Name: quantitative micro-ct analysis of aortopathy in a mouse model of β-aminopropionitrile-induced aortic aneurysm and dissection
Uploaded: 2018-07-16T13:00:00.000+00:00
Duration: 6 min 46 s
Description: Watch this Scientific Journal Video about Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of Î²-aminopropionitrile-induced Aortic Aneurysm and Dissection at JoVE.com

我们要感谢马克. 史密斯在射线成像方面的帮助。这项工作得到了 nih T32 赠款的支持, 为心血管疾病 (蟒蛇) 的跨学科研究, 美国心脏协会 (SMC) 和 nih R35 补助金 (拿 dk)。

《动物处理议定书》由马里兰州马里兰大学机构动物护理和使用委员会 (0116024 号动物议定书) 批准, 并按照 AAALAC 国际标准进行。
1. 试剂的制备
<ol><li>
肝 素
	<ol>
<li>稀释250µL 的 1000年 U/毫升肝素硫酸酯50毫升的磷酸盐缓冲盐水, 使最终浓度的 5 U/毫升。</li>
		<li>温暖的血气 (5 U/毫升) 磷酸盐缓冲盐水, 这将取代血液在血管中的水浴设置为37°c。</li>
		<li>通过连接所需油管和2个空10毫升注射器, 1 血气盐缓冲液和1用于造影剂, 准备压力控制泵。</li>
		<li>用温暖的血气磷酸盐填充油管, 从压力泵的油管中取出气泡。</li>
	</ol>
</li>
	<li>
对比剂 注: 请参阅对比剂套件成分的材料表。<ol>
<li>将色素化合物与稀释剂混合, 以达到1:6 染料稀释率。</li>
		<li>在使用前 (步骤 2.3.12), 添加200µL 的固化剂, 每5毫升整除稀释色素化合物和混合他们好 (4% 按体积)。 注: 制造商报告工作时间为40分钟。当硅橡胶造影剂在添加固化剂后开始聚合20分钟后, 在输液前立即准备溶液是很重要的。</li>
	</ol>
</li>
	<li>
BAPN 饮用水
	<ol>
<li>在饮用水中溶解β-aminopropionitrile (BAPN), 以创建最终浓度为3克/升 (根据文献中先前描述的协议改编)9,16,17。</li>
		<li>将含 BAPN 的饮用水管理到一组小鼠, 一旦它们4周的年龄, 直到微 CT 灌注的时间。</li>
	</ol>
</li>
</ol>2. 手术方法
<ol><li>
动物制剂
	<ol>
<li>在3周的时间里, 将老鼠戒掉, 维持在12小时 light/12 的黑循环, 并喂养他们标准的啮齿动物周。对于 BAPN 治疗组, 管理新鲜准备 BAPN 饮用水 16-26 周广告随意。为控制动物提供标准的饮用水广告随意。</li>
	</ol>
</li>
	<li>
麻醉技术 注:24 h 在 CT 分析前, 执行以下步骤。手术程序的制定, 以准备标本的死后心脏灌注。<ol>
<li>通过一个精确的蒸发器提供 100% O2和3% 异氟醚的感应罐进行麻醉。麻醉诱导后, 停止异氟醚, 并冲洗室与 O2。用 2-2.5% 异氟醚和1升/分2通过鼻锥维持麻醉。</li>
		<li>将感应室和面罩连接到木炭清除剂中, 用于废气吸附保护人员。通过证明没有对有害刺激 (脚趾捏) 的反应, 确保足够的麻醉飞机。</li>
		<li>准备一个由手术托盘和必要的手术器械组成的手术场。</li>
		<li>将动物转移到手术领域, 并将其定位在背卧床。</li>
	</ol>
</li>
	<li>
手术技术
	<ol>
<li>使用剪刀, 使中线切口通过皮肤和软组织从中间的耻骨联合到胸骨切口, 延伸通过皮肤和软组织覆盖胸骨。</li>
