Name: subcutaneous angiotensin ii infusion using osmotic pumps induces aortic aneurysms in mice
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The research work presented in this manuscript was supported by a grant (HL107319 to Alan Daugherty and HL107326 to Lisa A. Cassis) from the National Institutes of Health of the United States of America. The content in this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The publication of this manuscript was sponsored by DURECT Corporation.

伦理声明:鼠标研究与肯塔基州机构动物护理和使用委员会（:2006-0009 IACUC协议号）的大学批准执行。小鼠用氯胺酮（〜210毫克/千克）和赛拉嗪的过量鸡尾酒（〜30毫克/千克）在安乐死终止。 对血管紧张素Ⅱ金额1.计算注:此协议使用输液血管紧张素Ⅱ（1000毫微克/公斤/分钟）的例子，连续4周在4个男性低密度脂蛋白受体 - / - 小鼠喂食饱和脂肪，丰富的饮食。 <ol><li>称重计算需要输液血管紧张素Ⅱ的量前研究小鼠。 </li><li>使用的模板（表1）来计算所需要的实验的AngII的质量。使用"平均泵送率"中的泵中的模板的步骤4的"抽气速率"的指令表示。手动5和步骤6 - - 在模板中，记录步骤1 10自动计算。 <ol><li>在模板中，假设老鼠会获得1克体重注射的血管紧张素Ⅱ1000纳克/ kg / min的4周期间。 注意:各鼠标可以具有非常不同的体重增加，这将取决于许多变数，如小鼠品系和饮食。我们经常使用"0"或"1克"的基础上从先前的研究我们自己的经验。 </li><li>计算血管紧张素Ⅱ溶液300μl总体积为每只小鼠因为每个泵需要约250微升。 </li></ol></li></ol> 2.溶解血管紧张素Ⅱ <ol><li>商店冻干血管紧张素Ⅱ小瓶在-20℃。开幕前平衡血管紧张素Ⅱ小瓶RT。 </li><li>称重所计算的AngII质量（7.3如表 1所示毫克）到无菌塑料管。 注:每默克索引，不使用玻璃试管进行溶解，因为血管紧张素Ⅱ的水溶液具有用于结合到玻璃有很强的亲和力。 </li><li>添加无菌盐水的计算的体积（1200μ升）放入塑料管含有冻干的AngII，帽，并通过倒置调匀直到溶液澄清。 </li><li>标签鼠标号＃1，＃2，＃3，和＃4上个别无菌塑料试管与盖（0.5 - 1.5毫升）。作为计算在步骤1.2中和表1中准备下一个层流罩基于体重各小鼠的AngII溶液。 <ol><li>例如，吸管3.6微升无菌盐水到管＃1中，然后296.4微升血管紧张素Ⅱ溶液，并通过上下抽吸轻轻混匀。 </li></ol></li><li>在塑料管与瓶盖标签鼠标数字（4毫升，无菌）。这些将被用于如步骤3.13中所述孵育泵。 </li></ol> 3.渗透泵灌装<ol><li>泵的主体和流动调节剂（图1）:在两个独立的部分获得泵。每个包厢有10泵体和流量主持人被独立包装。记录批号。 注:始终穿手套，因为从手中转移到泵的外部套管油将产生不利泵血功能的影响。使用无菌手套，纱布，管，填充针，并称量皿制备的泵，避免从植入物感染的危险。 </li><li>打开所需的研究泵体和流量主持人只有数字，因为这些不能被存储一旦打开。如果需要超过10个水泵，保证了泵的批号是相同的一项研究显示，由于来自不同批号泵具有不同的平均灌装量和泵速。 </li><li>称量每个泵（包括本体和流动调节剂），并注意重量到4位小数（例如，1.1443克小鼠＃1）。这个重量，模板（表1）在被称为"泵空重"，将被用于计算填充比率。 </li><li>附加泵填充针到1毫升无菌注射器，并仔细填充从适当的AngII溶液注射器编号的塑料管。以避免空气吸入注射器是非常重要的。 </li><li>从注射器仔细除去所有的气泡，而针的方向为向下。保持针头/注射器在这个位置上，以防止引入气泡进入泵内。 </li><li>轻轻插入填充针进入泵体。推进针的尖端进入泵。不休息的针的尖端紧紧在泵的底部。 </li><li>推注射器推杆慢慢填充血管紧张素Ⅱ溶液泵。一个黑影泵内指示填充水平。灌装量大约是246微升，每说明。 </li><li>停止填充泵，并仔细尽快取出针作为流体的珠升起泵。 </li><li>通过对泵本体的顶部的孔插入流量调节剂进入泵，直到没有间隙看到的流动调节剂的头部和泵本体的顶部（图1）之间。 </li><li> moderat的插入或进入泵体内导致从流动调节剂的开口一些流体泄漏。仔细涂抹，可能主持人置入过程中已经泄漏的所有额外的液体。 </li><li>称量充填泵。记录下的重量在模板中"泵重填充"。 </li><li>计算充填率（％）=（泵重"装" - "空"）×1000 /平均补量×100。 <ol><li>计算填充比如表1所示理想的情况下，填充比应等于或大于100％。再填充泵，如果填充率&lt;95％（牵连气泡可能存在于泵）。 </li></ol></li><li>将填充泵入标记为4毫升管（步骤2.5）与主持人头部朝上。添加无菌盐水的足够体积，以覆盖泵。保持泵的盐水管，直到植入。 </li><li>将管在37ºC孵化器。孵育泵O / N（至少12小时），以允许部分启动，然后植入到小鼠。泵ING血管紧张素Ⅱ的开始植入后约24小时，这使得小鼠从手术之前，期间输液血管紧张素Ⅱ所引起的任何潜在的压力中恢复。 </li></ol> 4.准备泵植入<ol><li>高压灭菌器（重力模式，干燥周期，15分钟）的纱布，棉签和手术工具，包括剪，止血钳，镊子，订书钉，订书机和至少1天在手术前。 </li><li>在操作室，准备蒸发器使用异氟烷麻醉。打开无菌单在层流罩，然后将鼻锥异氟醚麻醉。放置优碘，70％的乙醇，无菌水，珠灭菌，杀菌擦手，拭子，纱布，并填充泵在层流罩。 </li><li>唐口罩和礼服，然后在层流罩用干净的手打开外悬垂性。戴上无菌手套，打开无菌内包。 </li></ol>泵植入5.手术过程<ol><li>鼠标放置在感应湛BER与异氟烷以1.5的流速流入 - 2％。监测小鼠另外2 - 斜卧后3分钟。剃须面积约四分之一的大小，在左边或右边肩膀。 </li><li>放置鼠标在层流罩，用它的鼻子平齐与连接到异氟烷流出（图2A）的锥体。将鼠标的头部朝着外科医生的惯用手。使用鼠标的眼睛兽医药膏，以防止干燥，而在麻醉下。确保鼠标有之前手术的疼痛刺激无反应。例如，踏板响应是对于疼痛的良好指标。 </li><li>拭子并用优碘接着是三个擦拭物用70％乙醇擦拭剃的区域。唐或更改无菌手套。 </li><li>使用手术刀，使耳朵上前腿的肩胛骨背后〜1厘米的切口。这个切口应垂直于尾部。请小心，只切开皮肤，而不是深层组织。 </li><li>保持FORC在一方面EPS以打开切口，并用另一只手使用止血钳（图2B）在皮肤下一个皮下隧道。 </li><li>推进止血钳尖端朝向尾部，并创建一个口袋泵。这是通过小心地打开止血钳钳夹皮肤下开辟一个袋子来完成。退出切口止血。 </li><li>插入泵入切口与所述主持头定位到小鼠（图2C）的后部。轻轻泵完全推入口袋。应该有足够的自由空间，以无张力或拉伸皮肤的闭合伤口。 </li><li>一旦泵已经插入，紧紧捏住切口的两侧，矫直所以边缘会合处，并放置1或2个伤口夹关闭（图2D）。检查切口部位，以确保有伤口完全闭合，并且该泵被直接在网站上不加压。 </li><li>局部应用利多卡因乳膏（4％重量/重量），用干净的棉布swab.Remove鼠标从鼻锥，并将其放置在一个加热垫，直到它恢复意识。回收后，将小鼠返回其笼中。 </li><li>放置手术器械成珠灭菌对小鼠之间10秒。允许仪器使用前冷却。干净的手套与小鼠之间防腐剂擦手。监视所有小鼠，直到完全恢复为止。 </li><li>手术后密切监测小鼠。注射无菌盐水的大丸剂（0.2 - 0.3毫升）皮下如果小鼠显示窘迫，脱水或表观重量损失的迹象。每天至少一次随后在第10天观察小鼠每天至少两次，和。立即执行尸检如果在血管紧张素Ⅱ输液任何小鼠死亡。手术后14天 - 在7取出伤口剪辑。 </li></ol> 6.收集，固定，清洗和主动脉的成像<ol><li>切腹侧鼠标打开胸腔和腹腔，切开OPEn右心房，灌注生理盐水通过心脏的左心室清除血液中的主动脉，然后收获主动脉27。 </li><li>将塑料管含24至少3毫升4％多聚甲醛和10％的中性缓冲福尔马林的收获主动脉- 48小时27。 </li><li>小心取出外膜组织。销主动脉黑蜡引脚。获取主动脉图像相同的放大倍率。包括在每一个图像用于校准的标尺， 如图3。 </li></ol>主动脉7.恩面对成像<ol><li>通过主动脉弓的外和内曲率切开主动脉纵向，并切开主要分支包括无名，左颈动脉和左锁骨下动脉。引脚主动脉持平外外膜铺设毗邻黑蜡。 </li><li>获得带连接主动脉的内膜表面在相同的放大倍数脸图像。包括在每个图像校准一把尺子，作为表演中的n 图4。 </li></ol>

