Name: methods for acute and subacute murine hindlimb ischemia
Uploaded: 2016-06-21T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

This study was supported by NIH grants R21HL118661, R56HL124444, and R01HL124444 to CDK, and by NIH grants R00HL103797 and R01HL125695 to JMM.

所有动物实验根据由杜克机构动物护理和使用委员会批准的方案进行。作为研究的科学目的指示在这项研究中雄性小鼠使用，虽然任一性别可以使用。 1.脱毛<ol><li>前麻醉诱导，建立由设定在37℃的覆盖加热垫和连接到异氟醚的连续流鼻锥口的手术前准备区。 </li><li>放置在麻醉诱导室鼠标。将O 2流量计1升/分和异氟醚设置为1 - 3％。 注:麻醉通常诱导将25g小鼠用2％异氟醚。 </li><li>检查轻摇室和观察缺乏翻正反射鼠标的刺激反应。 </li><li>同花顺O 2室以清除在打开之前的异氟醚。迅速将鼠标移动到加热p广告并通过鼻锥连接到异氟醚。 </li><li>调节异氟烷1.5％。检查由踏板反射（趾捏）刺激反应。 </li><li>宽松应用眼科润滑剂双眼，以避免手术过程中干燥。 </li><li>刮胡子使用小型电动微调从两个后肢的头发。保持剃须时避免划破皮肤绷紧皮肤。 </li><li>应用预热的脱毛膏，让坐1分钟。轻轻擦去用湿纱布垫。 </li><li>对于以后的外科手术，关闭异氟醚和鼠标移动到空纸巾内衬回收笼，保证了鼠标不吸笼床上用品。监测动物，直到它能够保持胸骨斜卧。否则，将鼠标移动到手术台。 注:脱毛过程会刺激皮肤，影响灌注测量。建议等待1 - P前去除动物的毛发后第2天erforming预外科灌注扫描或执行手术。 </li></ol> 2.术前准备<ol><li>在此过程中使用以下工具;小直手术剪，2细尖钳成角，小镊子格雷夫，针驱动镊子，拉钩3，小弹簧剪刀和细尖的棉签。 </li><li>用消毒手术前的启动高压釜的所有工具。之前和每个外科手术过程之间使用热珠灭菌，最多5只动物。对于大于5组手术消毒额外的手术工具包。 </li><li>准备一个无菌手术领域包括盖加热垫和异氟醚端口。 10立体声解剖显微镜下完成所有的工作。 </li><li>麻醉和步骤1.1〜1.5的介绍准备鼠标。 </li><li>检查鼠标完全镇静并放入上手术台仰卧位置。安全使用医用胶带双腿。&lt;/ LI&gt; <li>如果使用温度控制的加热垫，附加温度探针和将它固定到用外科胶带，以确保它不会意外在手术过程中拔出外科平台的基础。 </li><li>清洁用3交替碘伏和酒精擦拭切口部位。用无菌手术单动物切一个洞，露出切口部位。 </li></ol> 3.肢体缺血诱导<ol><li>使用手术刀进行初始切口沿着大腿内侧的中心，从朝腹部膝盖运行，并且延长切口约1cm细剪刀（ 图1A）。 </li></ol><img alt="图1" src="/files/ftp_upload/54166/54166fig1.jpg" /> 图1.手术部位及血管解剖标志的鼠标后肢缺血手术。（A）的外观图鼠标在仰卧位后肢。的阴影线表示切口部位适当地进行下肢缺血的程序。（B）近端鼠标后肢血管观。股动脉（FA）的近端源于腹股沟韧带（IL）的下方。在FA的前端位于其分叉成腘动脉（PA）和隐动脉（SA）。主要的侧支动脉关闭的FA是旋股外侧动脉（LCFA），近侧尾股动脉（PCFA）和浅尾腹壁动脉（SCEA）。股静脉（FV）运行邻近于FA，和静脉分支可以看出平行于主动脉分支。星号（*）表示用于AMEROID蟒蛇或结扎的放置的近端和远端站点，这取决于亚急性或急性缺血是否会被诱导。 <a href="https://www.jove.com/files/ftp_upload/54166/54166fig1large.jpg" target="_blank">请CLI</a>这里完蛋查看此图的放大版本。 <ol start="2"><li>使用镊子，打开切口暴露覆盖腹股沟脂肪组织（IFT）膜。 </li><li>使用封闭镊子，刺穿膜进入IFT和腹部之间的分离。轻轻松开钳压向IFT从腹部肌肉分开，露出下面的神经血管束。观察近端和肤浅的尾鳍分支作为重要解剖标志（ 图1B）。 </li><li>插入的牵开器和近侧拉动腹部组织以暴露近端AMEROID缩或结扎部位，只是近端旋股外侧动脉（ 图1B）。横向回旋支位于约5毫米近端近端和肤浅的尾动脉。 <ol><li>插入两个更多牵开器插入切口的远端部分，一是内侧和一个横向，拉IFT从远侧远手术部位扩大手术视野。 </li></ol></li><li>用两个细钳去除最外面的覆盖膜神经血管束。轻轻地插入静脉和动脉之间的细镊子尖的一半，结合在一起的膜下滑动钳尖。关闭镊子轻轻撕掉膜。 </li><li>插入静脉和动脉之间的闭合钳子的前端，并通过在钳子释放压力创建它们之间的间隙。重复此技术来创建动脉和神经之间的间隙。 </li><li>亚急性肢体缺血，放置在近端股动脉（ 图2）的AMEROID缩。 <ol><li>安装近侧AMEROID缩，股动脉下滑动的钳子的前端，使其与神经血管束隔离。使用第二组angled-镊子夹住缩的边缘和引导它股动脉之下。 </li><li>莱股动脉成S很多大蟒。重复远侧缩，它定位立即近端股动脉的分岔进腘动脉和隐动脉（ 图2）。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/54166/54166fig2.jpg" /> 图2.放置AMEROID蟒蛇和连字。（A）放置在股动脉引起亚急性下肢缺血2 AMEROID蟒蛇的例子。近侧缩放置只是近端旋股外侧动脉（长链脂肪酸）。远端缩放置刚好接近腘（PA）和隐动脉（SA）的分支。蟒蛇中装朝上，以保证动脉槽被适当缩内。（B）的股动脉结扎的实施例，以诱发急性后肢缺血。 LigaturES（黄色箭头）被放置成使得它们侧翼蟒蛇在面板（B）中的位置，并在股动脉被各组两个连字（星号）之间切断。酒吧，1毫米。 <a href="https://www.jove.com/files/ftp_upload/54166/54166fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="8"><li>对于急性肢体缺血，结扎和横切股骨近端动脉。 <ol><li>向横切股动脉，螺纹7 - 动脉刚好接近近侧缩的位置的输入输出缝合（参照步骤3.7）和结扎。扎约1mm远离第一第二结扎。 </li><li>用弹簧剪刀横切两间结扎动脉。对于远端动脉横断，重复这些步骤，将1毫米左右2连字拆开，只是近端股动脉分叉处到腘动脉和隐动脉，但要确保他们远离肤浅caud人腹壁下动脉（ 见图1） </li></ol></li><li>关闭切口用中断5 - Ø薇乔缝线。 </li></ol> 4.灌注成像<ol><li>将鼠标移动到设定的激光多普勒血流灌注成像仪（LDPI）下37℃加热垫，并通过鼻锥连接到异氟醚源。如果没有温度显示器可用，允许5分钟的鼠标温热至37℃。 </li><li>打开相机并启动图像捕捉软件。 </li><li>点击"新建单幅图像"图标，打开"扫描仪设置"窗口。将"扫描尺寸"到"大"和"扫描速度"到"4毫秒/像素"。由"扫描区域（单位）"窗格下改变x和y值设定扫描区域。 </li><li>点击"视频和距离"选项卡，查看视频输入信号，并安排鼠标适合红框所示的扫描区域。点击"自动距离"校准从激光到对象的距离。点击"下一步"，打开"主题详细信息"窗口。 </li><li>输入主题信息和任何相关评论。点击"下一步"按钮以进入扫描窗口。 </li><li>点击"开始测试"按钮，打开"确认或重写扫描距离"对话框。点击&#39;确定&#39;以确认扫描距离。观察扫描过程开始并运行4 - 根据扫描区的大小8分钟。 </li><li>扫描完成后，观察"另存为"窗口。为文件命名并保存。 </li><li>关闭异氟醚和鼠标移动到一个空笼子的恢复和监控，直到动物能够保持胸骨斜卧。切勿将鼠标从麻醉恢复与其他老鼠的笼子。 </li><li>打开的图像分析软件。点击"打开"图标，浏览和打开图像文件进行分析。在文件窗口，观察流量，鼠标的照片，和彩色图像。 </li><li>为了纪念通量图像感兴趣区域（ROI）的区域，单击"显示投资回报率"图标。接下来，单击"添加多边形"按钮，拖动光标周围的非手术肢体得出投资回报率。右键单击关闭的形状。再次选择"添加多边形"周围画了手术肢体匹配的投资回报率。 </li><li>点击"统计信息"图标，打开"图像的ROI统计结果（PU）"窗口。注意在"流量％"列在不断变化的百分比差异。 注意:画将作为参考的第一个投资回报率。 注:为每个后续灌注扫描之前按照第1节概述麻醉鼠标的步骤和步骤4.1至4.11图像动物。 </li></ol>

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The authors have no conflicts of interest to disclose.

或许在此过程中的最具挑战性的步骤是从股静脉股动脉的分离。的较大直径和相比，这些动脉的股静脉的壁更薄提高其敏感性穿刺和手术操作过程中撕裂。可以通过使用用PBS浸湿的无菌拭子保持伤口湿润减少破坏静脉的可能性。同样重要的是，以确保所有的钳子削尖，对齐，并且为了使血管和周围组织的精确操纵自由断裂。在该出血确实发生的情况下，直到出血停止施加压力的区域用无菌纱布。?...

外周动脉疾病（PAD）是心血管发病率和死亡率在发达国家1的主要原因。 PAD结果从外周动脉，导致肢体缺血与产生劳累或休息痛，偶尔不愈合使得需要截肢溃疡和坏疽的动脉粥样硬化阻塞。针对PAD疗法涉及主要针对血管内2或血管重建手术3，本质上是没有有效的药物治疗存在4。 不幸的是，血管重建术往往有限的好处是，由于旁路移植具有高失败率（可达50％5年内）5，其在某些人群（ 如吸烟者，妇女，非隐静脉移植）6,7-更糟。血管内的方法，如血管成形术和支架置入术，也被高再狭窄率受到影响（超过50％，1年内），我格外ñ股腘疾病8，虽然使用药物洗脱球囊和支架有所提高9-11的结果。为了开发新的治疗的PAD它是开发的动物模型能可靠​​地再现人类疾病至关重要。 迄今为止，PAD的最常见的模式是后肢缺血模型（HLI），其在小鼠中12,13最常进行的。在其最常见的表现，该模型引起该近端和远端股骨动脉的手术结扎并随后在容器的切除其居间侧分支，从而导致血流和急性肢体缺血诱导的闭塞。 HLI已主要用来研究在周肢体肌肉组织中的血管生成和动脉生成反应和各种疗法（ 例如 ，药物，基因递送，干细胞）对这些反应的影响。最近，我们的团队已经用这个模型来研究骨骼肌细胞的作用，我n个回应肢体缺血和对结果的14遗传差异的影响。 该HLI模型促进了我们目前的理解是，血管和肌肉响应缺血取决于遗传学（ 即 ，近交系）15，16岁，及其它疾病或相关的动脉粥样硬化的条件，包括糖尿病17和存在或不存在高胆固醇血症18。然而，传统的挡拆模型的一个重要的弱点是，它是急性肢体缺血12,13的典范，而人PAD引起慢性缺血作为外周动脉闭塞性动脉粥样硬化的逐步发展的结果。 在试图绕过这个弱点，唐和他的同事最初开发利用AMEROID蟒蛇逐渐19股动脉闭塞的大鼠模型，同组随后ðeveloped类似的小鼠模型20。 AMEROID蟒蛇是在慢性心肌缺血21,22的犬最初描述20世纪50年代。这些设备有一个外金属套包围吸湿材料的内层，通常是酪蛋白，并且当周围放置一个动脉它们诱导逐渐血管闭塞，因为它们吸收来自周围组织的水分。在他们的模型中的修饰，Yang等放置在近端和在类似于手术结扎网站的网站远侧股动脉都蟒蛇，和它们连接的股动脉的侧分支，如在传统模式。相比急性HLI，AMEROID缩诱导缺血导致炎症和剪切应力依赖性的基因的低表达，降低血流量恢复4 -第5周后，可操作地，少肌坏死20。根据这些意见，有人认为，渐进的动脉闭塞可能提供P的模型广告更贴近人类疾病。 值得注意的是，在原有的报告中，只在C57BL / 6小鼠19，这是对缺血诱导的肌肉坏死15相对耐审查AMEROID缩引起缺血的效果。我们最近修改的逐渐缺血模型进一步探索和其在缺血更为敏感的BALB / c小鼠品系23的效果。在该模型的第一表现，我们放置在近端和股骨远端动脉既蟒蛇但留下的所有侧分支完好。在第二，较温和的变形例中，我们只放在近端股动脉单缩并再次左动脉的所有侧设有分公司完好无损。在该模型中的两个变型，我们发现，BALB / c小鼠，但不C57BL / 6小鼠，尽管具有相似的血流和血管密度显示显著肌肉坏死。类似我们以前的研究14，这些研究结果表明，肢体肌肉伤不仅仅由血流的影响，但是部分取决于遗传背景。此外，我们发现，肢体血流3天之内下降到最低点，因此该模型似乎更&#39;亚急性&#39;，而不是渐进的肢体缺血之一。 根据这些以前的研究，似乎明显，用于诱导后肢缺血一个单一的方法可能不适合于所有情况。因为各种条件（ 例如 ，遗传差异和存在或不存在的共同病态条件）影响既血管和骨骼肌肉特异性反应，调查可能发现需要修改慢性和/或后肢缺血的最好的严重性适合自己的目的。此外，该模型的现有描述通常缺乏合适的解剖学界标，以促进技术的可靠-研究者间重现性。在本文中，在小鼠诱导的急性或亚急性下肢缺血的方法被描述，并提供精确的解剖标志。