		<li>使用剪刀, 在剑突过程中的隔膜上创建一个孔进入胸腔。</li>
		<li>用剪刀解剖腹侧胸壁的隔膜, 双边。</li>
		<li>穿过肋软骨, 将肋骨从胸骨的右胸骨边界分开。</li>
		<li>在胸骨尖端 (靠近剑突的过程) 应用一个细止血钳, 移动止血 cranially, 使其定位在鼠标头上。这将收回胸腺和胸骨远离心脏, 暴露心脏和伟大的血管进一步操纵。</li>
		<li>尖锐地解剖心脏和胸壁之间的任何附着物。</li>
		<li>将27口径 IV 导管针连接至注射器预装10毫升的血气磷酸盐缓冲盐水 (5 U/毫升), 并填写所有油管与缓冲, 以消除空气气泡从管的压力泵。</li>
		<li>当液体线上的气泡在准备液体时使用小心可能会阻碍较小容器的填充。限制在动物准备过程中损坏的船只数量, 因为这会导致造影剂泄漏出被切断的容器, 改变完整填充所需的体积, 并将工件引入最终成像。</li>
		<li>用直角钳固定的27口径针刺穿左心室。立即切开右心室或下腔静脉, 排出肝素溶液和血液。 注: 肝素可作为抗凝剂, 防止动物死后血管凝固。</li>
		<li>用单个注射器泵灌注动物以2毫升/分钟的恒定速率。注意器官的可见烫漂。继续灌注, 直到灌流液从静脉循环排出的血液没有血 (约 5-6 毫升)。停止泵。</li>
		<li>从10毫升注射器中拔下静脉注射导管, 注意不要干扰针在左心室的位置。</li>
		<li>在完全放血后立即将造影剂溶液分离成5毫升整除数, 并在此时添加固化剂 (见步骤 1.2)。把它们搅拌好。将5毫升的造影剂混合成一个10毫升的注射器, 并用它灌注动物。</li>
		<li>对于完整的血管充填 (动脉和静脉), 继续输液通过点, 当它可以看到退出静脉解决方案。寻找成功灌注的迹象, 包括在冠状动脉、肺动脉、肠道和肝脏血管中的铸造剂的可视化。</li>
		<li>造影剂在室温下大约20分钟后会痊愈。固化后, 根据需要, 收获各器官, 并将其固定在10% 中性缓冲福尔马林中。如果样品不用于第二天的显微 CT 扫描, 则修复整个尸体。如果尸体将在随后的一天使用, 把他们放置在一个金属托盘, 并把他们放在冰箱4摄氏度, 以治愈过夜。</li>
	</ol>
</li>
</ol>3. 显微 CT 扫描及参数
注意: 特定的图像采集参数将取决于使用的机器。
<ol><li>获取每只老鼠的 x 射线计算机断层扫描图像在使用微 CT 扫描器进行灌注后的第二天, 使用 X 射线管电压为 55 kVp, 电流为150μA, 系统放大系数为 2.19, CCD 相机像素 binning 因子2。这将生成一个有效的像素大小为29µm。<ol>
<li>将小鼠胴体仰卧在微 CT 扫描台上, 获得侦察 X 线扫描。</li>
		<li>在躯干上聚焦57.4 毫米 (轴向) x 37.1 毫米 (transaxial) 的探测器视野, 以图像的主动脉全长。</li>
		<li>获取180个图像投影, 其旋转增量为2度, 每次投影的时间为2800毫秒。</li>
	</ol>
</li>
	<li>用改进的 Feldkamp 算法重建图像;重建的体素大小是 29 x 29 x 29 µm3 (切片厚度 = 29 µm) 使用的多模式3D 可视化插件的软件在这里使用。</li>
</ol>4. 后处理和渲染
<ol><li>使用适当的软件将 CT 数据转换为 DICOM 格式。</li>
	<li>分析图像以确定是否存在动脉瘤。测量主动脉弓、降胸主动脉和腹主动脉最宽点的小轴直径 (如前所述18 ) (图 1)。 注意: 在我们的研究中, 图像被两个独立的观察者 (一个盲人) 分析, 利用 DICOM 查看器来确定动脉瘤是否存在。在主动脉弓、降胸主动脉和腹主动脉的最宽点测量了小轴直径, 如前所述19 (图 1)。BAPN 未治疗小鼠的平均非动脉瘤性动脉段建立正常血管直径作为年龄匹配的控制值。</li>
	<li>动脉瘤被定义为主动脉段的局部或弥散扩张, 直径大于参考直径的50%。根据上述测量结果找到这些数据。</li>
</ol>

