN y aislamiento de las proteínas. </li></ol>

<ol>
	<li>Dooley, W. C., 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=Ductal+lavage+for+detection+of+cellular+atypia+in+women+at+high+risk+for+breast+cancer.">Ductal lavage for detection of cellular atypia in women at high risk for breast cancer.</a> J Natl Cancer Inst. 93, 1624-1632 (2001).</li><li>Jacobs, L., Sukumar, S., Stearns, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+therapy+for+the+prevention+of+breast+cancer.">Intraductal therapy for the prevention of breast cancer.</a> Curr Opin Investig Drugs. 11, 646-652 (2011).</li><li>King, B. L., Love, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraductal+approach+to+the+breast:+raison+d'etre.">The intraductal approach to the breast: raison d'etre.</a> Breast Cancer Res. 8, 206 (2006).</li><li>Murata, S., 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=Ductal+access+for+prevention+and+therapy+of+mammary+tumors.">Ductal access for prevention and therapy of mammary tumors.</a> Cancer Res. 66, 638-645 (2006).</li><li>Stearns, V., 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=Preclinical+and+clinical+evaluation+of+intraductally+administered+agents+in+early+breast+cancer.">Preclinical and clinical evaluation of intraductally administered agents in early breast cancer.</a> Sci Transl Med. 3, 106ra108 (2011).</li><li>Flanagan, M., Love, S., Hwang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+of+Intraductal+Therapy+for+Ductal+Carcinoma+in+Situ.">Status of Intraductal Therapy for Ductal Carcinoma in Situ.</a> Curr Breast Cancer Rep. 2, 75-82 (2010).</li><li>Nguyen, D. -. A., Beeman, N., Lewis, M., Schaack, J., Neville, M. C., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in mammary gland biology and breast cancer research. , 259-270 (2000).</li><li>Behbod, F., 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=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Res. 11, R66 (2009).</li><li>Bu, W., Xin, L., Toneff, M., Li, L., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+vectors+for+stably+introducing+genes+into+mammary+epithelial+cells+in+vivo.">Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 401-404 (2009).</li><li>Reddy, J. P., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS-TVA+system+for+introduction+of+oncogenes+into+selected+somatic+mammary+epithelial+cells+in+vivo.">The RCAS-TVA system for introduction of oncogenes into selected somatic mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 405-409 (2009).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mammary+gland+development+and+function+in+mouse+models.">Evaluation of mammary gland development and function in mouse models.</a> J. Vis. Exp. , e2828 (2011).</li></ol>

Los autores no tienen accesos financieros.

Los ratones transgénicos y knockout son herramientas muy valiosas para estudiar el papel in vivo de genes individuales en el cáncer de mama. Sin embargo, es costoso y requiere mucho tiempo para generar estos animales y la caída gen es por lo general ubicua que a veces nos impide el reconocimiento de los efectos específicos de ese gen en la glándula mamaria. Por lo tanto, una caída dirigida aliviaría los problemas de efectos secundarios y toxicidades no específicas de otros órganos. La inyección intraductal de ARNsi que aquí se presenta permite la caída gen localizado en la glándula mamaria de ratón. Es un método rápido para una entrega dirigida de medicamentos y localizada a la glándula mamaria no limitado a la entrega de siRNA. Tal administración de fármacos específico de la glándula mamaria evita efectos secundarios inespecíficos y toxicidad a otros órganos y permite el estudio de las alteraciones de genes en puntos de tiempo específicos durante el desarrollo de la glándula mamaria y la formación de tumores de mama. No hay evidencia de SiPartículas de ARN en las glándulas no inyectados, el hígado u otros órganos que proporcionan evidencia adicional de que este método presenta una forma preferida de caída gen localizado en la glándula mamaria. Además, las inyecciones intraductal pueden repetirse semanalmente o cada dos semanas durante varios meses para permitir la monitorización a largo plazo de los agentes terapéuticos. Esta técnica abre la posibilidad para el ensayo de la entrega intraductal de diversos reactivos para los conductos mamarios de ratón. Los avances en la entrega localizada en modelos de ratón bien caracterizados deben acelerar la aplicación de la no-invasivo, dirigido estrategias terapéuticas en seres humanos.