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发布这篇文章是由赞助的Alzet。

渗透泵输送皮下血管紧张素Ⅱ是一种常规的方式来诱导主动脉瘤小鼠。基于从许多实验室数据，已经出现了一致的发现，这是研究既腹主动脉瘤3,4-和22-26的TAA小鼠的可靠和可再现的方法。因此，这种小鼠模型被认为是一个模型，概括人类主动脉瘤的一些特性，并提供机械的见解这些破坏性的疾病。 虽然衰老是人类的危险因素腹主动脉瘤，它并没有被系统地研究了小鼠的血管紧张素Ⅱ致腹主动脉瘤。然而，似乎发生率和血管紧张素Ⅱ致腹主动脉瘤的严重性是在8岁小鼠相似- 48周4,5,7。目前，只有少数研究报告在小鼠血管紧张素Ⅱ诱导的TAA在8岁时- 24周22-26，并没有显示对TAA形成明显的年龄差异。 雌性小鼠有腹主动脉瘤低得多的发病率较注入血管紧张素Ⅱ4,28雄性小鼠。还值得一提的是，血管紧张素Ⅱ诱导的腹主动脉瘤的发病率在hyper-比量正常cholesterolemic小鼠，这是超过50％，而小于30％，分别为高得多。此外，主动脉破裂是常见的 - 在AngII的输液既normo-和高胆固醇血症小鼠中（约10 30％）。血管紧张素Ⅱ输液在1000纳克/千克/ min的速率为高胆固醇血症小鼠，如低密度脂蛋白受体 - / - 小鼠喂食西方饮食或载脂蛋白（载脂蛋白E） - / - 小鼠喂食正常或西方饮食，对AAA最大效应发展3,4,29。此输注速度是最佳的一项研究中，操纵在高胆固醇血症小鼠中的目的基因有望降低腹主动脉瘤。如果一个操作在高胆固醇血症小鼠中预计将增加腹主动脉瘤时，建议注入的AngII在500纳克/千克/分钟或更低的30的速率。在对比腹主动脉瘤，没有演示男性或高胆固醇血症和血管紧张素Ⅱ诱导的TAA 25之间nstrated关联。然而，类似于腹主动脉瘤中，如果预期操纵感兴趣的基因，以增加的TAA，我们建议1000纳克/千克/分钟的AngII的输液更低的输注速率。 同样重要的是要知道的血管紧张素Ⅱ诱导的主动脉瘤是发生率和严重性而变化个体小鼠中和研究之间。如果小鼠不发展主动脉瘤，一个潜在的可能性是可能的AngII尚未成功递送到小鼠中。为血管紧张素Ⅱ的高输注率的验证，如1000纳克/千克/分钟，测量血压，建议之前和期间，血管紧张素Ⅱ输注使用非侵入性的尾套法31。血管紧张素Ⅱ输注在1000纳克/千克/分钟的速度增加小鼠收缩压。此外，血浆肾素浓度可能会在血管紧张素Ⅱ输注或终止，因为血管紧张素Ⅱ对为r的负反馈来衡量ENIN分泌。因此，血管紧张素Ⅱ输注导致减少血浆肾素浓度。如果小鼠输注血管紧张素Ⅱ没有明显的主动脉病状，没有增加血压，并没有降低血浆肾素浓度，这将表明，血管紧张素Ⅱ尚未交付有效地通过植入渗透微泵。我们建议从研究中删除此鼠标。同样重要的是要注意，一些小鼠不发展尽管增加收缩压主动脉瘤和降低血浆肾素浓度。这些小鼠应保持在研究中。 总之，血管紧张素Ⅱ输注是通过皮下植入用渗透泵来诱导小鼠主动脉瘤实现。这种方法提供了血管紧张素Ⅱ不断地按照规定的速率为用来研究既腹主动脉瘤和肿瘤相关抗原指定的持续时间。