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methods for acute and subacute murine hindlimb ischemia

外周动脉疾病（PAD）是心血管发病率和在发达国家死亡率，和动物模型可靠地再现人类疾病是需要开发新的治疗这种疾病的一个主要原因。小鼠后肢缺血模型已广泛用于这一目的，但通过股动脉结扎诱导急性肢体缺血的标准做法可能会导致大量的组织坏死，损害研究者的研究血管和骨骼肌组织反应缺血能力。另一种方法，以股动脉结扎是渐进的股动脉闭塞，通过使用AMEROID蟒蛇的诱导。当在如股动脉结扎的位点相同或不同的位置周围放置股动脉，这些装置阻塞动脉用1 - 3天，导致更渐进的，亚急性缺血。这导致更少实质性骨骼肌组织坏死，WHICH可能更接近地模拟人类的PAD看到的响应。由于遗传背景的影响在这两个急性和亚急性缺血模型成果，考虑到小鼠品系正在研究是选择最好的模型非常重要的。本文介绍了适当的程序和鼠标股动脉结扎或AMEROID蟒蛇解剖位置，以引起亚急性或急性下肢缺血的鼠标。

鼠标后肢血管的正确识别是确保的技术重现诱导两种亚急性和急性下肢缺血，如这里所描述的关键。除了 ​​在动物研究中所固有的变型中，其他因素可以在激光多普勒灌注成像（LDPI）引入可变性，包括麻醉的类型，（俯卧仰卧对 ）的动物的位置，和体温（ 见图3）。此外，亚急性后肢缺血模型依赖于AMEROID蟒蛇，它可以在给定批次内广泛地变化的质?...

Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

Methods for Acute and Subacute Murine Hindlimb Ischemia

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

对急性和亚急性鼠下肢缺血的方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

methods-for-acute-and-subacute-murine-hindlimb-ischemia

Research

JoVE Journal

Medicine

16.3K 次观看.  Duke University Medical Center. Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion. 

视频: Methods for Acute and Subacute Murine Hindlimb Ischemia

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

结扎冠状动脉内注射在小鼠模型梗死

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

科研

医学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

生物学

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

缺血性中风和缺血再灌注小鼠的诱导使用梗塞区的中心动脉闭塞技术和可视化

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

远端中动脉闭塞开发缺血性卒中小鼠模型

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

作者聚焦：在小鼠中建立可靠的远端 MCA 闭塞模型用于中风研究

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...