Die Autoren möchten Dr. Michael Easterling (Social and Scientific Systems, Inc., Durham, NC) für seine Unterstützung bei der Validierung dieser Methode und Dr. Tiago Ferreira (McGill University, Montreal, Quebec, Kanada) für seine kontinuierliche ansprechen Unterstützung bei der Sholl-Anmeldung.

<ol>
	<li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+of+the+neurons+in+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization of the neurons in the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Ferreira, T. A., Iacono, L. L., Gross, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+receptor+1A+modulates+actin+dynamics+and+restricts+dendritic+growth+in+hippocampal+neurons.">Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons.</a> Eur J Neurosci. 32 (1), 18-26 (2010).</li><li>Stanko, J. P., Easterling, M. R., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Sholl+analysis+to+quantify+changes+in+growth+and+development+in+rat+mammary+gland+whole+mounts.">Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts.</a> Reprod Toxicol. 54, 129-135 (2015).</li><li>Assis, S., Warri, A., Cruz, M. I., Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+Mammary+Gland+Morphology+and+Breast+Cancer+Risk+in+Rats.">Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats.</a> J. Vis. Exp. (44), e2260 (2010).</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. (53), e2828 (2011).</li><li>ImageJ User Guide. IJ 1.46r. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/docs/guide/146.html">https://imagej.nih.gov/ij/docs/guide/146.html</a> (2012)</li><li>Tucker, D. K., Foley, J. F., Hayes-Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+High-quality+Hematoxylin+and+Eosin-stained+Sections+from+Rodent+Mammary+Gland+Whole+Mounts+for+Histopathologic+Review.">Preparation of High-quality Hematoxylin and Eosin-stained Sections from Rodent Mammary Gland Whole Mounts for Histopathologic Review.</a> Toxicol Pathol. 44 (7), 1059-1064 (2016).</li><li>Fenton, S. E., Hamm, J. T., Birnbaum, L. S., Youngblood, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+abnormalities+in+the+rat+mammary+gland+following+gestational+and+lactational+exposure+to+2,3,7,8-tetrachlorodibenzo-p-dioxin+(TCDD).">Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).</a> Toxicol Sci. 67 (1), 63-74 (2002).</li><li>Murray, T. J., Maffini, M. V., Ucci, A. A., Sonnenschein, C., Soto, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+gland+ductal+hyperplasias+and+carcinoma+in+situ+following+fetal+bisphenol+A+exposure.">Induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol A exposure.</a> Reprod Toxicol. 23 (3), 383-390 (2007).</li><li>Moral, R., Santucci-Pereira, J., Wang, R., Russo, I. H., Lamartiniere, C. A., Russo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+exposure+to+butyl+benzyl+phthalate+induces+modifications+in+the+morphology+and+the+gene+expression+profile+of+the+mammary+gland:+an+experimental+study+in+rats.">In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats.</a> Environ Health. 10 (1), 5 (2011).</li><li>Johnson, M. D., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dimensional+multiphoton+imaging+of+fresh+and+whole+mount+developing+mouse+mammary+glands.">Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.</a> BMC Cancer. 13, 373 (2013).</li><li>Enoch, R. R., Stanko, J. P., Greiner, S. N., Youngblood, G. L., Rayner, J. L., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development+as+a+sensitive+end+point+after+acute+prenatal+exposure+to+an+atrazine+metabolite+mixture+in+female+Long-Evans+rats.">Mammary gland development as a sensitive end point after acute prenatal exposure to an atrazine metabolite mixture in female Long-Evans rats.</a> Environ Health Persp. 115 (4), 541-547 (2007).</li><li>Hovey, R. C., Coder, P. S., Wolf, J. C., Sielken, R. L., Tisdel, M. O., Breckenridge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+mammary+gland+development+in+female+Long+Evans+rats+following+in+utero+exposure+to+atrazine.">Quantitative assessment of mammary gland development in female Long Evans rats following in utero exposure to atrazine.</a> Toxicol Sci. 119 (2), 380-390 (2011).</li><li>Mandrup, K. R., Hass, U., Christiansen, S., Boberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+ethinyl+oestradiol+alters+mammary+gland+development+in+male+and+female+Wistar+rats.">Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats.</a> Inter J of Androl. 35 (3), 385-396 (2012).</li></ol>

Die Autoren haben nichts zu offenbaren.