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作者没有什么可透露的。

微 CT 成像可用于提供高度详细和三维的血管病理重建动物模型。通过使用血管造影增强的培养基, 非增强的软组织, 如血管的流明, 可以区别于那些加强。虽然激光多普勒, microangiography, 磁共振血管造影, 组织学与共焦, 或双光子显微镜可用于评估血管床, 他们通常侧重于有限的研究领域和/或仅限于二维评估。微 CT 提供了一种成本效益高的方法来获得详细的血管结构成像, 这有助于了解潜在的血管生成和血管生物学。其他成像小动物的方法包括活体微 CT 和数字减影血管造影。与这里描述的技术类似,体内微 CT 依赖于外源造影剂来增加分辨率。孔 vc 和 Isovue-370 是2这样的血池造影剂, 可用于微 CT. 孔 vc 已被使用, 并报告有最大平均增强 620 Hounsfield 单位在主动脉20分钟后注射20。其他基于纳米微粒的造影剂也显示了 CT 和 MRI 的潜力。左心室插管不开胸是另一种方法, 整个小鼠灌注显微成像, 已被描述21。超声成像对血管直径的纵向测量是一种可用于生存研究的最终方法。
在这个过程中, 充分的灌注需要高度重视细节。这是重要的血管在灌注前完全 exsanguinated 与造影剂。如果肝脏不漂白, 并保持其深红色, 在灌注前可能需要额外的冲洗与血气盐水溶液的对比。不完全放血的血管可能影响最终的图像质量, 出现填补缺陷的血管或 artifactual 远端血管闭塞。此外, 针插入左心室是一个微妙的程序, 可以撕裂心室壁或切割进入右心室。如果针插入右心室, 肺部将迅速灌注, 并会在灌注过程中早期变黄, 导致血管灌注不完全。血管破裂是导致不完全灌注的最终并发症。通过动脉树启动灌注前, 确保充分的静脉循环引流, 可以最大限度地减少血管破裂的几率。逆行注射是一种灌注血管床的替代方法, 以前被描述为冠状动脉11的影像。我们选择了一种顺的灌注方法, 因为它更准确地代表了胸和腹主动脉的生理血流。微 CT 图像的质量限制了动脉内血栓或血肿的评估能力。然而, 根据 CT 协议的制定情况, 软组织的可视化程度也很好。在我们的研究中, 我们注意到主动脉部分的充填缺陷可能是由于狭窄腔内血栓所致。所描述的技术是评估血管内空间的最佳方法。
总之, 本协议为大鼠主动脉瘤和解剖模型的血管的高分辨率检测提供了一种安全的技术。定量分析可以很容易地实现, 通过显微 CT 图像, 能够提供一个精确的投射血管。为了本出版物所示实验的目的, 我们选择测量最大的主动脉直径, 以模拟在临床实践中对主动脉瘤的测量。如果适当的话, 可以利用解剖地标和切片位置距离的组合来测量主动脉的特定段。结果表明, 灌注的动物血管可以成像和数字分离的骨骼背景, 以达到预期的数量, 以创建一个三维表示的主要血管及其较小的远端分支。本协议保留感兴趣的血管组织, 可嵌入石蜡, 用于组织学切片进一步检查。
微 CT 是研究血管生物学的一种很有前景的成像方式。它具有较高的空间分辨率, 为评估血管的结构、组织和病理提供了机会。本手稿所描述的程序需要多种技能和技术, 包括动物处理和准备、图像采集和结果量化。本文成功地证明了血管灌注在小鼠 aortopathies 中的应用。需要进一步的研究来评估它在其他动物模型和疾病过程中的适用性。

