使用Sholl分析方法量化大鼠乳腺组织中的分支密度
Summary
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。
Abstract
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。
Introduction
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 。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Dissecting board | NA | NA | A piece of styrofoam roughly 10"x12" is suitable. |
| Dissecting T-Pins | Daigger | EF7419A | |
| Spray bottle with ethanol | NA | NA | 70% ethanol is suitable. |
| Curved dissecting scissors | Fine Science Tools | 14569-09 | |
| Straight dissecing scissors | Fine Science Tools | 14568-09 | |
| Curved forceps | Fine Science Tools | 11003-12 | |
| Superfrost Plus 24x75x1 mm microscope slides | ThermoFisher Scientific | 4951PLUS-001 | Thermo Scientific Superfrost Plus & 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. https://www.thermofisher.com/order/catalog/product/4951PLUS4. |
| Fisherfinest Premium Cover Glass 24x60x1 mm | Fisher scientific | 12-548-5P | |
| Bemis Parafilm M Laboratory Wrapping Film | Fisher scientific | 13-374-12 | |
| Chloroform | Sigma-Aldrich | C2432 | |
| Glacial acetic acid | Sigma-Aldrich | A9967 | |
| Ethanol absolute, ≥99.8% (GC) | Sigma-Aldrich | 24102 | |
| Xylene | Sigma-Aldrich | 214736 | |
| Carmine alum | Sigma-Aldrich | C1022 | |
| Aluminum potassium sulfate | Sigma-Aldrich | A6435 | |
| Permount mounting media | Fisher Scientific | SP15 | |
| Macroscope | Leica | Z16 APO | This is the image capturing hardware and software used in this laboratory. As there are many different options, the methods and applications may vary between laboratories. |
| Digital camera | Leica | DFC295 | |
| Camera software | Leica | Leica Application Suite v3.1 | |
| ImageJ software | Open source | http://imagej.net/Welcome | |
| Sholl analysis | Open source | http://imagej.net/Sholl_Analysis |
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