我们承认凯文·麦克劳德使用他的创作共用授权的音乐相关的视频，包括（按出场顺序），他的曲目"机场贵宾室"，"墙纸"和"晚报情节剧。"我们也想与手术视频他们的协助，承认Ndubisi Okeke和弗雷德里克Torstrick的。

1。 Spinotrapezius饲料动脉结扎术<ol><li>腹腔注射麻醉鼠标。在0.5ml注射器，制定:0.06毫升，100毫克/毫升氯胺酮 0.03毫升，20.0毫克/毫升甲苯噻嗪中 0.01毫升，0.4毫克/毫升阿托品 0.30毫升0.9％的生理盐水。 剂量:0.01毫升*每克的体重，IP </li></ol> *的剂量变量后，小鼠品系和股票，进行相应的调整，使鼠标恢复是在一个小时内从感应。 <ol start="2"><li>应用标准眼药膏，以防止角膜干燥。 </li><li>清除头发从后面使用微调快船队，随后进行了简短的脱毛霜中的应用。 </li><li>洗必泰或碘伏消毒的医疗器械领域。替代聚维酮碘与乙醇三次，用碘伏结束。 </li><li>将鼠标事先准备手术的工作表面上，加热垫和无菌悬垂性在的地方。 </li><li>通过背部皮肤约5毫米的尾鳍骨突出的肩胛骨使用虹膜的剪刀和标准模式镊子（ 例如 FST 11271-30）的隆起的组织和切割平行于创建一个线性切口（3-5毫米）脊椎。目标切口部位可确定通过透皮可视化背脂肪垫，如果皮肤色素沉着许可证;切割的尾部边界的脂肪垫。根据需要扩大切口，穿过继发性皮肤层春风似剪刀。 <ol><li>解剖学注 - 的spinotrapezius肌肉位于背侧，并从T4到L3沿脊柱两侧延伸。前缘横向延伸，约在肩胛骨。逐渐变细的尾部，使最后缘位于内侧前缘，并奠定冲洗与脊柱的肌肉。两种脂肪垫也是很重要的是要注意，直接铺设背部和腹部的spinotrapezius对颅一半的肌肉。 </li><li>已被证明因炎症切口血管重构响应没有影响，作为假结扎手术在重塑的spinotrapezius脉管的显示没有改变。两个空间的因素促成了这个结果。首先切口除去5毫米，从感兴趣的区域通常是检查在肌肉的血管反应颅方向。另外，被分离的背部皮肤上的切口部位的血管结扎站点由腹侧脂肪垫，顶部的spinotrapezius肌颅第三在于。 </li></ol></li><li>温热林格氏液，以防止肌肉干燥，冲洗切口。应用血管扩张剂如罂粟碱，将有助于可视化的目标动脉结扎。 </li><li>在解剖显微镜下，用血管钳钝性解剖和分离（但不删除）背的白色脂肪垫的基础MUSCular可视化的血管组织，以进入和离开它的横向边缘上的spinotrapezius。 </li><li>找到目标动脉，以及具有一个或二个成对的静脉，通过脂肪垫是在它的途中到的横向边缘的肌肉的spinotrapezius腹侧。此动脉馈送可以确认通过反射和更换观察的路径的动脉内的肌肉的肌肉的肌肉，尾部的一半的主要部分。结扎的目标位置段的动脉退出腹部的脂肪垫和进入肌肉，并是相当在这一地区。的动脉静脉对可能需要轻轻分离从在这个区域的腹侧的脂肪垫。请注意，spinotrapezius被送到的多动静脉对，包括船舶，横向的背部和腹部的脂肪垫。小心不要破坏这些船只。 <ol><li>多个指标可能被用来区分动脉从静脉。其中一个最好的方法是轻轻地阻碍的探针和上游距离的肌肉的​​血流量，流量将不会恢复在动脉的阻塞，直到被释放。颜色也可以被使用，如静脉壁包含一个白色的结缔组织鞘动脉缺乏。此外，一个可以考虑的直径前应用血管扩张剂，静脉通常规模较大，。 </li></ol></li><li>动脉通常是在接近其配对静脉;使用钝性分离与探针和＃5钳子隔离约5毫米的分部，背后的动脉，通过微探针和使用的前端，使在自然的间隙更宽裂的动脉和静脉之间。这是通过缝合线将螺纹的间隙。 </li><li>使用10-0单一线程的缝合线和缝合针夹持器的地方围绕饲料动脉结扎（〜70微米直径）向近端的解剖段使用的外科医生的结。解放军策的解剖动脉段朝末端，一个额外的结扎横断的动脉之间的连字，以允许有足够的分离。 <ol><li>的小规模与进料的spinotrapezius动脉导致手术困难增加，相比的一个更大的容器如股动脉结扎。没有经验的外科医生，特别是有很高的撕裂，但由于我们的疏漏，某些出血，及其他手术并发症。特别值得关注的是识别和切割无色动脉，后流受到阻碍，只是在切割前的难度。出于这些原因，将2连字建议对于不习惯的程序，作为外科医生具有视觉引导定位在哪里横切动脉以及一个确认的切口与容易观察到分离的2个连字。然而，一个更有经验的外科医生可以找到一个单一的绑带足够的，只要它们是能够清楚地识别和，切断动脉结扎下游的。虽然这种技术的，意外割伤附近的船只的急剧结扎缝合降低了风险，但也不是没有缺点，其中包括增加了难度，确认动脉横断。 </li></ol></li><li>验证结扎，通过观察血流量缩减（阻碍RBC列）下游的结扎部位（ 即朝向肌肉）。 </li><li>用解剖剪横切结扎动脉，两者之间的连笔字。如果只有一个连字被放在（11A），切在下行方向格外小心。 </li><li>药物加载纯膜（1毫米）可放置下游（尾端）实验程序的一部分。 </li><li>重新流离失所的筋膜和脂肪组织原来的方向。 </li><li>关闭使用8-0非吸收性缝合皮肤切口。根据观察，然后将鼠标在一份准备好的温水恢复笼和管理的APpropriate镇痛药，如布比卡因，0.25％，0.02〜0.05毫升局部浸润。 </li></ol> 2。 Spinotrapezius活体显微镜，原位 <ol><li>麻醉鼠标的感应室中，用3-4％异氟烷在100％氧气中蒸发在3-4升的流速·分钟-1。 <ol><li>吸入麻醉的使用功能测量是最好的，因为那里是减少心血管疾病的抑郁症与注射麻醉剂。如果无法使用的，长效注射麻醉剂，如戊巴比妥钠的吸入麻醉是优选的，以避免重复注射一次的动物已被定位。 </li></ol></li><li>麻醉后，通过不断提供异氟醚麻醉面罩（又名鼻锥）的的维护浓度（一般为1〜2％）和流量的0.5-1 L·分钟-1。 <ol><li>小鼠为主，通过他们的鼻腔通风，所以它是只有NEC埃森捂鼻子的麻醉面罩隔膜。 </li></ol></li><li>如果有必要，将头发从后面使用修剪剪及脱毛霜。 </li><li>运输的动物的热垫。 <ol><li>重要的是要保持在适温状态的小鼠，以防止冷诱导的血管收缩，这可以通过灯完成，循环水热垫，微波炉加热垫等。 </li></ol></li><li>插入直肠的温度探测器和热控制器设置到35℃。 </li><li>使皮肤切口尾端的使用的虹膜剪和标准模式钳spinotrapezius。 </li><li>延长切口头部的脂肪垫，创建一个马蹄切开，并覆盖保鲜膜，防止干燥的皮瓣。 </li><li>钝性解剖皮下结缔组织，用钳子和春风似剪刀，最大限度地提高知名度。 </li><li>将刺激电极接近山口进一步地，最大限度地减少当前字段的大小，并将其放置在暴露的肌肉外侧脊柱尾端。 </li><li>执行一个测试刺激以确认使用的电极的数据采集系统，电极刺激隔离器，和计算机控制器，具有一个200微秒的持续时间，振幅2毫安，交付在1赫兹的方波的电极位置。 </li><li>将保鲜膜覆盖在外露的肌肉，以防止干燥。 </li><li>开始计时的30分钟期间，在此期间，容器直径将平衡。 </li><li>的活体显微镜以上的spinotrapezius的肌肉，以查看血管结构。 <ol><li>如果使用一个浸没透镜，PBS中的目标和保鲜膜之间放置一个下拉。 </li></ol></li><li>从上面的主小动脉（最大的船只可见的）开始，操作阶段在XY平面内定位感兴趣的容器中。 <ol><li>可视化小arteriolES反射光显微镜需要更高的对比度，比传统的红细胞列。我们主要利用侧流暗场成像显微镜增强微血管的对比度，但相反也可以提高与反射光的荧光显微镜后，静脉注射高分子量荧光葡聚糖。 </li></ol></li><li>经过30分钟的平衡，拍摄图像/视频的船只的兴趣。 </li><li>与方波的持续时间200微秒，振幅2毫安，于8Hz，交付时间:90秒，如上所述，刺激肌肉。 </li><li>捕捉另一个图像/视频后立即刺激，继续抓住每分每秒，直到船又回到了休息直径〜10分钟。 </li><li>重复步骤6-18使用对侧肌肉。 </li><li>数据分析可以实时执行的，与使用图像分析软件的视频卡钳或脱线。 </li><li>安乐死的鼠标跟随ING IACUC协议。 </li></ol> 3。 Spinotrapezius组织收获和固定<ol><li>麻醉小鼠腹腔注射，制定在Spinotrapezius饲料动脉结扎术步骤1。 </li><li>进行颅骨切开几毫米的使用的虹膜剪和标准模式钳鼠标的肩胛骨骨突出。横向扩大切口，然后尾端两侧。 </li><li>背躯干轻轻地反映了皮肤和切割浅筋膜，皮肤释放。 </li><li>使用钝性分离，以去除的背脂肪组织使用标准镊子，然后反映的肌肉和类似的解剖腹部的脂肪组织。 </li><li>删除筋膜覆盖的肌肉使用产钳和春风似剪刀。此步骤可以是困难和费时的，但重要的是要提高免疫荧光染色和成像。保持组织的水分可以缓解去除筋膜。 </li><li>定位外侧缘的肌肉，使用钝性分离分离的spinotrapezius的脂肪垫位于腹侧肌肉。 </li><li>继续在颅尾方向钝性分离。注意几个血管进入和离开肌肉这个腹面;如果需要灌注，它之前，应执行横切这些船只。还请注意，在尾侧的肌肉的一半，其外侧边缘的肌肉插入躺在腹侧。沿着这条边界完全定义和释放的边缘，可能需要一些前沿。 </li><li>海关的spinotrapezius肌肉。要做到这一点: <ol><li>免的横向边缘的肌肉，如上面所述。 </li><li>横向切割穿过肌肉的最颅的程度。 </li><li>剪切沿内侧缘（沿脊柱），在矢状面中。 </li></ol></li><li>重复该过程，步骤4-9对侧肌肉，然后安乐死的m乌斯IACUC批准的方法。 </li><li>修复明胶包被的载玻片上的组织通过浸没在冷却的甲醇（4℃），或在4％多聚甲醛/去离子水的溶液在RT下20分钟。 <ol><li>可替换地，可以采用灌流固定。第2步之前，进行开胸手术，在右心房做一个切口，和灌注脉管用5毫升1×Tris盐缓冲液，然后用0.1mM CaCl 2和2％肝素通过左心室由5毫升4船舶固定，最后5毫升1×Tris缓冲盐水用0.1mM CaCl 2的 7％多聚甲醛。 </li></ol></li><li>紧随固定术，洗20分钟四次，每个试样用0.01M的磷酸盐缓冲盐水（PBS）的0.1％皂苷。 </li></ol>