主动脉瘤具有永久性管腔扩张，这预示着破裂，通常导致患者死亡的主动脉。这种疾病发生在两个腹部和胸部主动脉区域，这些区域被称为腹主动脉瘤（AAAS）和胸主动脉瘤（TAA）为例，分别。由于分子机制和病理生理过程不完全的理解，没有经过验证的药物治疗，可以防止扩张任一类型的主动脉瘤的或破裂。由于很难获得患者样品和在直接人类进行的实验中，研究重点是定义腹主动脉瘤的机制已被频繁外推从动物模型。常用的动物模型是皮下输液血管紧张素Ⅱ（血管紧张素Ⅱ）的入小鼠。相对于其他手术方法在小鼠，如主动脉内弹性蛋白酶灌注或氯化钙围主动脉的应用程序，需要剖腹探查1,2，这种实现方法具诱导腹主动脉瘤D不要求进入体腔，并且需要最小的手术专门知识3,4。 通过渗透泵皮下输注血管紧张素Ⅱ诱导腹主动脉瘤是最初报道在低密度脂蛋白（LDL）受体- / -小鼠中的apoE喂食饱和脂肪富集饮食3，并且随后- / -小鼠喂食正常实验室饮食4。最近的许多研究还表明，血管紧张素Ⅱ诱导腹主动脉瘤在血脂正常小鼠5-7。注入血管紧张素Ⅱ的方法已经应用于诱导腹主动脉瘤，并探讨的分子机制，以及潜在的治疗策略（ 如 5-15）的发展，因为这一模式概括人类腹主动脉瘤观察到的许多功能。例如，人类的腹主动脉瘤如吸烟，老化，男性的危险因素也增加小鼠16,17血管紧张素Ⅱ致腹主动脉瘤。高胆固醇血症与腹主动脉瘤在人类中的关联需要澄清。但是，它有可连接一致，即高胆固醇血症增强血管紧张素Ⅱ诱导腹主动脉瘤小鼠18。血管紧张素Ⅱ诱导的腹主动脉瘤的小鼠的病理是高度异质，其特点是深刻巨噬细胞浸润，胶原降解，血栓形成和分辨率，和新血管形成19-21。在对比腹主动脉瘤在人体中的最常见的下腹主动脉位置，血管紧张素Ⅱ诱导的小鼠腹主动脉瘤发生在肾上主动脉区域。血管紧张素Ⅱ致腹主动脉瘤的另一个普遍存在的特点是透内侧突破，导致透血栓形成。目前还不清楚透弹性断裂是否发生在人类身上，因为美国科学促进会在人类病理发育尚未完全，由于缺乏来自早期阶段动脉瘤组织的研究。 血管紧张素Ⅱ输注到老鼠体内，也导致了胸主动脉区域的深刻扩张，即主要限制在升主动脉这是最常见的区域的TAA在人类<s向上&gt; 19,22-26。类似的血管紧张素Ⅱ致腹主动脉瘤，肿瘤相关抗原中血管紧张素Ⅱ灌注诱导还概括人类的TAA 25的许多特征。然而，相对于血管紧张素Ⅱ诱导的腹主动脉瘤，血管紧张素Ⅱ诱导的TAA不与高胆固醇血症相关联，并且没有性别差异。 皮下输液血管紧张素Ⅱ的总体目标到小鼠是研究病理特征及腹主动脉瘤和肿瘤相关抗原的分子机制。