La glándula mamaria de ratón se compone de un árbol-ductal alveolar complejo que converge en un conducto central en el pezón. En los ratones, así como los seres humanos, la mayoría de los tumores de mama se originan en las células epiteliales que recubren los conductos de la leche. Tratamientos actuales incluyen la quimioterapia intravenosa, la radioterapia y la cirugía. Tratamientos intraductal localizadas se han explorado 1-3 y ofrecer importantes ventajas en términos de reducción de la toxicidad y efectos secundarios. Sin embargo, varios desafíos quedan antes de que estos pueden ser aplicados más ampliamente. La capacidad de realizar procedimientos comparables en la glándula mamaria de ratón facilita la caracterización de las intervenciones intraductales. Aquí presentamos un método no invasivo para la entrega de reactivos directamente en el conducto mamario de ratón. Entrega intraductal de agentes quimioterapéuticos en ambos pacientes de cáncer de mama y modelos de ratón ha sido previamente demostrado ser muy eficaz sin evidencia detoxicidad sistémica o histopatológico a largo plazo cambia 3-6. Además, la inyección de células tumorales y de entrega de vector lentiviral de oncogenes han sido previamente demostrado ser factible a través de inyección intraductal 7-10. Además de la administración de fármacos, este procedimiento es útil para el silenciamiento de los genes local de la glándula mamaria a través de la inyección intraductal de ARNsi. La inyección de pequeños volúmenes de solución de ARNsi es eficaz sin alterar la anatomía y fisiología de la glándula mamaria. En nuestro laboratorio hemos demostrado recientemente silenciamiento exitosa de genes clave mediador en ratones transgénicos que desarrollan espontáneamente tumores de mama que resulta en la prevención de la progresión del tumor (Brock et al., No publicado). Hemos sido capaces de repetir las inyecciones cada dos semanas en los mismos pezones hasta 7 veces en intervalos de 2 semanas sin ningún daño tisular visible o toxicidad. Inyecciones intraductal pueden ser programados en un ratón adulto para abordar desarrolloeventos opmentally específicos relacionados con el desarrollo de la glándula mamaria durante el embarazo, la lactancia y la involución o para apuntar últimas etapas durante el desarrollo y progresión del tumor. Las hembras vírgenes de tan sólo 8 semanas de edad se han inyectado de forma no invasiva en nuestro laboratorio.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>meloxicam</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5-10 mg/ml</td>
		</tr>
		<tr>
			<td>Evans blue dye</td>
			<td>Sigma-Aldrich</td>
			<td>E2129-10G</td>
			<td>0.2% in PBS</td>
		</tr>
		<tr>
			<td>hair removing cream</td>
			<td>Veet</td>
			<td>&nbsp;</td>
			<td>Choose sensitive option</td>
		</tr>
		<tr>
			<td>stereoscope</td>
			<td>Zeiss</td>
			<td>Stemi DV4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>micro-dissecting tweezers</td>
			<td>Roboz</td>
			<td>rs-4976</td>
			<td>With a 0.10 x 0.06 mm tip made of dumostar</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7637-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>33-gauge needle</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>point style 4, 12 degree bevel (standard)</td>
		</tr>
		<tr>
			<td>siGLO cyclophilin B control siRNA</td>
			<td>Dharmacon</td>
			<td>D-001610-01-05</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

intraductal injection for localized drug delivery to the mouse mammary gland

Aquí se describe un protocolo para entregar diversos reactivos para la glándula mamaria de ratón a través de inyecciones intraductales. Entrega de la droga localizada y knock-down de los genes en el epitelio mamario ha sido difícil de lograr debido a la falta de moléculas diana apropiadas que son independientes de las etapas de desarrollo, como el embarazo y la lactancia. Aquí se describe una técnica para la administración localizada de los reactivos a la glándula mamaria en cualquier etapa en la edad adulta a través de inyección intraductal en los pezones de los ratones. Las inyecciones se pueden realizar en ratones vivos, bajo anestesia, y permiten una administración de fármacos no invasivo y localizada a la glándula mamaria. Además, las inyecciones se pueden repetir durante varios meses sin dañar el pezón. Colorantes vitales como el azul de Evans son muy útiles para aprender la técnica. Tras la inyección intraductal de colorante azul, todo el árbol ductal se hace visible para el ojo. Por otra parte, los reactivos marcados con fluorescencia también permitenla visualización y la distribución dentro de la glándula mamaria. Esta técnica es adaptable para una variedad de compuestos que incluyen ARNsi, agentes quimioterapéuticos, y pequeñas moléculas.

