Name: quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method
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Description: Watch this Scientific Journal Video about Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method at JoVE.com

作者要感谢Michael Easterling博士（社会和科学系统公司，达勒姆，NC），他对这种方法的验证和Tiago Ferreira博士（加拿大魁北克蒙特利尔的麦吉尔大学）持续不断的协助协助Sholl申请。

本研究的所有动物使用和手术均由NIEHS实验室动物护理和使用委员会批准，并在实验动物护理认证机构评估和认证协会进行。 消费乳腺<ol><li>使用二甲苯防止方法（铅笔效果最佳）预先标记所有的幻灯片。在末端覆盖安装解决方案以保护标签。 </li><li>通过机构动物保护和使用委员会批准的方法对动物进行安乐死。 </li><li>安乐死后，将动物放在解剖板上（ 图1 ）。拉伸并扣住所有四肢，后肢形成倒V形（保持针或小规格针工作良好）。 </li><li>用70％乙醇充分喷雾腹部，以保持头发脱离乳房样品。 </li><li> 使用镊子，拉起腹部皮肤在中线，并做一个小切口解剖剪刀，小心不要穿刺腹膜。 <ol><li>从切口开始，将皮肤切割至颈部区域，然后远离前肢。切割到腹股沟区域，然后向远侧方向延伸到同一侧后肢的第一关节;该切口通常分离乳腺5和6.​​避免切割腹膜。 </li><li>使用镊子将附着的乳房脂肪垫拉回皮肤，使用解剖剪刀的平头端将其从腹膜轻轻分离。单独作为远背侧尽可能以完全暴露4和第 5腹股沟乳腺在皮肤上的下侧;将皮肤固定在解剖板上（ 图2 ）。 </li><li>轻轻抓住第 5腺体的乳腺组织用弯钳的圆形侧慢慢地修剪腺远离皮肤，小心不要划伤腺或皮肤。 </li><li>继续修剪，直到第4和第 5 个腺体完全从皮肤上解离出来。确保乳头区域与腺体的其余部分一起被去除，因为它作为Sholl分析的起点。切割乳腺组织远离其附着于腺体的身体4。 </li></ol></li><li>一旦将腺体从动物中取出，将其均匀分布在带静电的25 mm x 75 mm x 1 mm显微镜载玻片上，边缘与皮肤相邻。 注意:对于老年或哺乳动物的腺体，可能需要51 x 75 x 1毫米的幻灯片。 </li><li>戴手套时，请轻轻挤出任何气泡。用塑料石蜡膜和另一个显微镜载玻片覆盖腺体，并压缩腺体以将其粘附到载玻片上。 注意:充满水的50mL锥形管适合用作重量。粘附到载玻片上所需的时间取决于腺体的厚度。薄，postna天天（PND）4腺应压缩至少30分钟，而较厚的成体腺体可能需要长达2-5小时。 </li></ol> 2.准备乳房整体<ol><li> 至少提前24小时准备胭脂红矾溶液，因为它需要煮沸和制冷。 <ol><li>将1克胭脂红明矾和2.5克硫酸铝钾（AlK（SO 4 ） 2 ·12H 2 O）溶解在500毫升蒸馏水中，并在1升烧瓶中煮沸20分钟。使用蒸馏水使最终体积达到500 mL。 </li><li>在真空下通过滤纸过滤溶液并冷藏保存。 注意:胭脂红矾溶液可以重复使用，但当颜色开始褪色时应该丢弃。 </li></ol></li><li>在固定前，将石蜡剥离腺体，小心不要将腺体从载玻片上拉出。将幻灯片放置在玻璃载玻片上，并将其浸入固定剂（100％乙烷）中醇，氯仿和冰醋酸，比例为6:3:1），取决于腺体的厚度。 </li><li>倾倒固定剂，并将腺体浸泡在70％乙醇中15分钟。通过倒出1/3的乙醇溶液并用蒸馏水代替，逐渐使腺体再次水化。浸泡5分钟重复此过程三次。 </li><li>最后冲洗后，倒出所有乙醇/水溶液，并用100％蒸馏水代替。浸泡腺体5分钟。 </li><li>倒出蒸馏水，将腺体浸入胭脂红矾溶液中。根据厚度，将腺体染色12-24小时。 注意:腺体不能过度染色，但多批次染色时间应相同，染色强度相同。 </li><li>倒出胭脂红色明矾溶液，并在100％蒸馏水中冲洗腺体30秒。在70％乙醇中浸泡15分钟，95％乙醇浸泡15分钟，逐渐脱水腺体和100％乙醇20分钟。 </li><li>根据厚度，将其浸泡在二甲苯中12-72小时，清除脂肪腺体。 注意:腺体应为半透明（透明）。如果任何不透明（白色）区域保留，继续浸泡在二甲苯直到半透明。如果泌乳或其他非常厚的腺体被染色和清除，则二甲苯可能需要更换一次才能完全清除腺体。 </li><li>通过移液足够的介质只覆盖腺体，用二甲苯安装介质安装载玻片。添加盖玻片，确保不会形成气泡。 </li><li>让幻灯片干燥。当安装介质干燥时，它将在盖玻片下收缩，可能需要添加额外的安装介质。一旦不需要额外的介质，允许滑块完全干燥;这可能需要2-3周。 </li><li>一旦载玻片完全干燥，可以用棉签和少量二甲苯除去任何残留的固定介质。小心不要使用太多的二甲苯，因为这可以溶解安装介质并松开盖玻片。如果发生这种情况并且气泡聚集在盖玻片下面，则盖玻片应该在二甲苯中除去，并且重复安装过程。 </li></ol> 3.准备整体图像进行分析<ol><li>使用宏观显微镜或解剖显微镜和数码相机使用适当的软件捕获整体安装的图像（ 图3 ）。 注意:虽然可以选择捕获整个腺体上皮的任何放大倍数，但是必须捕获将以相同放大倍数彼此进行比较的所有全贴图像。 </li><li>下载ImageJ软件（或FIJI软件） 6 。 </li><li> 通过点击"文件"→"打开"打开ImageJ中的乳腺整体图像。选择手写工具并追踪腺体上皮。选择"编辑"＆＃8594; "外界清晰"。 <ol><li>通过跟踪节点并使用手写工具和"编辑"→"剪切"来删除淋巴结。 </li></ol></li><li>通过选择"图像"→"颜色"→"分割通道"来分离颜色通道。选择具有最佳对比度的通道，通常为蓝色通道。 注意:RGB图像由该图像的红色，绿色和蓝色分量的堆叠组成。此操作将这些组件分为三个8位灰度图像。 </li><li>通过选择"处理"→"减去背景"来减去背景。选择所需的参数，然后单击"预览"以预览更改。 注意:"减去背景"可以消除平滑，连续的背景。另外，可以使用"过程"→"过滤器"→"无刷面罩"来创建对比度。 </li><li> 选择其中之一<html> e自动消除噪音的以下方法:去除斑点或去除异常值。 注意:ImageJ提供了自动消除噪声的第三种方法:去除NaN。但是，删除NaNs命令不适用，因为它使用32位映像，而当前方法使用8位映像。 <ol><li>通过选择"处理"→"噪声"→"去斑点"，使用去斑指令去除噪声。 注意:这相当于添加一个中值滤波器，它将每个像素替换为其3×3邻域中的中值。 </li><li>通过选择"处理"→"噪声"→"删除异常值"，使用"删除离群值"命令消除噪点。 注意:如果偏离中位数超过某个值（阈值），此过程将替换像在周围环境中像素的中值的像素。 </li></ol></li><li> 手动清除任何剩余噪音（lass ="xfig"&gt;图4）。 <ol><li>打开原始图像的副本，并使用它作为指导，什么是什么和什么是不噪音。单击工具栏最右侧的双重红色箭头按钮。选择绘图工具;绘图工具按钮现在将显示在工具栏中。 </li><li>单击橡皮擦工具。通过右键单击橡皮擦工具按钮来调整橡皮擦直径。按住鼠标左键消除噪点。 注意:只有一个删除会话可以撤消。一旦鼠标左键被释放并再次点击，上一次的擦除将无法撤消。 </li></ol></li><li> 通过选择"图像"→"调整"→"阈值"来调整阈值。移动滑块以调整最小（上滑块）和最大（下滑块）阈值，直到达到压盖的适当描述。 注意:设置阈值将灰度图像分段为感兴趣的和背景的特征。<ol><li>单击应用。如有必要，请按照步骤3.6.1和3.6.2删除此处的附加噪声。 </li></ol></li><li> 重建通过阈值和噪声去除去除的腺体上皮的部分（ 图5 ）。 <ol><li>仔细进行图像重建，并保持原始图像的完整性。参考原始图像，以了解什么是什么和什么不是上皮。 </li><li>单击喷雾工具（在工具栏上使用绘图工具）。通过右键单击Spray Can工具按钮调整喷雾直径和速率。通过点击或按住鼠标左键，小心地填入腺体的缺失部分。 </li></ol></li><li> 创建一个用于进行Sh​​oll分析的腺体的骨架图像。通过选择"处理"→"二进制"→"制作B"，确保阈值图像为二进制图像<html> inary"。 <ol><li>如果图像在黑色背景上为白色，请选择"处理"→"二进制"→"选项"，然后取消选中"黑色背景"。通过选择"进程"→"二进制"→"骨骼化"来对图像进行骨骼化。 注意:这将重复从二进制图像的边缘删除像素，直到其缩小为单像素宽的形状。 </li><li>通过选择"过程"→"二进制"→"扩张"来一次填充图像。 注意:这通过将像素添加到二进制图像的边缘来填补由阈值化和骨架化创建的间隙。 </li></ol></li><li>选择"文件"→"另存为"保存图像。选择图像类型（通常为jpeg），输入文件名，然后单击"确定"。 </li><li> 通过将骷髅图像叠加到原始图像上来检查骷髅图像的准确性（ass ="xfig"&gt;图6）。 <ol><li>通过打开原始图像和骨架化图像来创建叠加层。选择"图像"→"覆盖"→"添加图像"。在"添加图像"对话框中，从"图像添加"下拉菜单中选择骨架图像，并将不透明度设置为30％。 </li><li>通过选择"文件"→"另存为"，将覆盖的骨架图像的图像保存到原稿上。选择图像类型，输入文件名，然后单击"确定"。 </li></ol></li></ol> 4.进行Sholl分析<ol><li> 打开骨架图像，并通过选择"进程"→"二进制"→"制作二进制"使图像二进制。 <ol><li>在设置放大倍率之前，使用千分尺测量拍摄图像的放大倍率的像素数/ mm。 </li><li>设置测量的倍率s选择"分析"→"设置比例"。输入像素数/ mm。将"已知距离"和"像素长宽比"设置为1.为"单位长度"输入"mm"，并选中"全局"（每个图像保持相同的比例）。 </li></ol></li><li> 通过从主导管开始（分析中心）到腺体上皮的最远端点绘制一条线，确定Sholl分析的结束半径（包围半径）（ 图7 ）。 <ol><li>使用工具栏中的线条绘图工具在感兴趣的点之间画一条线。按"M"键进行测量，并注意结果窗口将打开。 注意:"长度"列中的值是以mm为单位的行长度。设置Sholl参数时，该值将自动输入为结束半径。 Sholl插件将使用起点的线作为中心环的中心。 </li></ol></li><li> 运行Sholl分析。 <ol><li>通过选择"插件""高级分析"来运行分析;将出现一个参数窗口。 </li><li>将"I.壳体定义"中的起始半径设置为0.00 mm;在步骤4.2中测量的线的长度将自动输入为"结束半径"。 </li><li>将"半径步长"设置为0.1 mm。 注意:半径步长确定环数（有效地迭代次数）;较小的步距将增加环的数量，而较大的步长将减少环数。半径不能太小，虽然半径过小会导致不必要的环数。然而，它可以设置得太大，这将产生较少的环，并且随后低估了交叉点的真实数量。参考Stanko 等（2015），以获取有关确定步长的更多信息。 </li><li>在"II。每个半径多个样本"中将"＃样本"设置为1，将"积分"设置为"均值"。 </li><li>将"封闭半径截止"设置为1，并在"III。描述符和曲线拟合"中选择"起始半径推断"为"＃主分支"。 注意:可以根据需要检查"适合配置文件和计算描述符"和"显示配件详细信息"。 </li><li>检查"线性"，并在"不符合标准化的配置文件"中选择"最佳配合度";检查"最有信息"。在"IV。Sholl方法"中选择"规范化配置文件的区域"。 </li><li>在"V.输出选项"中选中"创建交点掩码"，以创建交点的热图（可选）。 </li><li>单击"Cf. Segmentation"生成具有振铃的图像的预览窗口以确认该区域的分析。 </li></ol></li><li>单击"确定"运行分析。 </li></ol> 5.测量MEA <ol><li> 通过追踪上皮树周围的最短距离来测量MEA（ 图8 ）。 <ol><li>在ImageJ中，打开压盖的骨架图像，单击多边形工具。点击上皮树周边的一个点，开始多边形，围绕腺体的周边移动，然后单击以添加一个线段。 </li><li>当整个上皮区域被绕过时，双击关闭多边形。按"M"键打开结果窗口; "区域"列中的值是MEA。 </li></ol></li><li> 在腺体上皮超过淋巴结的情况下，在计算分支密度时，从MEA中减去淋巴结面积（LNA）。 <ol><li>通过跟踪同一个人的淋巴结来测量LNA呃为上皮树，按"M" 注意:在这些情况下，必须从MEA中减去LNA，因为淋巴结阻止分析计算LNA内的交点。当上皮未达到淋巴结时，LNA为零。 </li></ol></li></ol>报告数据<ol><li> 报告包围半径MEA，N， k和分支密度的值。 注意:在步骤4.2中确定包围半径，并在步骤5.1中确定MEA。 <ol><li>运行分析生成一个Sholl结果窗口。 注意:报告的N是和相的值。报告的k是Sholl回归系数（半对数）的值。 Sholl分析将在腺体上皮的任何测量区域上返回k值。为了在整个上皮上获得k的准确值，必须存在导管末端。 </li><li>修改th通过使用N的输出值计算分支密度（该方法的基本终点），对乳腺进行分析。使用公式N /（MEA-LNA）计算分支密度，并将该值报告为N / mm 2 。 </li></ol></li></ol>

