Os autores gostariam de agradecer a Michael Goldberg para preparar solução siRNA estabilizado e Heather Tobin por sua assistência com os animais. Este estudo foi financiado pelo Departamento de Defesa Innovator Award W81XWH-08-1-0659 para DEI. O treinamento de pós-doutorado de SK foi apoiado pela fundação Susan G Komen (KG101329). Os autores gostariam de agradecer a Kristin Johnson para a preparação do esquema.

1. Preparação de Solução de injecção <ol><li> Prepare a 0,2% de corante azul de Evans em tampão fosfato salino (PBS) ou outra solução aquosa de interesse. Este corante vital é útil para avaliar o sucesso de injeções intraductais e é recomendado como auxiliar de visualização ao desenvolver conhecimentos na técnica. </li></ol> O volume necessário dependerá do número e localização das glândulas para ser injectado. Todos os 10 glândulas mamarias de ratos fêmeas pode ser injectado, mas, devido ao menor tamanho da glândula, pares de glândulas 1 e 5 são tipicamente injectados com 10 ul de solução. Todos os outros pares mamarias foram injectados com 20 ul. Esses volumes são suficientes para preencher toda a árvore ductal. 2. Preparação pré-operatória <ol><li> Registe o peso do corpo de cada rato. Tal como acontece com todos os estudos pré-clínicos, pesos dos animais devem ser monitorizados regularmente (duas vezes por semana ou mais) para avaliar a toxicidade potencial. </li><li> Anestesiar a mouse usando uma câmara de isoflurano e aplicar lubrificante ocular. Durante o procedimento, os ratos continuarão a ser anestesiados com inalação de concentração de 2-4% de isoflurano em oxigênio através de um cone do nariz. Acompanhar atentamente o mouse para mudanças na freqüência respiratória, ajustando o nível de isoflurano em conformidade. </li><li> Injectar meloxicam (5-10 mg / kg) subcutaneamente antes do procedimento como analgesia. </li><li> Aplicar um creme sobre a remoção de balcão de cabelo para a área do bocal. Espere 5 minutos e retire cuidadosamente o cabelo solto com um aplicador de algodão de ponta usando um movimento circular. Retire o creme de usar toalhas de papel úmidas molhadas com água morna. Barbear não é recomendada devido ao risco de danos aos mamilos. </li><li> Fixe o mouse sob o estereoscópio delicadamente gravando as extremidades. </li><li> Locais de injeção limpos com compressas com álcool. </li></ol> 3. Injeção Intraductal <ol><li> Localize mamilos apropriadas para ser injectado sob a estereoscópio. Use bem micro-dissecando tweezers para remover qualquer pele morta que cobre a abertura do mamilo. </li><li> Carga de 10-20 mL de solução de injeção em uma seringa de 50 mL com um 33 G hub agulha de metal afixada. Às vezes, após a injeção, pequenas quantidades (1 mL ou menos) pode vazar para fora do mamilo após retirar a agulha. Portanto, recomenda-se a injectar 11 ou 21 ul, respectivamente, para ter em conta a perda de potencial. </li><li> Segure o mamilo suavemente com a pinça fina e levante-o ligeiramente para posicioná-la para a injecção. Não é necessário cortar o mamilo. </li><li> Injetar a solução lentamente para minimizar os danos potenciais causados ​​por um fluido movendo-se rapidamente dentro dos lumens ductal. A taxa de injecção deve ser mantido a cerca de 40 mL / min. </li></ol> 4. Cuidados no pós-operatório <ol><li> Observe o local da injeção. Não deve haver nenhum sinal de trauma para a região do mamilo ou tecido circundante. Inchaço na área em torno do mamilo provavelmente indica uma almofada de gordura mamária injeção em vez de sinr do que uma injecção intraductal bem sucedida. </li><li> Remover o animal do cone do nariz e se mudar para uma gaiola separada para recuperação. Coloque a gaiola, sob uma lâmpada de aquecimento para evitar a hipotermia e ajudar na recuperação. Os ratos são alojados individualmente e será acompanhada de perto, até que recuperar a consciência e mobilidade. </li></ol> 5. Análise da glândula mamária Tissue <ol><li> Após a conclusão do estudo, os ratinhos foram sacrificados por deslocamento cervical seguinte CO 2 de gás comprimido numa câmara de isolamento. Glândulas mamarias são excisadas e pode ser utilizado para a preparação toda suporte 11, histologia, ou RNA e isolamento da proteína. </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>

Os autores não têm divulgações financeiras.

