Gli autori desiderano ringraziare Michael Goldberg per la preparazione della soluzione siRNA stabilizzato e Heather Tobin per la sua assistenza con gli animali. Questo studio è stato finanziato dal Dipartimento della Difesa Innovator Award W81XWH-08-1-0659 al DEI. La formazione post-dottorato di SK è stato sostenuto dalla Fondazione Susan G Komen (KG101329). Gli autori desiderano ringraziare Kristin Johnson per la preparazione dello schema.

1. Preparazione della soluzione per l&#39;iniezione <ol><li> Preparare 0,2% Evans blu colorante in tampone fosfato salino (PBS) o altra soluzione acquosa di interesse. Questo colorante vitale è utile per valutare il successo delle iniezioni intraduttali ed è consigliato come un aiuto di visualizzazione durante lo sviluppo di competenze nella tecnica. </li></ol> Il volume richiesto dipenderà dal numero e la posizione delle ghiandole da iniettare. Tutti 10 ghiandole mammarie di topi femmina possono essere iniettati ma a causa delle dimensioni della ghiandola minore, coppie ghiandola 1 e 5 sono tipicamente iniettati con 10 ml di soluzione. Tutte le altre coppie mammarie sono iniettati con 20 microlitri. Questi volumi sono sufficienti a riempire l&#39;intero albero duttale. 2. Preparazione preoperatoria <ol><li> Registrare il peso corporeo di ciascun topo. Come per tutti gli studi preclinici, i pesi degli animali devono essere controllati regolarmente (due volte alla settimana o più) per valutare la potenziale tossicità. </li><li> Anestetizzare il mOuse utilizzando una camera di isoflurano e applicare lubrificante occhio. Durante la procedura di topi continueranno ad essere anestetizzato con inalatoria concentrazione 2-4% di isoflurano in ossigeno attraverso un cono di naso. Monitorare attentamente il mouse per i cambiamenti nella frequenza respiratoria, regolando il livello di isoflurano conseguenza. </li><li> Iniettare meloxicam (5-10 mg / kg) per via sottocutanea prima della procedura come analgesia. </li><li> Applicare una crema di rimozione over-the-counter capelli per la zona del capezzolo. Aspettare 5 minuti e rimuovere delicatamente i capelli sciolti con un applicatore di cotone punta con un movimento circolare. Togliere la crema usando tovaglioli di carta umidi bagnate con acqua tiepida. Rasatura non è raccomandato a causa del rischio di danneggiare i capezzoli. </li><li> Fissare il mouse sotto lo stereoscopio con nastro adesivo dolcemente verso le estremità. </li><li> Siti di iniezione puliti con tamponi imbevuti di alcool. </li></ol> 3. Iniezione intraduttale <ol><li> Individuare capezzoli adeguate per essere iniettato sotto lo stereoscopio. Utilizzare multa micro-dissezione tweeZers per rimuovere tutta la pelle morta che copre l&#39;apertura capezzolo. </li><li> Carico 10-20 ml di soluzione iniettabile in una siringa da 50 ml con un 33 G hub metallo ago apposto. A volte, dopo l&#39;iniezione, piccole quantità (1 ml o meno) possono fuoriuscire dal capezzolo su estraendo l&#39;ago. Pertanto, si raccomanda di iniettare 11 o 21 ml rispettivamente per tenere conto della perdita potenziale. </li><li> Tenere il capezzolo delicatamente con le belle pinzette e sollevarlo leggermente per posizionarla per l&#39;iniezione. Non è necessario tagliare il capezzolo. </li><li> Iniettare la soluzione lentamente per ridurre al minimo i danni potenziali causati da rapido movimento fluido all&#39;interno dei lumi duttali. La velocità di iniezione deve essere mantenuta a circa 40 microlitri / min. </li></ol> 4. Cura postoperatoria <ol><li> Osservare il sito di iniezione. Non ci dovrebbero essere segni di trauma alla regione capezzolo o tessuto circostante. Gonfiore nella zona che circonda il capezzolo probabilmente indica un mammaria cuscinetto adiposo piuttosto c iniezioner che un&#39;iniezione intraduttale successo. </li><li> Rimuovere l&#39;animale dal cono e trasferirsi in una gabbia separata per il recupero. Posizionare la gabbia sotto una lampada di calore per prevenire l&#39;ipotermia e assistere nel recupero. I topi sono alloggiati singolarmente e saranno monitorati attentamente fino a che riprendano conoscenza e la mobilità. </li></ol> 5. Analisi della ghiandola mammaria Tissue <ol><li> Al termine dello studio, i topi sono sacrificati mediante dislocazione cervicale seguente CO 2 gas compresso in una camera di isolamento. Ghiandole mammarie vengono asportati e possono essere utilizzati per tutta la preparazione mount 11, istologia, o RNA e isolamento delle proteine. </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>

Gli autori non hanno informazioni finanziarie.