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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>

作者没有什么可以披露的。

从出生到青春期，乳腺生长是异速生长的。青春期后，乳腺通过广泛的导管分支和伸长发展，直到乳腺上皮占据整个脂肪垫。分支特征是乳腺发育的一个重要方面，客观量化这些特征的能力对于评估正常乳腺发育和识别早期暴露于乳腺有毒物质的异常发育是非常有用的。 评估形态特征，量化基本尺寸测量和计数乳腺结构是评估乳腺发育的典型方法。然而，由于啮齿动物乳腺?...

乳腺分支是通常被评估为腺体发育指标的特征，但难以客观量化。 1953年，Sholl 1描述了一种用于测量猫的视觉和运动皮质中的神经元树突状结构的方法，Ferriera 等人 2开发了一种用于该技术的插件。因为神经元和乳腺都具有类似的树状结构，所以该插件用于定量乳腺上皮分支密度在青春期大鼠乳腺的2D图像中。选择年龄阶段进行分析，因为断奶是在学术实验室和测试指南研究中经常评估的生命阶段。 Sholl分析是与FIJI一起分发的插件，它是开源图像处理软件包ImageJ，并附加了一些插件。该插件创建了一系列围绕predef的同心环（通常是神经元的神经元或乳腺的主要管道的起源）并延伸到物体的最远部分（包围半径）。然后计算每个环上发生的交点数（N）。该插件还返回一个Sholl回归系数（ k ），它是上皮分支衰减速率的量度。 使用ImageJ，创建乳腺整体的骨架图像，并测量乳腺上皮区域（MEA）。使用Sholl分析插件分析图像，并返回N和k的值以及其中未使用的其他值。通过计算N / MEA确定乳腺上皮分支密度。分支持续在腺上皮的外部区域的程度是分支复杂性，并且是均匀的远端上皮生长的指标。因为k是海拔远端下降的量度elial分支，它是分支复杂性的有效措施和乳腺发育的可靠指标。 该协议描述了一种计算机辅助方法，用于创建乳腺整体的骨架图像，并定量评估年龄男性和雌性大鼠的乳腺分支特征。这种方法相对较快，不需要使用专门的显微镜设备。该方法的开发和验证在Stanko 等人 （2015） 3 。本报告还介绍了大鼠乳腺整体的制备方法。 de Assis 等人已经描述了类似的乳腺整体手术（2010） 4和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

越来越多的研究正在利用啮齿动物乳腺作为评估化学物质暴露的发育毒性的终点。这些暴露对乳腺发育的影响通常使用基本尺寸测量或通过评定形态学特征来评估。然而，解释发展变化的广泛方法可能导致实验室之间的翻译不一致。需要一种常见的评估方法，以便从研究中进行比较的数据形成适当的解释。本研究描述了Sholl分析方法应用于定量乳腺分支特征。 Sholl方法最初开发用于量化神经元树突状模式。通过使用ImageJ，开源图像分析软件包和为此分析开发的插件，乳腺分支密度和复杂性确定来自年龄大的雌性大鼠的乳腺。这里描述的方法将能够使用Sholl分析作为定量乳腺发育重要特征的有效工具。

在本方案中分析的乳腺的测量封闭半径，MEA，N， k和计算的分支密度的值报告在表1中 。 Sholl分析产生每个半径处的交点数（ 图9 ）的线性和半对数图 ，如果选择，则生成交点的热图（ 图10 ）。欠发达的腺体在同一MEA内表现出较少的交叉点，因此具有较低的支化密度。发育良好的腺体?...

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

通常使用描述性评估或通过测量基本物理属性来评估啮齿动物中的乳腺发育。分支密度是乳腺发育的一个指标，难以客观量化。该方案描述了乳腺分支特征定量评估的可靠方法。

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

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 次观看.  National Institute of Environmental Health Sciences. 通常使用描述性评估或通过测量基本物理属性来评估啮齿动物中的乳腺发育。分支密度是乳腺发育的一个指标，难以客观量化。该方案描述了乳腺分支特征定量评估的可靠方法。 

视频: 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.

切片乳腺全身为病变鉴别

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

科研

癌症研究

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.

大鼠乳腺内皮细胞移植到间膜白脂肪垫

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.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

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

发育生物学

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.

导管内递送和X射线可视化乙醇烧蚀溶液在小鼠模型中预防和局部治疗乳腺癌

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.

导管内乙醇消融治疗在大鼠模型中预防乳腺癌的X射线可视化

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.

变化在大鼠乳腺形态和患乳腺癌的风险

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 ...

生物学

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维组织学音量和其研究小鼠乳腺腺体的应用改造

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.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

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.

使用带有可更换盖子的乳腺成像窗口进行纵向活体腔内显微镜检查

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>乳腺活体亚细胞显微镜检查

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

乳腺活体亚细胞显微镜检查

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