支持这项工作是由美国心脏协会的博士前奖学金788808-41910，NIH-NINDS NS-066001和NS-055832，该中心的听力研究NIH训练1T32DC010775，-01。

1。入门:需手术器械参见图4 <ol><li>牙钻（卡瓦牙科设备，型号:UMXL-TM），2位钻头，钻和3位</li><li> 〜30号注射针头</li><li>镊子，弯尖锯齿可选（可以是有益的，但​​不是必需的） </li><li>两个精尖镊子</li><li>钢丝钳</li><li>缝合线</li><li>微型剪刀</li></ol> 2。创建外科窗口<ol><li>麻醉:程序是在遵守与美国国立卫生研究院的指导方针，并已通过加州大学欧文分校的动物护理和使用委员会。实验对象是295-400克雄性SD大鼠（查尔斯河实验室，威尔明顿，MA，USA），应使用以下的麻醉过程: <ol><li>大鼠腹腔注射用戊巴比妥钠静脉推注（55毫克/公斤体重），其次是肌注阿托品（0.05毫克/千克体重）在H移民局腿，施用3.0毫升的5％葡萄糖水皮下注射。 </li><li>补编根据需要注射戊巴比妥钠（27.5毫克/公斤体重）。管理眼科抗生素眼膏保护角膜的眼睛在下列程序。辖5％葡萄糖（3毫升），阿托品（0.05毫克/千克体重），每六小时减少呼吸道分泌物，在麻醉过程中。通过直肠探头测量体温，保持体温在37℃，通过自我调节的热毯。 </li></ol></li><li>找到马华要么: <ol class="lalpha"><li>减薄2毫米×2毫米成像/可视化窗口使用的大小HP 3钻头在躯体感觉皮层，直到颅骨几乎是透明的，然后变薄充分的透明度，使用大小HP钻头。看到，MCA的位置然后可以通过这个窗口，其近端用于近似的初始段的位置的轨迹。马华将一般斜对面在吻端至尾/腹侧到背侧方向（例如，左/右底部顶部查看时从医生的角度来看左半球）窗口。然后，可以在上面创建的手术窗口观察者估计位于M1段（近端皮质分支）的基础上通过第一窗口可见的远端分支。为了尽量减少头骨的量被去除，以获得MCA，成像/可视化窗口应当位于接近，但是从外科手术窗分开。 或</li><li>一个小的手术窗口，应放置约3毫米的前部和1毫米卵圆孔或下颌神经外侧，靠近到拱主席台30,33,34。为了有效地访问的MCA干（也被称为M1段），颞肌暂时反射相差从颅骨表面。 （注:在长期生存手术的情况下，我们实验室的经验一直THA吨允许颞肌保持附着在其锚定，肌肉会重新退火的颅骨表面，使健康的饮食习惯和有效地维持体重。 </li></ol></li><li>按照民政部延髓腹角的成像窗口（如果使用此作为参考），以估算其初始皮质支所在。 </li><li>稍微薄头骨创建一个新的区域（请参阅到这个手术视窗）延髓腹侧成像窗口（如果使用此作为参考），其中M1段（预皮质分支）马华应该。重要注意事项:如果使用此作为参考）和手术窗口，留出约2毫米的间隙之间的影像窗口（。 </li><li>找到干（也被称为M1段）的MCA前皮质的动脉分支，如在图1A和1B所示。 </li><li>使用HP-3钻头大小，薄上面的头骨估计M1段位置。当头骨变得有些透明，切换到HP-2钻头和薄的头骨尺寸更加细腻，直到它是完全透明的。目视确认手术窗口区域变得足够薄，以查看脉管系统，并评估M1的位置，在这一点上，完成的窗口，使得有2-3毫米的任一侧上的长度M1段（这允许插入的空间和退出的缝合针的任一侧上的MCA）。 </li></ol> 重要注意事项:停止变 ​​薄颅骨的厚度时，是类似于保鲜膜。容器会破裂，如果钻颅骨和硬脑膜突破。如果头骨是不足够薄，另一方面，将取出闭塞是困难的，并可能导致损坏皮质或动脉。 <ol start="7"><li>以30号（30 G）的皮下注射针和弯针的尖端，使用锯齿形镊子。 </li><li>使用30 G针头刺穿颅骨护理完全动脉的正上方的区域中。用这种穿刺孔镊子夹住头骨和小心地取出手术窗口减薄。 </li><li>采取新的30 G的需要，其尖端弯曲的步骤6，并小心地取出硬脑膜。 </li></ol> 注:切割硬脑膜将导致它向后剥离和MCA将更加突出，作为减压的结果。 3。阻断MCA <ol><li>使用钢丝钳修剪半曲线反向切割缝合针（圆3/8，16毫米缝合针）下降到约3-5毫米。 </li><li>线程修剪缝合针图4E中的图片所示。注意事项:重要的是，针，使缝合线两端的等效长度，螺纹。这使拉动两个线程根据M1在同一时间结束时，针可以被自由切割，留下两个长度的螺纹配合的两个结周围MCA。 </li><li>使用锯齿形镊子M1下滑的缝合针。插入从MCA约0.5-1毫米的距离，保持尽可能浅，以尽量减少皮质受损，但避免太多的压力马华。 </li><li>当缝合针的另一侧，例如，它是根据MCA出来，使用细尖镊子（如下所示），同时继续喂饱或推缝合线的另一端​​，从相反侧的缝合针的前端拉针锯齿尖镊子。 </li><li>一旦缝合针被完全通过下MCA被拉出，继续上拉的缝合针或线程，直到线的长度等于任一侧上的MCA。向下按压的线程，因为它是美联储通过马华，最小的应力，可以有助于防止该线程会传递下动脉破裂。 </li><li>剪断线接近的缝合针。 </li><li>同时使用细点镊子解开两个产生的缝合线如此，有两个独立的线程串成根据MCA没有接触。理想的线程会相距1毫米左右，他们通过在马华。 </li><li>同时使用细点镊子，以配合两个独立的结（两连字）与马华试图保持横断空间〜1毫米的空间之间的疙瘩，让周围的线程。 </li></ol> 注意:如果假内部控制需要，准备闭塞离开闭塞结的松动，使他们不收缩马华和收集数据之前，拧紧结，切割船只。剪线，以防止它在任何醒目闭塞前，但留下足够的线程允许收紧疙瘩后。这种方式，任何基线成像或数据收集可以进行所有相同的外科侵袭为闭塞和紧固在适当的时间点上的结，与小的延迟。 <ol start="9"><li>一旦疙瘩连接拽紧，使用微型剪刀在两个节点之间的断面M1。 </li><li>在的情况下，长期生存的研究: <ol class="lalpha"><li>缝合到位无菌手术线切开头皮皮瓣或确保组织使用无菌伤口剪辑。 </li><li>使用抗生素局部伤口区域（如杆菌肽软膏）和系统的预防性注射氨苄青霉素（150毫克/千克IM）。 </li><li>虽然主体仍然是麻醉管理的眼科抗生素软膏的眼睛。 </li><li>管理补充阿托品（0.05毫克/千克IM），以减少呼吸道分泌物，在麻醉过程中。 </li><li>在手术结束，并再次次日早晨（约12小时后）疼痛控制注入皮下氟尼辛葡甲胺（1.1毫克/千克）。 </li><li>将动物干燥，温暖的，倾斜的表面，如动物的鼻子是高于它的尾巴上的倾斜（这有利于呼吸，直到动物是清醒的）。/ LI&gt; <li>监测动物，直到它是清醒和自身的安全。 </li><li>的vivarium一旦动物，动物的活动，外观，发声，喂食和饮水行为应每日监测。 </li></ol></li></ol> 4。 "安乐死" <ol><li>每个实验结束时，大鼠用戊巴比妥钠（2-3毫升，腹膜内）安乐死。 </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ministrokes+in+rat+barrel+cortex.">Ministrokes in rat barrel cortex.</a> Stroke. 26, 1459-1462 (1995).</li><li>Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J., Pulsinelli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+brain+ischemia+in+the+rat:+methods+for+reproducible+neocortical+infarction+using+tandem+occlusion+of+the+distal+middle+cerebral+and+ipsilateral+common+carotid+arteries.">Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries.</a> J. Cereb. Blood Flow Metab. 8, 474-485 (1988).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J. Cereb. Blood Flow Metab. 1, 53-60 (1981).</li><li>Dittmar, M., Spruss, T., Schuierer, G., Horn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+carotid+artery+territory+ischemia+impairs+outcome+in+the+endovascular+filament+model+of+middle+cerebral+artery+occlusion+in+rats.">External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats.</a> Stroke. 34, 2252-2257 (2003).</li><li>Bederson, J. B., Germano, I. M., Guarino, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+blood+flow+and+cerebral+perfusion+pressure+in+a+new+noncraniotomy+model+of+subarachnoid+hemorrhage+in+the+rat.">Cortical blood flow and cerebral perfusion pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat.</a> Stroke. 26, 1086-1091 (1995).