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作者宣称，他们有没有竞争的金融利益。

这里提出的模型的小鼠spinotrapezius结扎是一种有效的小动物模型，研究动脉闭塞的功能和结构的调整所造成的。此模型是被广泛使用的后肢缺血模型4互补的，因为它提供了一个完整的微血管网络的高空间分辨率的整个肌肉的视图。此外，由于位于下方的背部皮肤肌肉，它是访问序列成像与活体显微镜，并通过灌流局部给药或薄膜植入2。这些特点使它成为一个有吸引力的小动物模型研究新的治疗靶点动脉闭塞的血管重构和功能的血管舒张的影响。 请注意结扎后肢模型获得的数据，由于几个键d的spinotrapezius结扎模型获得的数据进行比较时，应谨慎使用ifferences。首先，spinotrapezius肌肉稳定的肌肉，他们从不同的功能和纤维类型分布的腿部肌肉。我们的组先前已表明，小动脉结扎在spinotrapezius肌创建少或可忽略不计的缺氧菌株具有发达的抵押品小动脉的网络相比，在后肢观察后肢模型1,8股骨动脉结扎后的缺氧- 10。我们还表明，的spinotrapezius结扎模型产生不同的重构在Balb / c小鼠中的响应相比，C57BL / 6小鼠，Balb / c小鼠和C57BL / 6重塑通过扩大现有的小动脉连接3通过毛细管侧支重塑网络。这些意见和后肢结扎模型之间有一个有趣的平行，其中BALB / c小鼠经历长时间的灌注恢复结扎后相比，C57BL / 6小鼠9,11,12 &lt;/ SUP&gt;。在组织缺血，鼠标年龄13性别14和存在的疾病15其他型号的，也会影响组织的小动脉闭塞，虽然我们还没有测试这些预测在spinotrapezius结扎模型。 其次，动脉结扎在spinotrapezius模型的大小基本上是小于股动脉，因此，结扎影响较小的组织体积，是进一步的下游， 即在较低的水平循环系统树，在spinotrapezius相比的后肢3。因此，结扎的位置，在这两个模型中的解剖差异时，应考虑本干预比较各自的生理反应（ 例如血管重构）。 第三，可准许氧气到达unperfused的部分的组织从邻近的薄的spinotrapezius组织相比，在后肢的模型，如上面指出3合并缺血和缺氧。第四，恢复流的部分结扎下游spinotrapezius是比在后肢结扎更快。结扎模型的spinotrapezius是一个独特的慢性模型，不应该混淆与其他型号的短暂动脉闭塞或缺血再灌注损伤模型。我们认为这是可能的，以适应这种模型，以容纳这些类型的条件，但是，这是本文提出的工作范围之外。最后，我们提醒，反对直接相关的临床观察，在这个模型中，在人类缺血性疾病的临床表现，尤其是在利用年轻，健康的老鼠，由于固有的生理极限，如心脏率5。然而，我们认为这种新的模式作为一个有价值的工具发现的组织反应的基本机制连接和识别潜在的在体内设置rapeutic目标。 除了设置程序（解剖附注（6a）的，区分从静脉动脉（9a）中，并使用一个单一的绑带（11a）的）在spinotrapezius饲料动脉结扎手术票据，其他几个方面的程序受益于的讨论。 首先，边框（或尾到肩胛骨的几mm，如果透皮可视化是不可能的）在背侧脂肪垫的初始切口理想做得尽可能小为外科医生是舒服的，在操作过程中如果需要的话和扩大。这种做法最大限度地减少所需的缝合关闭切口，这是方便的外科医生，降低手术时间，并且也是在恢复过程中的动物的刺激性较小。另外，一个较大的皮肤切口可能是适当的，根据外科医生的偏好，由于肌肉具有扩展字段的定位可以更容易。在这种情况下，切口可受益于一个马蹄形状，内侧开脸，对脊柱进行折叠。同时使皮肤切口和延伸通过二级皮肤层，如果表面容器损坏，并导致出血，用无菌纱布和施加轻的压力，提供时间止血。 另外值得注意的，这是非常重要的处理与所需的最低要求的力的组织，并尽可能形体用钳子接近的肌肉的外侧边缘，并远离预期的缺血区。这种做法可以帮助避免挤压伤，这可能会导致额外的炎症介导的组织的反应，可能会混淆动脉结扎诱发的血管重塑响应。 总之，我们已经展示了作为模型研究血管和组织反应小动脉结扎​​手术的的小鼠spinotrapezius肌。这种模式是适合活体评估血管的变化（例如，例如，功能性血管舒张），以及用于体外血管变化的评估（例如，免疫荧光成像和定量血管网络）。这两种预-临床和临床研究已经证明，一个人的响应小动脉梗阻（ 例如，通过结扎在小鼠或动脉粥样硬化斑块在人类中）是依赖于他们的年龄的存在或不存在的疾病（ 如糖尿病）的阻塞动脉的直径，个人的基因构成和代谢需求的组织11-15。小鼠模型中，如这里介绍的，利用应变特定解剖和遗传差异和疾病特异性的表型，这有利于这些复杂的关系调查研究者。