Transgenici e topi knockout sono strumenti preziosi per studiare il ruolo in vivo dei singoli geni nel cancro al seno. Tuttavia, è costoso e di generare questi animali richiede tempo e knockdown gene è solitamente onnipresente che a volte ci impedisce di riconoscere gli effetti specifici di tale gene sulla ghiandola mammaria. Pertanto, un atterramento mirata allevierebbe questioni di non-specifici effetti collaterali e tossicità in altri organi. L&#39;iniezione intraduttale di siRNA qui presentata consente knockdown gene localizzato nella ghiandola mammaria mouse. Si tratta di un metodo rapido per una somministrazione di farmaci mirati e localizzato alla ghiandola mammaria non limitato alla consegna siRNA. Tale somministrazione di farmaci specifici ghiandola mammaria evita aspecifiche effetti collaterali e la tossicità verso altri organi e consente lo studio di alterazioni geniche in punti temporali specifici durante lo sviluppo della ghiandola mammaria e formazione del tumore al seno. Non c&#39;è evidenza di SiParticelle di RNA in ghiandole non iniettati, fegato o di altri organi che forniscono ulteriore prova che questo metodo presenta un modo preferito per knockdown gene localizzato nella ghiandola mammaria. Inoltre, le iniezioni possono essere ripetute intraduttale settimanale o bisettimanale diversi mesi per permettere il monitoraggio a lungo termine degli agenti terapeutici. Questa tecnica apre la possibilità per saggiare la consegna intraduttale di vari reagenti ai dotti mammari del mouse. Advances in erogazione localizzata in modelli murini ben caratterizzati dovrebbero accelerare l&#39;applicazione di non invasiva, mirate strategie terapeutiche nell&#39;uomo.

La ghiandola mammaria mouse è composto da un albero duttale-alveolare complesso che converge in un condotto centrale al capezzolo. Nei topi che nell&#39;uomo, la maggior parte dei tumori al seno hanno origine nelle cellule epiteliali che rivestono i dotti galattofori. Trattamenti attualmente disponibili comprendono la chemioterapia endovenosa, radioterapia e chirurgia. Intraduttali trattamenti localizzati sono state esplorate 1-3 e offrono vantaggi significativi in termini di tossicità ed effetti collaterali ridotti. Tuttavia, molte sfide restano prima che queste possano essere applicate più ampiamente. La possibilità di eseguire procedure comparabili nella ghiandola mammaria topo facilita la caratterizzazione degli interventi intraduttale. Presentiamo qui un metodo non invasivo per la fornitura di reagenti direttamente nel condotto mammaria mouse. Consegna intraduttale di agenti chemioterapici in entrambe le pazienti con carcinoma mammario e modelli di topo è stato precedentemente dimostrato di essere altamente efficace senza evidenza ditossicità sistemica o istopatologico cambiamenti a lungo termine 3-6. Inoltre, l&#39;iniezione di cellule tumorali e consegna vettore lentivirale di oncogeni hanno dimostrato in precedenza di essere realizzabile tramite iniezione intraduttale 7-10. Oltre alla somministrazione di farmaci, questa procedura è utile per silenziamento locale di geni della ghiandola mammaria mediante iniezione intraduttale di siRNA. L&#39;iniezione di piccoli volumi di soluzione siRNA è efficace senza alterare l&#39;anatomia e la fisiologia della ghiandola mammaria. Nel nostro laboratorio abbiamo recentemente dimostrato con successo il silenziamento di geni mediatore chiave in topi transgenici che sviluppano spontaneamente tumori mammari con conseguente prevenzione della progressione tumorale (Brock et al., Inedito). Siamo stati in grado di ripetere le iniezioni bisettimanali negli stessi capezzoli fino a 7 volte a intervalli di 2 settimane, senza danni ai tessuti visibili o tossicità. Iniezioni intraduttali possono essere a tempo in un topo adulto per affrontare develeventi opmentally specifici rilevanti per lo sviluppo della ghiandola mammaria durante la gravidanza, l&#39;allattamento, e involuzione o di indirizzare fasi successive durante lo sviluppo e progressione tumorale. Femmine vergini giovani come 8 settimane di età sono state iniettate in modo non invasivo nel nostro 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

Qui descriviamo un protocollo per consegnare vari reagenti al mouse ghiandola mammaria tramite iniezioni intraduttali. Consegna della droga localizzata e knock-down di geni all&#39;interno dell&#39;epitelio mammario è stato difficile da raggiungere a causa della mancanza di adeguati molecole di targeting che sono indipendenti di stadi di sviluppo come la gravidanza e l&#39;allattamento. Qui, descriviamo una tecnica per la consegna localizzata dei reagenti alla ghiandola mammaria in qualsiasi fase adulta mediante iniezione intraduttale nei capezzoli di topi. Le iniezioni possono essere eseguite su topi vivi, sotto anestesia, e consentono una consegna non invasiva e localizzata farmaco alla ghiandola mammaria. Inoltre, le iniezioni possono essere ripetute per diversi mesi senza danneggiare il capezzolo. Coloranti vitali come il Blu di Evans sono molto utili per imparare la tecnica. Una volta iniettato intraduttale del colorante blu, l&#39;intero albero duttale diventa visibile all&#39;occhio. Inoltre, reagenti fluorescente consentono anchevisualizzazione e distribuzione all&#39;interno della ghiandola mammaria. Questa tecnica è adattabile per una varietà di composti compresi siRNA, agenti chemioterapici, e piccole molecole.

