저자는이 방법과 Tiago Ferreira (McGill University, Montreal, Quebec, Canada) 박사의 지속적인 도움을 받아 Dr. Michael Easterling (Social and Scientific Systems, Inc., Durham, NC)을 인정하고 싶습니다. Sholl 응용 프로그램에 대한 지원.

<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>

저자는 공개 할 것이 없습니다.

출생부터 사춘기까지 유선의 성장은 알로 메트릭입니다. 사춘기 후, 유선은 광범위한 지방 분지 및 신장을 통해 발생하며 유방 상피가 전체 지방 패드를 차지할 때까지 계속됩니다. 분지 특성은 유선 개발의 중요한 측면이며, 이러한 특성을 객관적으로 정량화하는 능력은 정상적인 유방 발달을 평가하고 유방 내 독성 물질에 일생 초기 노출 후 비정상적인 발달을 확인하는 데 매우 유용 할 수 있?...

유선 분지는 일반적으로 분비선 발달의 지표로 평가되는 특성이지만 객관적으로 정량화하기는 어렵습니다. 1953 년에 Sholl 1 은 고양이의 시각 및 운동 피질에서 신경 연결 돌기 arborization을 측정하는 방법을 설명했으며,이 기술을위한 플러그인은 Ferriera 등 2에 의해 개발되었습니다. 뉴런과 유선 모두 유사한 나무와 유사한 구조를 나타 내기 때문에,이 플러그인은 혈관 주위 쥐 유선의 2D 이미지에서 유방 상피 분지 밀도를 정량화하기 위해 사용되었습니다. 생후기가 학업 실험실 및 시험 가이드 라인 연구에서 종종 평가되는 생활 단계이기 때문에 주변기 단계는 분석을 위해 선택되었습니다. Sholl 분석은 추가 플러그인이 포함 된 오픈 소스 이미지 처리 패키지 인 ImageJ 인 FIJI와 함께 배포되는 플러그인입니다. 플러그인은 predef를 둘러싼 일련의 동심원 반지름을 만듭니다.ined center (전형적으로는 신경 세포의 soma 또는 유선의 1 차관의 기원)과 물체의 distal-most 부분 (enclosing radius)으로 확장된다. 그런 다음 각 링에서 발생하는 교차 수 (N)를 계산합니다. 플러그인은 Sholl 회귀 계수 ( k )를 반환하는데, 이는 상피 분지의 붕괴 속도를 측정합니다. ImageJ를 사용하여 유선 전체 이미지의 골격화 된 이미지가 생성되고 유방 상피 영역 (MEA)이 측정됩니다. Sholl 분석 플러그인을 사용하여 이미지를 분석하고 여기서 사용하지 않은 다른 값 중 N 및 k 값이 반환됩니다. 유방 상피 분화 밀도는 N / MEA를 계산하여 결정됩니다. 선 상피의 바깥 쪽 영역에서 분지가 계속되는 정도는 분기 복잡성이며 균일 한 원위 상피 성장의 지표입니다. k 는 epith에서 원위부 감소의 척도이므로그것은 분지 복잡성과 유방 발달의 확실한 지표의 효과적인 척도이다. 이 프로토콜은 유선 전체 마운트의 골격화 이미지를 생성하고 peripubertal 남성과 여성의 쥐의 유방 분기 특성을 정량적으로 평가하기위한 컴퓨터 보조 방법을 설명합니다. 이 방법은 상대적으로 빠르며 전문화 된 현미경 장비를 사용할 필요가 없습니다. 이 방법의 개발 및 검증은 Stanko et al. (2015) 3 . 이 보고서는 또한 쥐 유선 전체 = 마운트의 준비를 설명합니다. 유사한 유방 전체 마운트 절차가 Assis et al. (2010) 4 및 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

점점 더 많은 수의 연구가 설치류 유선을 화학 물질 노출의 발달 독성을 평가하기위한 종점으로 이용하고있다. 이러한 노출이 유선 분화에 미치는 영향은 일반적으로 기본 치수 측정을 사용하거나 형태 학적 특성을 채점하여 평가됩니다. 그러나 발달 변화를 해석하는 광범위한 방법은 실험실 간 일관성없는 번역을 초래할 수 있습니다. 연구를 통해 비교되는 데이터로부터 적절한 해석이 형성 될 수 있도록 일반적인 평가 방법이 필요합니다. 본 연구는 유선의 분지 특성을 정량화하기위한 Sholl 분석 방법의 적용을 기술한다. Sholl 방법은 원래 신경 돌기 패턴을 정량화하는데 사용하기 위해 개발되었습니다. 오픈 소스 이미지 분석 소프트웨어 패키지 인 ImageJ와이 분석을 위해 개발 된 플러그인을 사용하여 유선 분지 밀도와 am혈관 주위 암컷 쥐의 유선이 결정되었다. 여기에 설명 된 방법은 유선 개발의 중요한 특성을 정량화하기위한 효과적인 도구로 Sholl 분석의 사용을 가능하게합니다.

이 프로토콜에서 분석 된 유선에 대해 측정 된 밀폐 반경, MEA, N, k 및 계산 된 분기 밀도 값을 표 1 에보고합니다. Sholl 분석은 각각의 반경 (도 9)의 교점의 수의 선형 및 세미 - 로그 플롯을 생성하고, 선택된 경우, 교차점의 히트 맵 (도 10). 덜 발달 된 땀샘은 동일한 MEA 내에서 더 적은 교차점을 나타내므로 더 ...

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

설치류의 유선 선생 발달은 일반적으로 기술적 평가를 사용하거나 기본적인 신체적 속성을 측정하여 평가되었습니다. 분지 밀도는 객관적으로 정량화하기 어려운 유방 발달의 지표입니다. 이 프로토콜은 유선의 분기 특성의 정량 평가를위한 신뢰할 수있는 방법을 설명합니다.

Sholl 분석법을 이용한 유방 내 유선 마운트의 분지 밀도 정량화

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 조회수.  National Institute of Environmental Health Sciences.  설치류의 유선 선생 발달은 일반적으로 기술적 평가를 사용하거나 기본적인 신체적 속성을 측정하여 평가되었습니다. 분지 밀도는 객관적으로 정량화하기 어려운 유방 발달의 지표입니다. 이 프로토콜은 유선의 분기 특성의 정량 평가를위한 신뢰할 수있는 방법을 설명합니다. 

비디오: 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.

병변 식별을위한 유선 절개 전체 장착

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

연구

암 연구학

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.

쥐 유방 상피 세포 이식에 갑피 흰 지방 패드

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.

Murine Mammary Gland의 갭, 타이트 및 Adherens 접합부의 구성 요소 사이의 단백질 - 단백질 상호 작용 및 공동 위치 분석

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

발생학

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.

마우스 모델에서 유방암의 예방 및 지역 치료를 위한 에탄올 기반 담약 솔루션의 내배송 및 X선 시각화

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.

쥐 모델에서 유방암 예방을 위한 관내 에탄올 기반 절제 치료의 X선 시각화

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.

쥐에서 유선의 형태론과 유방암 위험의 변화

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 ...

생물학

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

골지 - 콕스 방법을 이용한 고령의 해마 상피 세포의 수지상 돌기 복잡성 평가

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

신경과학

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.

3 차원 조직학 볼륨 및 마우스 유방 땀샘을 연구에의 응용의 재구성

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.

세포 외 기질 식 및 선 형태 분석을 그대로, 전체 마우스 유 방 땀 샘의 절연

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.

교체 가능한 뚜껑이있는 유방 이미징 창을 사용하는 종방향 생체 내 현미경 검사

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