Von der Geburt bis zur Pubertät ist das Brustdrüsenwachstum allometrisch. Nach der Pubertät entwickelt sich die Brustdrüse durch umfangreiche duktale Verzweigung und Dehnung, die sich fortsetzen, bis das Mammaepithel das gesamte Fettkissen einnimmt. Verzweigungscharakteristiken sind ein wichtiger Aspekt der Entwicklung von Brustdrüsen, und die Fähigkeit, diese Eigenschaften objektiv zu quantifizieren, kann sehr nützlich sein, um die normale Entwicklung der Mamma zu beurteilen und eine abnormale Entwicklung nach f...

Die Brustdrüsenverzweigung ist ein charakteristisches Merkmal, das allgemein als Indikator der Drüsenentwicklung beurteilt wird, aber es ist schwer objektiv zu quantifizieren. Im Jahr 1953 beschrieb Sholl 1 eine Methode zur Messung der neuronalen dendritischen Arborisierung in den visuellen und motorischen Kortizes der Katze, und ein Plugin für diese Technik wurde von Ferriera et al 2 entwickelt . Da sowohl Neuronen als auch Brustdrüsen eine ähnliche baumartige Struktur aufweisen, wurde das Plugin zur Quantifizierung von Mamma-Epithel-Verzweigungsdichten in 2D-Bildern der peripubertären Ratten-Brustdrüse verwendet. Die peripubertäre Bühne wurde für die Analyse ausgewählt, weil die Entwöhnung eine Lebensphase ist, die oft in akademischen Laboratorien und Testrichtlinien untersucht wird. Die Sholl-Analyse ist ein Plugin, das mit FIJI verteilt wird, welches das Open-Source-Bildverarbeitungspaket ImageJ ist, mit zusätzlichen Plugins. Das Plugin schafft eine Reihe von konzentrischen Ringen, die ein Predef umkreisen(Typischerweise das Soma eines Neurons oder der Ursprung des Primärkanals einer Milchdrüse) und erstreckt sich bis zum distalsten Teil des Objekts (der umschließende Radius). Es zählt dann die Anzahl der Kreuzungen (N), die auf jedem der Ringe auftreten. Das Plugin gibt auch einen Sholl-Regressionskoeffizienten ( k ) zurück, der eine Messung der Rate des Zerfalls der Epithelverzweigung ist. Mit ImageJ wird ein skelettiertes Bild einer Brustdrüsen-Ganzmasse erstellt und der Mamma-Epithelbereich (MEA) gemessen. Das Bild wird mit dem Sholl-Analyse-Plugin analysiert und Werte für N und k , unter anderen hier nicht genutzten Werten, werden zurückgegeben. Die Mamma-Epithel-Verzweigungsdichte wird durch Berechnung von N / MEA bestimmt. Das Ausmaß, in dem die Verzweigung in den äußeren Regionen des Drüsenepithels fortdauert, ist die Verzweigungskomplexität und ist ein Indikator für ein gleichmäßiges distales Epithelwachstum. Da k ein Maß für die distale Abnahme des Epiths istEliale Verzweigung, ist es ein wirksames Maß für die Verzweigungskomplexität und ein zuverlässiger Indikator für die Entwicklung von Mamma. Dieses Protokoll beschreibt eine computergestützte Methode zur Erzeugung von skelettierten Bildern von Brustdrüsen-Vollkästen und quantitativen Auswerten von Mamma-Verzweigungseigenschaften bei peripubertalen männlichen und weiblichen Ratten. Diese Methode ist relativ schnell und erfordert nicht die Verwendung von spezialisierten Mikroskopie Ausrüstung. Entwicklung und Validierung dieser Methode sind in Stanko et al. (2015) 3 Dieser Bericht beschreibt auch die Vorbereitung der Ratten-Brustdrüsen-Ganzheit. Ähnliche Mamma-Vollmontageverfahren wurden in de Assis et al. Beschrieben . (2010) 4 und Plante et al. (2011) 5 .