I capezzoli possono essere facilmente localizzati utilizzando uno stereomicroscopio, una volta di capelli è stato rimosso nella zona circostante (Figura 1A). Per padroneggiare la tecnica di iniezione, si raccomanda di iniettare Evans colorante blu e monitorare l&#39;integrità della ghiandola (Figure 1B e C). Questo approccio permette anche per la determinazione dei volumi appropriati da iniettare in ogni ghiandola come si può valutare visivamente se il colorante raggiunge l&#39;intero sistema duttale (Figure 1C e 2A). Un&#39;immagine del Evans blu immagine iniettato può quindi essere confrontato a tutto montato ghiandola mammaria, in cui l&#39;intero albero duttale è macchiato con Carmine allume colorante (Figura 2B). Si noti che la robustezza di questo metodo di iniezione è altamente dipendente dall&#39;operatore. Ad esempio, perforando il condotto si tradurrà in una iniezione mammaria cuscinetto adiposo ed iniettare la soluzione troppo veloce potrebbe danneggiare il cel epiteliale duttaleLS e provocare una risposta infiammatoria. Iniezione intraduttale successo consente la somministrazione di farmaci localizzato alla ghiandola mammaria e riduce non specifici effetti collaterali spesso osservati diversamente. Un&#39;immagine esempio di una ghiandola iniettato con fluorescente etichettato siRNA rivolti cyclophilin (un gene non essenziale) è mostrato in Figura 3. <img alt="Figura 1" src="/files/ftp_upload/50692/50692fig1.jpg" /> Figura 1. Una ghiandola mammaria inguinale iniettato attraverso il capezzolo con Evans colorante blu. A) Aspetto del capezzolo prima dell&#39;iniezione e B) dopo l&#39;iniezione con 20 microlitri Evans colorante blu. Nessun gonfiore o danni ai tessuti si osserva. C) All&#39;apertura della pelle, il colorante permette la visualizzazione dell&#39;intero albero duttale mammario. <img alt="Figura 2" src="/files/ftp_upload/50692/50692fig2.jpg" /> Figura 2. Whole-mount analisi di Evans blue-inghiandola proiettata. A) l&#39;immagine rappresentativa di una ghiandola sezionato asportato dall&#39;animale e si sviluppa su un vetrino immediatamente dopo l&#39;iniezione. B) Tutta la montatura colorazione della stessa ghiandola con il carminio colorante dopo che il grasso era stato cancellato. Confronto con A) conferma che la soluzione iniettata riempie l&#39;albero duttale e la ghiandola mammaria è anatomicamente intatto. <img alt="Figura 3" src="/files/ftp_upload/50692/50692fig3.jpg" /> Figura 3. Consegna intraduttale di siRNA fluorescenti. Immagine rappresentativa del campione di tessuto fresco, asportato 48 ore dopo l&#39;iniezione.

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 protocollo per la consegna intraduttale non invasiva di reagenti acquosi alla ghiandola mammaria topo è descritto. Il metodo si avvale di iniezione localizzato nei capezzoli delle ghiandole mammarie destinati dotti mammari in particolare. Questa tecnica è adattabile per una varietà di composti compresi siRNA, agenti chemioterapici e piccole molecole.

Iniezione intraduttale per Localizzato Drug Delivery per il mouse ghiandola mammaria

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 Views.  Boston Children's Hospital and Harvard Medical School.  Un protocollo per la consegna intraduttale non invasiva di reagenti acquosi alla ghiandola mammaria topo è descritto. Il metodo si avvale di iniezione localizzato nei capezzoli delle ghiandole mammarie destinati dotti mammari in particolare. Questa tecnica è adattabile per una varietà di composti compresi siRNA, agenti chemioterapici e piccole molecole. 

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.

Topo cellule epiteliali mammarie forma Mammospheres durante la differenziazione lattogenica

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

Ricerca

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.

Cambiamenti nella morfologia della ghiandola mammaria e rischio di cancro al seno in topi

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.

Valutazione dello Sviluppo ghiandola mammaria e funzione in modelli murini

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.

Analisi fosfopeptide di roditori Epididymal spermatozoi

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.

Immunofluorescenza indiretta su sezioni congelate di mouse ghiandola mammaria

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.

Uno studio comparato Drug metodi di consegna mirate per l&#39;orecchio interno mouse: Bullostomy<em&gt; Versus</em&gt; Iniezione transtimpanica

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 delle ghiandole mammarie Mouse intatto, intero per l'analisi di espressione di matrice extracellulare e la morfologia della ghiandola

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.

Legatura del dotto biliare parziale nel topo: un modello radiocomandato di colestasi ostruttiva localizzata

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-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

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.