Transgênicos e knockout camundongos são ferramentas inestimáveis ​​para estudar o papel in vivo de genes individuais em câncer de mama. No entanto, isso é caro e para gerar estes animais demorado e o knockdown do gene é normalmente ubíquo que, por vezes, nos impede de reconhecer os efeitos específicos de gene em que a glândula mamaria. Portanto, um knockdown alvo seria aliviar problemas de efeitos colaterais não-específicos e toxicidades em outros órgãos. A injecção intraductal de siRNA aqui apresentada permite o knockdown gene localizado na glândula mamária do rato. É um método rápido para a entrega de drogas específicas e localizada para a glândula mamaria não se limitando a entrega de siRNA. Tal entrega mamária fármaco específico da glândula evita os efeitos secundários e toxicidade não específicos para outros órgãos e permite o estudo de alterações do gene em pontos de tempo específicos durante o desenvolvimento da glândula mamaria e a formação do tumor da mama. Não há nenhuma evidência de SiPartículas de RNA em glândulas não injectados, o fígado ou outros órgãos que fornecem evidências adicionais de que este método apresenta um modo preferido para knockdown gene localizado na glândula mamária. Além disso, as injecções intraductais pode ser repetido semanalmente ou quinzenalmente ao longo de vários meses, para permitir a monitorização de longo prazo de agentes terapêuticos. Esta técnica abre-se a possibilidade de ensaiar a entrega intraductal de vários reagentes para os ductos mamários de ratinho. Avanços na entrega localizada em modelos bem caracterizados rato deve acelerar a aplicação do não-invasiva, alvo estratégias terapêuticas em seres humanos.

A glândula mamaria do rato é composto por uma árvore ductal-alveolar complexo que converge para um tubo central, no mamilo. Em ratinhos, bem como os seres humanos, a maioria dos tumores da mama são originários das células epiteliais que revestem os ductos. Os tratamentos atuais disponíveis incluem quimioterapia intravenosa, radioterapia e cirurgia. Tratamentos intraductais localizadas foram exploradas 1-3 e oferecem vantagens significativas em termos de redução de toxicidade e contra. No entanto, vários desafios permanecem antes que estes podem ser aplicados de forma mais ampla. A capacidade de executar procedimentos comparáveis ​​na glândula mamária do mouse facilita a caracterização de intervenções intraductais. Aqui apresentamos um método não-invasivo para a entrega de reagentes diretamente no duto mamário mouse. Entrega Intraductal de agentes quimioterápicos em ambos os pacientes com câncer de mama e modelos de mouse tem sido mostrado previamente para ser altamente eficaz, sem evidência detoxicidade sistêmica ou histopatológico de longo prazo muda 3-6. Além disso, a injecção de células tumorais e de distribuição de vectores lentivirais de oncogenes têm sido mostrado previamente para ser possível por meio de injecção intraductal 7-10. Em adição à entrega da droga, este processo é útil para o silenciamento de genes locais de glândulas mamárias através de injecção intraductal de siRNA. A injeção de pequenos volumes de solução de siRNA é eficaz, sem alterar a anatomia e fisiologia da glândula mamária. No nosso laboratório mostraram recentemente o silenciamento dos genes bem sucedida mediador chave em ratinhos transgénicos que desenvolvem espontaneamente tumores mamários, resultando na prevenção da progressão do tumor (Brock et al., Não publicado). Fomos capazes de repetir injecções quinzenais para os mesmos bocais até sete vezes em intervalos de 2 semanas, com nenhum dano no tecido visível ou toxicidade. Injeções intraductais pode ser cronometrada em um rato adulto para lidar com desenvolvimentoeventos opmentally específicas relevantes para o desenvolvimento da glândula mamária durante a gestação, lactação e involução ou para atingir estágios mais avançados durante o desenvolvimento e progressão tumoral. Fêmeas virgens tão jovens quanto 8 semanas de idade foram injetados não-invasiva em nosso laboratório.

<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

Aqui nós descrevemos um protocolo para entregar vários reagentes para a glândula mamária do mouse através de injeções intraductais. Entrega da droga localizada e knock-down de genes dentro do epitélio mamário tem sido difícil de alcançar devido à falta de moléculas alvo apropriadas que são independentes da fase de desenvolvimento, tais como a gravidez ea lactação. Descrevemos uma técnica para a distribuição localizada de reagentes para a glândula mamaria, em qualquer fase na idade adulta através de injecção intraductal nos bocais de ratinhos. As injecções podem ser realizadas em ratinhos vivos, sob anestesia, e permitem uma entrega não-invasivo e localizada de drogas para a glândula mamaria. Além disso, as injeções podem ser repetidas ao longo de vários meses, sem danificar o mamilo. Corantes vitais, tais como azul de Evans são muito úteis para aprender a técnica. Após a injecção intraductal do corante azul, todo o dutal torna-se visível a olho nu. Além disso, os reagentes de marcação fluorescente permitem tambémvisualização e distribuição no interior da glândula mamaria. Esta técnica é adaptável para uma variedade de compostos, incluindo o siRNA, agentes quimioterapêuticos, e pequenas moléculas.