<table><tbody><tr><td>Angiotensin II</td><td>Bachem</td><td>H-1705</td><td>compound used to induce aortic aneurysms</td></tr><tr><td>Alzet Osmotic Pumps</td><td>DURECT Corporation</td><td>Alzet Model 2004</td><td>feasible for 28-day infusion in mice weighed &gt; 20 g</td></tr><tr><td>Saturated fat-enriched diet</td><td>Harlan Teklad</td><td>TD.88137</td><td>42% calories/calories to stimulate hypercholesterolemia in LDL receptor -/- mice</td></tr></tbody></table>

subcutaneous angiotensin ii infusion using osmotic pumps induces aortic aneurysms in mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

4男LDL受体 - / - 在协议节所述小鼠4周血管紧张素Ⅱ输注后安乐死。主动脉收获，清洗，并成像可视化主动脉扩张灶。 如图3，主动脉有几个不同的特性，包括扩张肾上区域（AAAS; 图3A），扩展的上升区域的（的TAA; 图3B），或膨胀这两个区域的（包括腹主动脉瘤和的TAA的存在;图图3C），而在形态一只小鼠被严重正常（图3D）。腹主动脉的扩张是通过测量肾上区域的离体的最大宽度量化，就说明了红线图 3A。为了测量升主动脉扩张，主动脉切开并固定，如图4。内膜表面积测定由第包围的升主动脉区域（区在图4A）e红色线，以定量的TAA。标尺被包括在每个图像标准化测量，如图图 3和4。 <img alt="图1" src="/files/ftp_upload/53191/53191fig1.jpg" /> 。填充渗透泵的图1代表图像中的每个泵包含两个独立的部分:一主体和流动调节剂。填充所述泵体具有血管紧张素Ⅱ后，流程主持人被插入以密封泵。 <img alt="图2" src="/files/ftp_upload/53191/53191fig2.jpg" /> 图2.流程泵植入手术的（A）的小鼠被放置在层流罩与鼻锥是连续释放异氟烷和氧气。 （B）的甲直止血插入皮肤切口，使皮下通道; （C）的泵是通过插入<html>皮肤切口轻轻的; （D）中的皮肤切口泵插入之后装订。 <img alt="图3" src="/files/ftp_upload/53191/53191fig3.jpg" /> 从注入小鼠图3.主动脉图像（ 离体 ）的AngII的AngII 1000纳克/千克/分钟输注在雄性LDL受体- / -小鼠28天。 （A）腹主动脉瘤伴有血栓形成;红线（2.05毫米）表示最大宽度主动脉在肾上区域中的测量。 （B）升主动脉扩张（TAA）与严重正常腹主动脉; （C）的同时在上升和肾上主动脉区域（的TAA和腹主动脉瘤）深层次扩张; （ 四）与升序或肾上主动脉地区没有明显的扩张非常正常的主动脉。 4.JPG"/&gt; 图胸主动脉区域从输注血管紧张素Ⅱ的AngII 1000纳克/千克/分钟输注在雄性LDL受体小鼠4.恩面部图像- / -小鼠28天。以红线概述表面积表示升主动脉区域包括主动脉弓的一部分。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 1 </td><td colspan="2">所需剂量</td><td></td><td> 1000 </td><td colspan="2">毫微克/公斤/分钟</td><td></td></tr><tr><td> 2 </td><td colspan="3">开始体重（最大鼠标） </td><td> 24.8 </td><td> G </td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3">预计总体重增加</td><td> 1 </td><td> G </td><td></td><td></td></tr><tr><td> 4 </td><td>抽气速率</td><td></td><td></td><td> 0.25 </td><td>微升/小时</td><td></td><td></td></tr><tr><td>五</td><td colspan="2">怒江小鼠MBER </td><td></td><td> 4 </td><td></td><td></td><td></td></tr><tr><td> 6 </td><td colspan="2">每小时剂量动物</td><td></td><td> 1518 </td><td> NG </td><td></td><td></td></tr><tr><td> 7 </td><td>浓需要</td><td></td><td></td><td> 6072 </td><td>纳克/微升</td><td></td><td></td></tr><tr><td> 8 </td><td colspan="2">对于300 UL的解决方案</td><td></td><td> 1.82 </td><td colspan="2">毫克/ 300微升</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td colspan="2">解决方案所需</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 9 </td><td colspan="2">总血管紧张素Ⅱ（毫克） </td><td></td><td> 7.3 </td><td>毫克</td><td></td><td></td></tr><tr><td> 10 </td><td colspan="2">溶于生理盐水＆＃160; </td><td></td><td> 1200 </td><td>微升</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="2"></td><td></td><td></td><td></td></tr><tr><td>鼠标器</td><td>体重</td><td>稀释因子</td><td colspan="2">卷（微升） </td><td colspan="2">泵重量（g） </td><td>填充比率</td></tr><tr><td> ＃ </td><td> （G） </td><td></td><td>血管紧张素Ⅱ </td><td>盐水</td><td>空洞</td><td>填充</td><td> （％） </td></tr><tr><td> 1 </td><td> 24.5 </td><td> 1.0 </td><td> 296.4 </td><td> 3.6 </td><td> 1.1443 </td><td> 1.3877 </td><td> 99 </td></tr><tr><td> 2 </td><td> 23.0 </td><td> 0.9 </td><td> 278.2 </td><td> 21.8 </td><td> 1.1677 </td><td> 1.4145 </td><td> 100 </td></tr><tr><td> 3 </td><td> 24.8 </td><td> 1.0 </td><td> 300.0 </td><td> 0.0 </td><td> 1.1438 </td><td> 1.3904 </td><td> 100 </td></tr><tr><td> 4 </td><td> 21.8 </td><td> 0.9 </td><td> 263.7 </td><td> 36.3 </td><td> 1.1438 </td><td> 1.3904 </td><td> 100 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="7">稀释因子​​=体重的鼠标/体重最大的鼠标</td><td></td></tr></tbody></table><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>鼠标器</td><td>体重</td><td>稀释因子</td><td colspan="2">卷（微升） </td><td colspan="2">泵重量（g） </td><td>填充比率</td></tr><tr><td> ＃ </td><td> （G） </td><td></td><td>血管紧张素Ⅱ </td><td>盐水</td><td>空洞</td><td>填充</td><td> （％） </td></tr><tr><td> 1 </td><td> 24.5 </td><td> 1.0 </td><td> 296.4 </td><td> 3.6 </td><td> 1.1443 </td><td> 1.3877 </td><td> 99 </td></tr><tr><td> 2 </td><td> 23.0 </td><td> 0.9 </td><td> 278.2 </td><td> 21.8 </td><td> 1.1677 </td><td> 1.4145 </td><td> 100 </td></tr><tr><td> 3 </td><td> 24.8 </td><td> 1.0 </td><td> 300.0 </td><td> 0.0 </td><td> 1.1438 </td><td> 1.3904 </td><td> 100 </td></tr><tr><td> 4 </td><td> 21.8 </td><td> 0.9 </td><td> 263.7 </td><td> 36.3 </td><td> 1.1438 </td><td> 1.3904 </td><td> 100 </td></tr></tbody></table> 表1:计算通过渗透泵28天输注。