<table><tbody><tr><td>Dissecting board</td><td>NA</td><td>NA</td><td>A piece of styrofoam roughly 10&quot;x12&quot; is suitable.</td></tr><tr><td>Dissecting T-Pins</td><td>Daigger</td><td>EF7419A</td><td></td></tr><tr><td>Spray bottle with ethanol</td><td>NA</td><td>NA</td><td>70% ethanol is suitable.</td></tr><tr><td>Curved dissecting scissors</td><td>Fine Science Tools</td><td>14569-09</td><td></td></tr><tr><td>Straight dissecing scissors</td><td>Fine Science Tools</td><td>14568-09</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Science Tools</td><td>11003-12</td><td></td></tr><tr><td>Superfrost Plus 24 x 75 x 1 mm microscope slides</td><td>ThermoFisher Scientific</td><td>4951PLUS-001</td><td>Thermo Scientific Superfrost Plus &amp;amp; Colorfrost Plus slides hold tissue sections on permanently without the need for expensive coatings in IHC and Anatomical Pathology applications. This treatment reduces tissue loss during staining as well as hours of slide preparation. Slides electro-statically attract frozen tissue sections and cytology preparations and feature a chemistry similar to silane, although optimized to improve application performance.&lt;br/&gt; https://www.thermofisher.com/order/catalog/product/4951PLUS4.&amp;nbsp;</td></tr><tr><td>Fisherfinest Premium Cover Glass 24 x 60 x 1 mm</td><td>Fisher scientific</td><td>12-548-5P</td><td></td></tr><tr><td>Bemis Parafilm M Laboratory Wrapping Film</td><td>Fisher scientific</td><td>13-374-12</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>A9967</td><td></td></tr><tr><td>Ethanol absolute, &amp;ge;99.8% (GC)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>24102</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>214736</td><td></td></tr><tr><td>Carmine alum&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C1022</td><td></td></tr><tr><td>Aluminum potassium sulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A6435</td><td></td></tr><tr><td>Permount mounting media</td><td>Fisher Scientific</td><td>SP15</td><td></td></tr><tr><td>Macroscope</td><td>Leica</td><td>Z16 APO&amp;nbsp;</td><td>This is the image capturing hardware and software used in this laboratory.&amp;nbsp; As there are many different options, the methods and applications may vary between laboratories.</td></tr><tr><td>Digital camera</td><td>Leica</td><td>DFC295</td><td></td></tr><tr><td>Camera software</td><td>Leica</td><td>Leica Application Suite v3.1&amp;nbsp;</td><td></td></tr><tr><td>ImageJ software</td><td>Open source</td><td>http://imagej.net/Welcome</td><td></td></tr><tr><td>Sholl analysis&amp;nbsp;</td><td>Open source</td><td>http://imagej.net/Sholl_Analysis</td><td></td></tr></tbody></table>

quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method

Eine zunehmende Anzahl von Studien nutzt die Nagetier-Brustdrüse als Endpunkt für die Beurteilung der Entwicklungstoxizität einer chemischen Exposition. Die Effekte, die diese Expositionen auf die Entwicklung von Brustdrüsen haben, werden typischerweise unter Verwendung von entweder grundlegenden Dimensionsmessungen oder durch Auswertung von morphologischen Merkmalen ausgewertet. Die breite Palette von Methoden zur Interpretation von Entwicklungsänderungen könnte jedoch zu inkonsistenten Übersetzungen in Laboratorien führen. Eine gängige Bewertungsmethode ist erforderlich, damit aus den Daten, die über Studien hinweg verglichen werden, richtige Interpretationen entstehen können. Die vorliegende Studie beschreibt die Anwendung der Sholl-Analyse-Methode zur Quantifizierung der Mamma-Drüsen-Verzweigungseigenschaften. Die Sholl-Methode wurde ursprünglich für die Verwendung bei der Quantifizierung von neuronalen dendritischen Mustern entwickelt. Durch die Verwendung von ImageJ, einem Open-Source-Bildanalyse-Softwarepaket und einem für diese Analyse entwickelten Plugin, der Verzweigungsdichte der Brustdrüse und der Komplexität von amAmmary Drüse aus einer peripubertären weiblichen Ratte wurden bestimmt. Die hier beschriebenen Methoden ermöglichen die Verwendung der Sholl-Analyse als wirksames Instrument zur Quantifizierung eines wichtigen Merkmals der Brustdrüsenentwicklung.

Die in diesem Protokoll analysierten Werte für den gemessenen eingeschlossenen Radius, MEA, N, k und die berechnete Verzweigungsdichte für die Brustdrüse sind in Tabelle 1 angegeben . Die Sholl-Analyse erzeugt lineare und halb-log-Plots der Anzahl der Kreuzungen bei jedem Radius ( Abbildung 9 ) und, falls ausgewählt, eine Wärmekarte der Kreuzungen ( Abbildung 10 ). Weniger entwickelte Drüsen weise...

Watch this Scientific Journal Video about Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method at JoVE.com

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

Die Brustdrüsenentwicklung im Nagetier wurde typischerweise mit deskriptiven Beurteilungen oder durch Messung von physikalischen Grundattributen ausgewertet. Die Verzweigungsdichte ist ein Indikator für die Entwicklung von Mamma, der objektiv schwer zu quantifizieren ist. Dieses Protokoll beschreibt eine zuverlässige Methode für die quantitative Beurteilung der Mamma-Drüsen-Verzweigungseigenschaften.

