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

16.2K 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

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

过期的CO 2测量气管插管或自主呼吸的病人从急诊科

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

科研

医学

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

研究方法检测疑似急性冠脉综合征患者短暂心肌缺血的连续ST段分析

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

糖尿病家兔欣德肢体缺血的临床前模型

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

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 ...