Os mamilos podem ser facilmente localizadas através de um microscópio estereoscópico, uma vez cabelo foi removida da área ao seu redor (Figura 1A). Para dominar a técnica de injecção, recomenda-se a injectar corante azul de Evans e controlar a integridade da glândula (Figuras 1B e C). Esta abordagem também permite a determinação de volumes adequados para ser injectada em cada glândula como se pode avaliar visualmente se o corante atinge a totalidade do sistema ductal (Figuras 1C e 2A). Uma imagem da imagem azul de Evans injectada pode então ser comparada a totalidade montado na glândula mamaria, em que toda a árvore ductal é corado com corante de carmim alúmen (Figura 2B). Por favor note que a robustez do método de injecção é altamente dependente do operador. Por exemplo, a perfuração da conduta vai resultar em uma injecção mamaria almofada de gordura e injectando a solução demasiado rápida pode danificar o cel epitelial ductalls e provocar uma resposta inflamatória. Injecção com sucesso intraductal permite a entrega de droga localizada para a glândula mamaria e reduz os efeitos colaterais não específicos, muitas vezes observados em contrário. Um exemplo de imagem de uma glândula injectados com fluorescentemente marcado siRNA alvejando ciclofilina (um gene não essencial) é mostrado na Figura 3. <img alt="Figura 1" src="/files/ftp_upload/50692/50692fig1.jpg" /> Figura 1. Uma glândula mamária inguinal injetado através do mamilo com corante azul de Evans. A) Aspecto do mamilo antes da injeção e B) após a injeção de 20 mL de corante azul de Evans. Sem inchaço ou dano tecidual é observado. C) Ao abrir a pele, o corante permite a visualização de toda a árvore ductal mamária. <img alt="Figura 2" src="/files/ftp_upload/50692/50692fig2.jpg" /> Figura 2. Análise de Evans azul-in Todo-mountglândula injetada. A) Imagem representativa de uma glândula dissecada retirado do animal e espalhar sobre uma lâmina de vidro imediatamente após a injeção. B) Total coloração monte da mesma glândula com corante carmim após gordura tinha sido apagada. Comparação com A) confirma que a solução injectada enche dutal mamaria e a glândula é anatomicamente intacto. <img alt="Figura 3" src="/files/ftp_upload/50692/50692fig3.jpg" /> Figura 3. Entrega Intraductal de siRNA fluorescente. Imagem Representante da amostra de tecido fresco, retirado 48 horas após a injecção.

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

Um protocolo para a entrega intraductal não-invasivo de reagentes aquosas para a glândula mamaria do rato está descrito. O método aproveita injecção localizada nos mamilos das glândulas mamárias destinadas especificamente ductos mamários. Esta técnica é adaptável para uma variedade de compostos, incluindo o siRNA, agentes quimioterapêuticos e moléculas pequenas.

Injeção Intraductal para localizada Drug Delivery ao Rato glândula mamária

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

34.9K visualizações.  Boston Children's Hospital and Harvard Medical School.  Um protocolo para a entrega intraductal não-invasivo de reagentes aquosas para a glândula mamaria do rato está descrito. O método aproveita injecção localizada nos mamilos das glândulas mamárias destinadas especificamente ductos mamários. Esta técnica é adaptável para uma variedade de compostos, incluindo o siRNA, agentes quimioterapêuticos e moléculas pequenas. 

Vídeo: 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.

Rato células epiteliais mamárias forma Mammospheres durante a diferenciação lactogênica

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

Biologia

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.

Alterações na morfologia da glândula mamária e risco de câncer de mama em ratos

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.

Avaliação do desenvolvimento da glândula mamária e Função em modelos de ratos

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.

Fosfopéptido Análise de Rodent epidídimo Espermatozóides

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.

Imunofluorescência Indireta em cortes congelados de Rato glândula mamária

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.

Um Estudo Comparativo de drogas métodos de entrega direcionados à orelha de rato Inner: Bullostomy<em&gt; Versus</em&gt; Injeção transtimpânica

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.

Isolamento de glândulas mamárias de Mouse intacto, todo para a análise da expressão de matriz extracelular e morfologia da glândula

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 do colédoco parcial no Mouse: um modelo controlado da colestase obstrutiva 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 pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas 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 ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Entrega de gene lentiviral assistida por laser em óvulos fertilizados de camundongos

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...