Watch this Scientific Journal Video about Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice at JoVE.com

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

渗透泵皮下植入提供了延长的和一致的输送化合物的便利的方法。这种方法已被广泛用于研究在小鼠中既腹部和胸部主动脉瘤。

使用渗透皮下血管紧张素Ⅱ输液泵诱导主动脉瘤小鼠

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

subcutaneous-angiotensin-ii-infusion-using-osmotic-pumps-induces

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

37.1K 次观看.  University of Kentucky. 渗透泵皮下植入提供了延长的和一致的输送化合物的便利的方法。这种方法已被广泛用于研究在小鼠中既腹部和胸部主动脉瘤。 

视频: Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

科研

免疫与感染

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

载脂蛋白 E 缺乏小鼠盐皮质激素受体的活化诱导动脉粥样硬化斑块的研究

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

医学

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 ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

局部性乳酸酶诱导下降胸腔主动脉瘤的毛尿外科模型

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the regulatory mechanism of RDN on hypertension remains elusive. Thus, it&#39;s essential to establish a simpler RDN model in mice. In this study, osmotic mini pumps filled with Angiotensin II were implanted in 14-week-old C57BL/6 mice. One week after the implantation of the mini-osmotic pump, a modified RDN procedure was performed on bilateral renal arteries of the mice using phenol. Age-sex-matched mice were given saline and served as sham group. Blood pressure was measured at baseline and every week subsequently for 21 days. Then, renal artery, abdominal aorta and heart were collected for histological examination using H&#38;E and Masson staining. In this study, we present a simple, practical, repeatable, and standardized RDN model, which can control hypertension and alleviate cardiac hypertrophy. The technique can denervate peripheral renal sympathetic nerves without renal artery damage. Compared to previous models, the modified RDN facilitates the study of the pathobiology and pathophysiology of hypertension.

Here, we present a protocol for renal sympathetic denervation (RDN) in mice with hypertension induced by Angiotensin II infusion. The procedure is repeatable, convenient and allows to study the regulatory mechanisms of RDN on hypertension and cardiac hypertrophy.

改善肾去神经支配缓解血管紧张素II.诱导的高血压

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

血管紧张素II诱导的腹主动脉瘤小鼠模型中中心静脉导管的药物治疗及3D超声监测

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

<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 ...

Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. Although mice are widely applied to shed light on the mechanisms of cardiovascular disease, mouse models of AR, especially those induced by surgery, are still paucity. Here, a mouse model of AR was described in detail which is surgically induced by disruption of the aortic valves under high-resolution echocardiography. In accordance with regurgitated blood flow, AR mouse hearts present a distinctive and clinically relevant volume overload phenotype, which is characterized by eccentric hypertrophy and cardiac dysfunction, as evidenced by echocardiographic and invasive hemodynamic evaluation. Our proposal, in a reliable and reproducible manner, provides a practical guide on the establishment and assessment of a mouse model of AR for future studies on molecular mechanisms and therapeutic targets of volume overload cardiomyopathy.

This protocol presents a practical guide on the surgery for creation of aortic regurgitation (AR) in the mouse. Assessment of the AR mouse by echocardiography and invasive hemodynamic measurement recapitulates its clinically relevant characteristics of volume overload-induced eccentric hypertrophy, suggesting its promising application in the study of cardiac hypertrophy.

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. ...

使用渗透皮下血管紧张素Ⅱ输液泵诱导主动脉瘤小鼠

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此视频中的章节

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

载脂蛋白 E 缺乏小鼠盐皮质激素受体的活化诱导动脉粥样硬化斑块的研究

小鼠胸腹主动脉超声检查确定动脉瘤大小

局部性乳酸酶诱导下降胸腔主动脉瘤的毛尿外科模型

改善肾去神经支配缓解血管紧张素II.诱导的高血压

血管紧张素II诱导的腹主动脉瘤小鼠模型中中心静脉导管的药物治疗及3D超声监测

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

Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse