动脉瘤高脂血症小鼠模型中油红O染色全主动脉病变的成像和分析
Summary
该协议提供了 分析小鼠动脉粥样硬化负担的分步程序。研究人员可以使用该协议来比较不同动物中动脉粥样硬化病变的丰度，位置和大小。
Abstract
载脂蛋白E（Apoe）或低密度脂蛋白受体（Ldlr）缺陷高脂小鼠是动脉粥样硬化研究最常用的两种模型。它们用于研究各种遗传因素和不同细胞类型对动脉粥样硬化病变形成的影响，以及测试新疗法的开发。分离、切除整个主动脉和定量油红 O 染色动脉粥样硬化病变是用于评估动脉粥样硬化负荷的基本形态测量方法。该协议的目标是描述一种优化的，循序渐进的手术方法，用于用油红O解剖，灌注固定，分离，染色，成像和分析小鼠主动脉中的动脉粥样硬化病变。由于动脉粥样硬化病变可以在整个主动脉树的任何地方形成，因此这种整个主动脉油红O染色方法具有评估单个小鼠中整个主动脉和所有分支中富含脂质的斑块的优点。除了油红O染色外，新鲜分离的全主动脉还可用于各种体外和体内实验以及细胞分离。
Introduction
冠状动脉疾病是美国死亡的主要原因，通常由动脉粥样硬化引起，这一过程导致斑块在动脉壁内积聚1。易患高脂血症的Apoe和Ldlr缺陷小鼠是动脉粥样硬化及其并发症和治疗开发的核心2，3，4，5。来自面部主动脉的动脉粥样硬化病变的定量是评估遗传操作对不同细胞类型的影响的重要终点分析。它还有助于研究旨在影响动脉粥样硬化性疾病发生，进展和消退的新疗法。动脉粥样硬化病变可在主动脉及其分支（即胸部的臂头动脉、颈动脉和锁骨下动脉，以及髂膜下方的肾脏、髂总动脉和股动脉）6的任何地方形成。对动脉粥样硬化负担的全面评估和适当的治疗需要评估不同地点的疾病负担，这是一个经常被忽视的挑战。
该协议描述了如何在单个小鼠中对动脉粥样硬化病变进行全面分析，从未打开的整个主动脉开始，然后进行面部准备。未开封的全主动脉油红O染色允许快速，定性地评估整个主动脉及其分支中富含脂质的斑块，而en面部制剂则提供小鼠主动脉中动脉粥样硬化病变分布的定量评估。
该技术使用8周龄的小鼠，在Apoe-/-高脂血症背景（MYH11-CreERT2;Tgfbr2f/f;mT/mGf/f;Apoe-/-;以下简称TGFβR2iSMC-Apoe小鼠）和凋落物Apoe-/-对照（MYH11-CreERT2;mT/mGf/f;Apoe-/-;以下简称为Apoe-/-小鼠）。这些动物以高胆固醇高脂肪饮食（HCHFD）作为研究材料饲养16周7。在研究终止时，对未打开的整个主动脉进行染色并用油红O成像（包括所有主要分支），以定性评估富含脂质的斑块。主动脉通过面部准备切开，所有动脉粥样硬化病变成像和定量。该协议可用于研究Apoe-/-或Ldlr-/-高脂血症小鼠模型中的动脉粥样硬化病变发展，并扩展到一般主动脉相关的血管生物学应用。
Protocol
Representative Results
Discussion
载脂蛋白E（Apoe）和低密度脂蛋白受体（Ldlr）缺乏小鼠可用于研究动脉粥样硬化的发展和治疗。研究人员可以使用整个主动脉的油红O染色来评估遗传学和治疗操作对动脉粥样硬化相关疾病的发生，进展和消退的影响9。主动脉油红O染色和病变定量是动脉粥样硬化研究的金标准终点。这种技术价格低廉，不需要特殊设备10。然而，获得高质量的油红O?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了联合生物学联盟微量资助的支持，该资助由NIH拨款P30AR070253（P.-Y.C.）和HL135582（M.S.）提供。我们感谢R. Webber和L. Coon维持本研究中使用的小鼠。
Materials
| 1.5 mL Eppendorf tube | DENVILLE | C2170 | |
| 10 mL syringe | BD | 302995 | |
| 16% Formaldehyde | Polysciences | 18814-10 | |
| 70% ethanol | VWR | RC2546.70-5 | To clean the dissection tools |
| Black dissection wax | CR Scientific | C3541 | |
| Corn oil | Sigma | C8267 | Solvent for Tamoxifen |
| DNeasy Blood & Tissue kit | QIAGEN | 69506 | To isolate DNA from mouse ear |
| Dulbecco’s Phosphate-buffered saline (1X DPBS), pH 7.4 | Gibco | 14190-144 | |
| Fine scissors | Fine Science Tools | 14059-11 | To cut the mouse skin and open the ribcage |
| Fisherbrand Economy Plain Glass Microscope Slides | Fisher Scientific | 12-550-A3 | |
| High cholesterol high fat diet | Research Diets | D12108 | To induce atherosclerosis |
| Imaging software | National Institutes of Health | Image J | Aortic lesion quantification |
| Isopropanol | VWR | JT9079-5 | |
| Kimwipes | Fisher Scientific | 06-666A | To clean the glass microscope slides |
| McPherson-Vannas Micro Dissecting Spring Scissors | ROBOZ | RS-5602 | To separate the heart and the aorta and to cut open the aorta and aorta branches |
| Microscope control software | Olympus | DP Controller | For aorta imaging |
| Minutien pins | Fine Science Tools | 26002-10 | |
| Needle-25G | BD | 305124 | |
| NonWoven Sponge | McKesson | 94442000 | |
| Oil Red O | Sigma | O-0625 | To stain the atherosclerosis lesions |
| Pall Acrodisc Sterile Syringe Filters with Super Membrane | VWR | 28143-312 | To filter working Oil Red O solution |
| Spring Scissors | Fine Science Tools | 15021-15 | To dissect and clean the aorta |
| Statistical software | GraphPad | Prism 8 | Statical analysis |
| Stereomicroscope | Nikon | SMZ1000 | For aorta dissection |
| Stereomicroscope | Olympus | SZX16 | For aorta imaging |
| Tamoxifen | Sigma | T5648 | To induce Cre-loxP recombination |
| Tissue-Tek O.C.T Compound, Sakura Finetek | VWR | 25608-930 | |
| Tweezer Style 4 | Electron Microscopy Sciences | 0302-4-PO | To cut the mouse skin and open the ribcage |
| Tweezer Style 5 | Electron Microscopy Sciences | 0302-5-PO | To dissect and clean the aorta |
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