Los pezones se pueden localizar fácilmente utilizando un estereomicroscopio una vez que el pelo se ha eliminado en el área que la rodea (Figura 1A). Para dominar la técnica de inyección, se recomienda para inyectar colorante azul de Evans y controlar la integridad de la glándula (Figuras 1B y C). Este enfoque también permite la determinación de volúmenes apropiados para ser inyectada en cada glándula como uno puede evaluar visualmente si el tinte llega a todo el sistema ductal (Figuras 1C y 2A). Una imagen de la imagen azul de Evans inyectado puede entonces compararse a toda montada la glándula mamaria, en la que todo el árbol ductal se tiñe con colorante carmín alumbre (Figura 2B). Tenga en cuenta que la robustez de este método de inyección depende en gran medida el operador. Por ejemplo, perforar el conducto resultará en una almohadilla de grasa mamaria de inyección e inyectar la solución demasiado rápido puede dañar el CEL epitelial ductalLS y provocar una respuesta inflamatoria. Inyección intraductal exitosa permite la entrega localizada de fármacos a la glándula mamaria y reduce los efectos secundarios no específicos observados a menudo lo contrario. Una imagen de ejemplo de una glándula inyectado con marcado con fluorescencia siRNA dirigidos a ciclofilina (un gen no esencial) se muestra en la Figura 3. <img alt="Figura 1" src="/files/ftp_upload/50692/50692fig1.jpg" /> Figura 1. Una glándula mamaria inguinal inyecta a través del pezón con colorante azul de Evans. A) Aspecto del pezón antes de la inyección y B) después de la inyección con 20 l colorante azul de Evans. No hinchazón o daño tisular se observa. C) Al abrir la piel, el medio de contraste permite la visualización de todo el árbol ductal mamaria. <img alt="Figura 2" src="/files/ftp_upload/50692/50692fig2.jpg" /> Figura 2. Análisis de azul de Evans en Todo el montajeglándula proyectada. A) Imagen representativa de una glándula diseccionado extirpado del animal y se extendió sobre un portaobjetos de vidrio inmediatamente después de la inyección. B) Total tinción de montaje de la misma glándula con colorante carmín después de grasa había sido despejado. Comparación con A) confirma que la solución inyectada se llena el árbol ductal de mama y de la glándula es anatómicamente intacto. <img alt="Figura 3" src="/files/ftp_upload/50692/50692fig3.jpg" /> Figura 3. Entrega intraductal de siRNA fluorescente. Imagen representativa de la muestra de tejido fresco, extirpó 48 horas después de la inyección.

Watch this Scientific Journal Video about Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland at JoVE.com

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Un protocolo para la entrega intraductal no invasiva de los reactivos acuosos para la glándula mamaria de ratón se describe. El método se aprovecha de la inyección localizada en los pezones de las glándulas mamarias dirigidos específicamente conductos mamarios. Esta técnica es adaptable para una variedad de compuestos que incluyen ARNsi, agentes quimioterapéuticos y moléculas pequeñas.

Inyección intraductal de entrega de la droga localizada a la glándula mamaria de ratón

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

intraductal-injection-for-localized-drug-delivery-to-mouse-mammary

Research

JoVE Journal

Biology

34.6K vistas.  Boston Children's Hospital and Harvard Medical School.  Un protocolo para la entrega intraductal no invasiva de los reactivos acuosos para la glándula mamaria de ratón se describe. El método se aprovecha de la inyección localizada en los pezones de las glándulas mamarias dirigidos específicamente conductos mamarios. Esta técnica es adaptable para una variedad de compuestos que incluyen ARNsi, agentes quimioterapéuticos y moléculas pequeñas. 

Video: Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Células epiteliales mamarias de ratón forma Mammospheres durante la diferenciación lactogénica

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Investigación

Biología

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Los cambios en la morfología de la glándula mamaria y el riesgo de cáncer de mama en ratas

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluación de la glándula mamaria de desarrollo y función en modelos de ratón

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Análisis Phosphopeptide de roedores epidídimo Espermatozoides

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Inmunofluorescencia indirecta en secciones congeladas de ratón Glándula Mamaria

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

Estudio comparativo de los métodos de entrega de fármacos dirigidos a la oreja del ratón interno: Bullostomy<em&gt; vs.</em&gt; Inyección transtimpánicos

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

Aislamiento de las glándulas mamarias del ratón intacto, todo para el análisis de expresión de la matriz extracelular y la morfología de la glándula

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

Ligadura del conducto biliar parcial en el ratón: un modelo controlado de la colestasis obstructiva localizada

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.