主动脉夹层的发生率为3例, 每10万年1。在美国, 主动脉夹层和动脉瘤性疾病每年超过1万人死亡, 占 1-2% 的死亡在西方国家2。主动脉夹层是由血管内膜层撕裂引起的, 在生理压力下通过主动脉壁层的血液传播而引发。患者脉压升高与解剖和并发症发生率增高有关。增加壁面剪应力与主动脉壁扩张有关, 导致动脉瘤形成3,4。主动脉夹层的后果包括阻塞血液流向远端器官, 包括大脑, 肾脏, 内脏和四肢, 形成慢性动脉瘤, 破裂, 或死亡5,6,7。
目前, 在主动脉瘤和解剖的启动和进展中所涉及的生物化学和细胞过程尚不清楚。可再生动物模型的主动脉瘤和解剖是了解其病理生理学的关键。β-aminopropionitrile (BAPN) 是一种赖氨酰氧化酶抑制剂, 它可以防止弹性蛋白和胶原蛋白的交联, 并已被证明大大改变了血管壁细胞外基质的结构及其生物力学完整性6, 8。用 BAPN 治疗的啮齿动物被用作主动脉瘤和解剖9、10的常见动物模型。
血管成像方法有助于识别血管病理, 确认血管通畅, 评价器官灌注。近年来, 微计算机断层扫描 (显微 CT) 已被用来研究小鼠和类似大小动物的血管。与骨不同的是, 血管的轴向成像是有限的, 因为腔内血液本身相对阴性。然而, 当与血管造影剂结合时, 微 CT 可以对动物 vasculatures 进行详细的三维重建, 用于研究宏观解剖血管病理学11。
所选的对比剂 (见材料表) 是一种含有铬酸铅和硫酸铅的显影硅橡胶。在催化剂的存在下灌注时, 它很快硬化形成一个血管的投射, 在血管的宏观解剖结构中最小的改变, 使血管高度显影与背景组织对比时,检查 x 线。这种造影剂是有利的, 因为它易于处理和避免组织退化和船舶损失由于断裂经常与血管铸型腐蚀有关。由于它治愈的最低萎缩12, 血管清除的血液仍然是专利, 并允许准确评估动物宏观血管在非生存实验。以前的工作已经成功地使用了显影硅橡胶-对比在各种动物研究。具体来说, 在可视化冠状动脉, 肾小球, 胎盘和脑循环11,12,13,14,15的适用性已经显示。本文详细介绍了以铅为基础的显影硅橡胶血管内灌注的开放性左心室穿刺方法, 以定量表征 BAPN 诱导的小鼠主动脉病变的显微 CT 表现。

<table><tbody><tr><td>Microfil</td><td>Flow Tech, Inc</td><td>MV-122</td><td>We use yellow, a different color can be ordered as desired. Kit includes MV-Compound, MV-Diluent, and MV-Curing Agent.</td></tr><tr><td>Heparin (1000 U/mL)</td><td>Sagent Pharmaceuticals</td><td>25021-400-10</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Isoflurane</td><td>Vet One, MWI</td><td>502017</td><td></td></tr><tr><td>3-Aminopropionitrile fumarate salt</td><td>Sigma-Aldrich</td><td>A3134</td><td></td></tr><tr><td>Single syringe pump</td><td>Fisher Scientific</td><td>14-831-200</td><td></td></tr><tr><td>27-gauge scalp vein set needle</td><td>Exel Int</td><td>26709</td><td>27G x 3/4&quot;, 12&quot; tube</td></tr><tr><td>Inveon Micro-CT scanner</td><td>Siemens Medical Solutions</td><td></td><td></td></tr><tr><td>Osirix MD</td><td>Pimxmeo SARL</td><td></td><td>Version 8.0.2</td></tr><tr><td>Inveon Research Workplace</td><td>Siemens Medical Solutions</td><td></td><td>Version 4.2</td></tr><tr><td>Rodent Chow</td><td>Harlan Teklad</td><td>2018sx</td><td></td></tr></tbody></table>

quantitative micro-ct analysis of aortopathy in a mouse model of &#946;-aminopropionitrile-induced aortic aneurysm and dissection

主动脉瘤和解剖与人口中严重的发病率和死亡率有关, 而且可能具有极高的致死性。虽然存在主动脉疾病的动物模型, 但在体内成像的血管已经受到限制。近年来, 微电脑断层扫描 (微 CT) 已成为一种在体内和体外成像大、小血管的首选方式. 结合血管铸型的方法, 我们成功地利用微 CT 来表征β-aminopropionitrile 治疗 C57/Bl6 小鼠主动脉病变的频率和分布。这种方法的技术局限性包括: 不良动物制剂所引入的灌注质量的变化, 适用于容器尺寸量化的适当方法, 以及本程序的不可生存性。本文详细介绍了一种以铅为基础的显影硅橡胶血管内灌注的方法, 用于 aortopathy 在动脉瘤和解剖小鼠模型中的定量表征。除了可视化的主动脉病理, 该方法可用于检查其他血管床在体内或血管床切除后的尸体。

为了评估这项议定书, 20 只成年小鼠, 以前描述的混合背景19和 20-30 周的年龄, 有或不 BAPN 治疗, 用铅基显影硅橡胶灌注 (见材料表) 使用上面详细说明的协议。他们在次日进行微 CT 扫描 (图 1和 图 2)。在对照组中, 小鼠的年龄没有显著差异。
小轴直径在这些小鼠被定量。BAPN 治疗小鼠升主动脉的平均直径明显大于未处理的年龄匹配对照组 (1.43 0.56 毫米至0.93 英寸0.11 毫米; p = 0.023, 未配对学生t测试)。与年龄匹配的对照组相比, BAPN 对赖氨酰氧化酶的抑制作用对平均降胸或腹主动脉直径没有显著影响 (p > 0.082, 未配对学生t检验,图 3)。
动脉瘤被定义为1.5x 未治疗组的平均直径。与 BAPN 未处理的对照组相比, BAPN 动脉瘤的小鼠数量显著增加 (50% 的 BAPN 治疗小鼠与0% 的未治疗小鼠; P = 0.042) 由费舍尔的确切测试。BAPN 治疗小鼠的动脉瘤仅在主动脉中被发现, 多数在胸主动脉中发现 (8 出10动脉瘤)。4只老鼠开发了超过1动脉瘤与 BAPN 治疗 (表 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57589/57589fig1.jpg"> 图 1: 代表性横断面显微 CT 图像。这是 BAPN 未经处理的小鼠的横断面成像。B. 这是 BAPN 治疗小鼠的横断面成像。小轴直径测量是在主动脉弓的水平。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57589/57589fig2.jpg"> 图 2: 三维的小鼠主动脉瘤的重建和显微 CT 鉴别。这是一个具有代表性的3维重建 BAPN 治疗小鼠与囊状腹主动脉瘤和主动脉弓动脉瘤。这个小组显示一个典型的降胸主动脉夹层在 BAPN 治疗的小鼠。真和假腔由内膜瓣分离。真正的流明 (TL) 是血液的正常通道, 假腔 (FL) 是新创建的通道。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57589/57589fig3.jpg"> 图 3: BAPN 治疗对主动脉直径的影响.这些面板显示的平均微小轴血管直径测量的微 CT BAPN 未经治疗和 BAPN 治疗雄性小鼠。那些确定有动脉瘤的老鼠用红色突出显示。未经处理的组的平均值表示为每个面板中的虚线。这是升主动脉/拱 (氧化铝/拱);0.93 ~ 0.11 vs 1.43 @ 0.56 毫米;p = 0.023。b. 这是降胸主动脉 (DTA);0.82 ~ 0.04 vs 1.11 @ 0.43 毫米;p = 0.0817。这是腹主动脉;0.66 ~ 0.11 vs 0.89 @ 0.58 毫米;p = 0.296。所有数据都显示为标准偏差。<a href="/files/ftp_upload/57589/57589fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>没有 BAPN (n = 8)</td>
			<td>BAPN (n = 12)</td>
		</tr>
<tr>
<td>总有动脉瘤的动物</td>
			<td>0</td>
			<td>6 *</td>
		</tr>
<tr>
<td>动脉瘤总数</td>
			<td>0</td>
			<td>10</td>
		</tr>
<tr>
<td>氧化铝/拱</td>
			<td>0</td>
			<td>5</td>
		</tr>
<tr>
<td>Dta</td>
			<td>0</td>
			<td>3</td>
		</tr>
<tr>
<td>和主动脉</td>
			<td>0</td>
			<td>2</td>
		</tr>
</tbody></table>表1。无 BAPN 治疗雄性小鼠动脉瘤的解剖分布。本表显示了微 CT 识别的动脉瘤的发生率 (n = 20)。阳极氧化铝 = 升主动脉;DTA = 降胸主动脉;主动脉 = 腹主动脉。* p < 0.05.

Watch this Scientific Journal Video about Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of Î²-aminopropionitrile-induced Aortic Aneurysm and Dissection at JoVE.com

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

本文描述了一个详细的方法, 使用显影铅基硅橡胶灌注小鼠血管的主动脉直径量化的主动脉瘤和解剖模型。

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

quantitative-micro-ct-analysis-aortopathy-mouse-model

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.5K 次观看.  University of Maryland School of Medicine. 本文描述了一个详细的方法, 使用显影铅基硅橡胶灌注小鼠血管的主动脉直径量化的主动脉瘤和解剖模型。

视频: Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

科研

医学

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

小鼠动脉粥样硬化的定量

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

免疫与感染

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

腹部主动脉瘤小鼠模型中脉冲传播速度、失散性和应变的测量

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

小鼠胸腹主动脉超声检查确定动脉瘤大小

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

<meta charset="utf8"></head><body>研究小鼠腹主动脉瘤的分子机制

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

<meta charset="utf8"></head><body>作者聚焦：腹主动脉瘤小鼠模型的开发和表征

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

血管周围应用弹性蛋白酶诱导的小鼠腹主动脉瘤模型

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

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通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型

通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型

通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型

血管周围应用弹性蛋白酶诱导的小鼠腹主动脉瘤模型