Quantifizierung der Verzweigungsdichte in Ratten-Mamma-Gland-Vollkonserven unter Verwendung der Sholl-Analysemethode

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

quantifying-branching-density-rat-mammary-gland-whole-mounts-using

Research

JoVE Journal

Developmental Biology

14.2K Aufrufe.  National Institute of Environmental Health Sciences.  Die Brustdrüsenentwicklung im Nagetier wurde typischerweise mit deskriptiven Beurteilungen oder durch Messung von physikalischen Grundattributen ausgewertet. Die Verzweigungsdichte ist ein Indikator für die Entwicklung von Mamma, der objektiv schwer zu quantifizieren ist. Dieses Protokoll beschreibt eine zuverlässige Methode für die quantitative Beurteilung der Mamma-Drüsen-Verzweigungseigenschaften. 

Serie: Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Sektion Mammary Gland Ganze Mounts für Läsion Identifizierung

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Forschung

Krebsforschung

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

Ratte Mammary Epithel Cell Transplantation in das interskapulierische weiße Fettpad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Analyse von Protein-Protein-Wechselwirkungen und Co-Lokalisierung zwischen Komponenten von Gap, Tight und Adherens Junctions in Murine Mamma Drüsen

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Entwicklungsbiologie

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

A method of intraductal injection of reagents for an ethanol-based ablative solution to the mouse mammary ductal tree for in vivo imaging and breast cancer prevention is described. Injection directly into the nipple opening allows for targeting mammary epithelial cells with minimal collateral tissue damage.

Intraduktale Verabreichung und Röntgenvisualisierung von Ethanol-basierter ablativer Lösung zur Prävention und lokalen Behandlung von Brustkrebs in Mausmodellen

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic ...

X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most effective intervention is prophylactic mastectomy. This is a drastic surgical procedure in which the mammary epithelial cells that can give rise to breast cancer are completely removed along with the surrounding tissue. The goal of this protocol is to demonstrate the feasibility of a minimally invasive intraductal procedure that could become a new primary intervention for breast cancer prevention. This local procedure would preferentially ablate mammary epithelial cells before they can become malignant. Intraductal methods to deliver solutions directly to these epithelial cells in rodent models of breast cancer have been developed at Michigan State University and elsewhere. The rat mammary gland consists of a single ductal tree that has a simpler and more linear architecture compared to the human breast. However, chemically induced rat models of breast cancer offer valuable tools for proof-of-concept studies of new preventive interventions and scalability from mouse models to humans. Here, a procedure for intraductal delivery of an ethanol-based ablative solution containing tantalum oxide nanoparticles as X-ray contrast agent and ethyl cellulose as gelling agent into the rat mammary ductal tree is described. Delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) by intraductal injection has been described previously in mouse and rat models. This protocol description emphasizes methodological changes and steps that pertain uniquely to delivering an ablative solution, formulation consideration to minimize local and systemic side effects of the ablative solution, and X-ray imaging for in vivo assessment of ductal tree filling. Fluoroscopy and micro-CT techniques enable to determine the success of ablative solution delivery and the extent of ductal tree filling thanks to compatibility with the tantalum-containing contrast agent.

A procedure for the delivery of a chemical ablative solution to the rat mammary ductal tree for image-guided preventive treatment of breast cancer is described. Mammary epithelial cells can be targeted with minimal collateral tissue damage through cannulation directly into the nipple opening and intraductal infusion of a 70% ethanol-based ablative solution.

Röntgenvisualisierung einer intraduktalen ethanolbasierten ablativen Behandlung zur Prävention von Brustkrebs in Rattenmodellen

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most ...

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.

Änderungen der Brustdrüsen Morphologie und Breast Cancer Risk in Ratten

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 ...

Biologie

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Die Rekonstruktion der 3-Dimensional Histologie Volume und seine Anwendung auf Maus Milchdrüsen Studieren

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

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.

Isolierung von intakt, ganze Maus Milchdrüsen zur Analyse der extrazellulären Matrix Ausdruck und Drüse Morphologie

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 ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

Längsschnittliche intravitale Mikroskopie unter Verwendung eines Brustbildgebungsfensters mit austauschbarem Deckel

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

<meta charset="utf8"></head><body>Intravitale subzelluläre Mikroskopie der Brustdrüse

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Intravitale subzelluläre Mikroskopie der Brustdrüse

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...