调查缺血性疾病的病理生理过程，如外周动脉疾病，冠状动脉疾病，脑血管疾病，慢性缺血的动物模型是有价值的工具。在啮齿类动物，如人类，动脉闭塞导致的血管网结构重构，包括侧支血管和血管生成。在健康和年轻的患者，重塑足以拯救组织缺血损伤，但疾病，如糖尿病，严重危及重塑和恢复。了解血管重塑事件的机制是必不可少的疗法，刺激这些内源性心肌血运重建术过程。 目前，在后肢股动脉结扎或切除是标准的技术研究慢性缺血诱导的血管重塑的小动物4,5。的直径，连通性和反应性分析微血管，使结扎股动脉的血管网的下游，是困难的，但是，由于厚度的肌肉。我们已经开发了一个小动脉结扎模型中的的鼠标spinotrapezius肌肉:单方面的横向输送动脉结扎术，在这种稳定的背部肌肉1。相对的薄spinotrapezius（60-200微米）适合于连接面染色为评估整个网络的拓扑结构与单细胞分辨率，可以在整个组织中血管重塑事件的详细检查。的的spinotrapezius也是肤浅的访问，并因而它的船只很容易观察活体显微镜，高效的表征重塑和小动脉结扎​​血管反应性的影响。 在这份报告中，我们详细描述和展示的的鼠标spinotrapezius小动脉结扎​​模型。无论是在体内和体外我thods评估以下手术进行了描述，包括功能性血管扩张，这已被证明为减值缺血性条件下6，和免疫荧光成像的整个肌肉的微血管网络的测量。我们还包括了两个独立的试验研究为例，证明了该模型的结果。首先，我们利用动脉结扎模型，以诱使统计上的显着增加，血管迂曲，在C57BL / 6小鼠（ 图2B）。曲折的增加在动脉形成的动脉的抵押品。在其他小鼠品系，更容易缺血，由于缺乏抵押品动脉（ 例如，BALB / C），毛细动脉观察10。毛细管动脉被检测到增加的直径和α-平滑肌肌动蛋白的反应性的发展。二，功能性电刺激的肌肉导致终端小动脉的spinotrapezi的血管舒张功能的，（ 图3B）。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>虹膜剪</td><td> FST </td><td> 14090-09 </td><td>类型:工具</td></tr><tr><td>尺寸7钳</td><td> FST </td><td> 11271-30 </td><td>类型:工具</td></tr><tr><td>尺寸5镊子</td><td> FST </td><td> 11251-20 </td><td>类型:工具</td></tr><tr><td>春风似剪刀</td><td> Roboz </td><td> RS5671 </td><td>类型:工具</td></tr><tr><td>探针</td><td> FST </td><td> 10140-03 </td><td>类型:工具 与可被取代的直链的探针</td></tr><tr><td>持针器</td><td> FST </td><td> 12500-12 </td><td>类型:工具</td></tr><tr><td>感应室</td><td>区JD医疗。株式会社</td><td> IC-1086 </td><td>类型:设备</td></tr><tr><td>眼部啫喱</td><td> Dechra </td><td> NDC 17033-211-38 </td><td>类型:试剂</td></tr><tr><td>散热垫</td><td> FST </td><td> 21060-01 </td><td>类型:设备</td></tr><tr><td>直肠温度探头</td><td> FST </td><td> 21060-01 </td><td>类型:设备</td></tr><tr><td>刺激电极</td><td> FHC </td><td> UEWSGCSE0N1M </td><td>类型:设备</td></tr><tr><td> Artisan的软陶</td><td>的Polyform </td><td> N / A </td><td>类型:设备</td></tr><tr><td> PowerLab数据采集系统</td><td>的ADInstruments </td><td> ML 845 </td><td>类型:设备</td></tr><tr><td>刺激隔离器</td><td>的ADInstruments </td><td> FE 180 </td><td>类型:设备</td></tr><tr><td> LabChart </td><td>的ADInstruments </td><td> ML S060 / 7 </td><td>类型:软件</td></tr&gt; <tr><td>反射光荧光显微镜</td><td>奥林巴斯</td><td> BFXM </td><td>类型:设备</td></tr><tr><td> MW荧光右旋糖酐</td><td>西格玛</td><td> FD250S-100MG </td><td>类型:试剂</td></tr><tr><td>视频卡钳</td><td>科罗拉多视频</td><td> 308 </td><td>类型:设备</td></tr><tr><td>自动血管分析（AVA） </td><td> Microvision公司医疗</td><td></td><td>类型:软件</td></tr><tr><td>反αSMA共轭荧光基团</td><td>西格玛</td><td> 1A4-CY3 </td><td>类型:试剂 克隆，1:100 </td></tr><tr><td>荧光显微镜</td><td>奥林巴斯</td><td> BFXM </td><td>类型:设备</td></tr><tr><td>高分子量荧光葡聚糖</td><td>西格玛</td><td> FD250S-100MG </td><td>类型:试剂</td></tr></tbody></table>

murine spinotrapezius model to assess the impact of arteriolar ligation on microvascular function and remodeling

的的小鼠spinotrapezius是薄的，肤浅的骨骼支撑肌肉，从T3到L4，是很容易通过背部皮肤切口。其独特的的解剖spinotrapezius有用的调查缺血性损伤和随后的血管重塑。在这里，我们展示了一个在我们的研究团队，是由先前公布的1-3的的小鼠spinotrapezius肌肉的小动脉结扎模型。对于某些脆弱的小鼠品系，如BALB / c小鼠，结扎手术可靠地创建骨骼肌缺血，作为一个平台，让调查疗法，刺激血管重建术。还证明，评估方法包括使用活体和共聚焦显微镜。非常适合spinotrapezius由于其可访问性（脊髓背解剖）和相对薄（60-200微米）等影像学检查。 spinotrapezius肌肉可以安装连接面，促进成像的无组织学切片的整个肌肉微血管网络。我们描述了使用的活体显微镜获取以下的功能性的血管舒张过程的度量，具体地，作为肌肉收缩的结果的增加在arterilar直径。我们还证明了收获和固定组织的程序，一个必要的前体，以免疫组化研究和激光共聚焦显微镜的使用。

在图1中所示的外科手术的主要动脉，供给spinotrapezius肌肉进行结扎视图，表示感兴趣的区域的标签。收获和免疫染色的区域的肌肉直接位于下游后结扎动脉1周从结扎在图2A中所示的一个例子的共聚焦图像。结扎后经扩大抵押品的动脉平滑肌α-肌动蛋白表达平滑肌细胞（红色）显示特性曲折。容器迂曲，报告作为容器的路径长度和线的距离（即，在相同的容器路径端点延伸的线）之间的比率，在连接后的肌肉（右）已被增强相到unligated对侧肌肉（ 图2B，每组8只） 图3示出的导频终端小动脉功能血管舒张的研究，在该研究中，使用活体显微镜测量的结果。正如预期的那样，小动脉二<html>直径显着增加后，电刺激引起的肌肉收缩。 <img src="/files/ftp_upload/50218/50218fig1.jpg" alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50218/50218fig1highres.jpg"/> 图1。外科领域的反射spinotrapezius肌肉和饲料动脉的饲料动脉结扎由A0表示在两个位置。这些连字之间，阻塞后的血流量，动脉横断在现场由A表示的目标段的动脉转换从腹侧脂肪垫，由C概述，到spinotrapezius肌肉，由D所概述，表示在网页与流量方向指示箭头B.由虚线箭头E. ghres.jpg"/&gt; 图2。共聚焦图像的spinotrapezius肌肉微血管网络的曲折的试验研究。（A）免疫组化染色收获和整个安装的肌肉平滑肌α-肌动蛋白和共聚焦显微镜成像使用。的视场示出的区域的肌肉直接位于下游的小动脉的结扎1周后结扎。弯曲的血管是明显在肌肉由于结扎。（B）的试验研究结果展示显著增加（P = 0.035; 1尾学生的t-试验）迂曲展出由微血管的结扎肌肉作为测定容器的路径长度，以线的距离比C57BL / 6小鼠的试验组（n = 8）到对侧的控制。 <img src="/files/ftp_upload/50218/50218fig3.jpg" alt="图3" fo:content-width="4.5in" fo:src="/files/ftp_upload/50218/50218fig3highres.jpg"/> Figu再3。活体显微成像功能spinotrapezius小动脉血管扩张， 体内 （A）（B）代表的显微照片（左）在休息spinotrapezius终端动脉，并立即停止8 Hz的肌肉收缩（右）。终端小动脉直径在休息（ 8.5±0.5微米，左）和紧随其后的（11±1微米，中间）90秒的肌肉收缩，这显着增加动脉直径（p值= 0.032; 1尾成对t-检验），在Balb / C小鼠（例6）。变动的百分比表示（右）。

Watch this Scientific Journal Video about Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling at JoVE.com

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

我们展示了一种新的动脉导管结扎术模型在小鼠spinotrapezius的肌肉，包括一步一步的程序和所需的仪器的描述。我们描述了外科手术和相关结果的测量，使用活体和共聚焦显微镜的血管网重构和功能的血管扩张有关。

小鼠Spinotrapezius模型来评估的影响小动脉结扎​​术，微血管功能与重塑

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

murine-spinotrapezius-model-to-assess-impact-arteriolar-ligation-on

Nanyang Technological University

Research

JoVE Journal

Medicine

12.1K 次观看.  University of Virginia. 我们展示了一种新的动脉导管结扎术模型在小鼠spinotrapezius的肌肉，包括一步一步的程序和所需的仪器的描述。我们描述了外科手术和相关结果的测量，使用活体和共聚焦显微镜的血管网重构和功能的血管扩张有关。

视频: Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

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

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 ...

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 ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

股动脉线损伤的小鼠模型的血管周围给药补丁植入

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

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 ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

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 ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

小鼠心肌梗死和心肌缺血再灌注损伤的诱导

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

ApoE小鼠后肢缺血的改进手术模型-/- 使用微型切口

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...

小鼠Spinotrapezius模型来评估的影响小动脉结扎术，微血管功能与重塑

摘要

探索更多视频

此视频中的章节

结扎冠状动脉内注射在小鼠模型梗死

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

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

股动脉线损伤的小鼠模型的血管周围给药补丁植入

对急性和亚急性鼠下肢缺血的方法

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

评估下肢缺血的Murine模型的治疗血管生成

使用左前部降冠动脉的永久结扎的月心肌梗死模型

小鼠心肌梗死和心肌缺血再灌注损伤的诱导

ApoE小鼠后肢缺血的改进手术模型^-/- 使用微型切口

小鼠Spinotrapezius模型来评估的影响小动脉结扎​​术，微血管功能与重塑

摘要

探索更多视频

此视频中的章节

结扎冠状动脉内注射在小鼠模型梗死

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

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

股动脉线损伤的小鼠模型的血管周围给药补丁植入

对急性和亚急性鼠下肢缺血的方法

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

评估下肢缺血的Murine模型的治疗血管生成

使用左前部降冠动脉的永久结扎的月心肌梗死模型

小鼠心肌梗死和心肌缺血再灌注损伤的诱导

ApoE小鼠后肢缺血的改进手术模型-/- 使用微型切口

小鼠Spinotrapezius模型来评估的影响小动脉结扎术，微血管功能与重塑

ApoE小鼠后肢缺血的改进手术模型^-/- 使用微型切口