</li><li>Kuge, Y., Minematsu, K., Yamaguchi, T., Miyake, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nylon+monofilament+for+intraluminal+middle+cerebral+artery+occlusion+in+rats.">Nylon monofilament for intraluminal middle cerebral artery occlusion in rats.</a> Stroke. 26, 1655-1657 (1995).</li><li>Laing, R. J., Jakubowski, J., Laing, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+without+craniectomy+in+rats.+Which+method+works+best?.">Middle cerebral artery occlusion without craniectomy in rats. Which method works best?.</a> Stroke. 24, 294-297 (1993).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20, 84-91 (1989).</li><li>Schmid-Elsaesser, R., Zausinger, S., Hungerhuber, E., Baethmann, A., Reulen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+reevaluation+of+the+intraluminal+thread+model+of+focal+cerebral+ischemia:+evidence+of+inadvertent+premature+reperfusion+and+subarachnoid+hemorrhage+in+rats+by+laser-Doppler+flowmetry.">A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry.</a> Stroke. 29, 2162-2170 (1998).</li></ol>

作者此时没有透露。

此协议是为了诱导缺血啮齿动物皮质内，这样做实验对象用最少的外围影响。双闭塞和横断法允许的视觉确认该船只已被永久封闭，并没有过多的入侵或组织损伤，可以进行，成活率高。此闭塞协议可能被应用到任何皮质船只可通过开颅手术，以促使在一个特定的域皮质缺血。此外，这样的闭塞时，可以执行一个动物是在立体定位仪上，允许同时使用各种研究的技术，如功能性成像或电生理记录?...

为了有效地模仿人类缺血性中风的诱发缺血性条件，广泛采用的几种动物卒中模型，不同卷梗死。在光化学模型中，脑是通过完整的头骨照射的光敏物质（如孟加拉玫瑰）静脉注射后，用激光照射导致光化学凝血的照射血管堵塞，缺血周围组织6内， 7。 photothrombosis可以导致在非常小的，孤立的梗死区域，通常被用作一种手段，建模"小冲程"，或"微招。 更广泛采用的技术诱导缺血性中风，尤其是在大脑中动脉（MCA），所述管腔内，灯丝手术引入颈外动脉的前端前进，直到阻塞MCA的基础上的单丝模型8。 PRI的线栓闭塞玛丽的挑战是高死亡率（70％MCA闭塞时3小时，有关中风研究的时间点）9。其他问题的方法包括可能的蛛网膜下腔出血，不完全闭塞，可变梗死体积10,11。这种模式的结果梗死的广泛程度在皮层和皮层下12，和一个巨大的人中风的模型。 虽然微观和严重的中风模型是重要的，但人类中风通常是介于两者之间。在大型临床研究，中风梗塞范围的大小从28-80厘米3，转换为4.5-14％，局部缺血性同侧半球9。相比较而言，我们的大鼠PMCAO梗死面积范围从约9-35毫米3，构成3〜12％的同侧局部缺血性大脑半球。我们的PMCAO模型，因此，类似于人类缺血性中风梗塞体积百分比脑音量。 除了造型结构损伤人的条件相似的功能和行为缺陷的的中风，PMCAO结果。至少，中风导致运动障碍，对侧13-15中风损伤，损失或中断的运动和感觉功能16,17，诱发神经元的活动16,18损失或中断，脑血流量减少19的有效模式， 20，梗死21,22。因此，我们PMCAO的模型严重的MCA闭塞导致身体残疾，丧失功能在感觉皮层（及邻近皮层），神经元活动的中断，严重的大脑中动脉的血流量减少，梗死标志属性缺血性中风23 -25，因此作为人类中风的有效模式。 在程序上，PMCAO涉及小的开颅手术中，我们小心地取出颅骨和硬脑膜2×2毫米的"外科手术式窗口"，只是在初始段（M1）马华前马华前部和后部皮质分支（ 图1A和1B）的主要分歧。我们通过半曲线反向切割缝合针和线（6-0丝绸），通过软脑膜层的脑膜，低于MCA和上述皮层的表面上（见表必要的外科用品的特定的试剂和设备进行PMCAO ）。我们再配合双重结扎，收紧马华两节左右，横切两节之间的船只。发生的双重结扎横断通过M1恰好远侧豆纹支化的，例如，皮质分支MCA的影响，从而发生皮质梗塞（无皮质下损伤）26,27（ 图2）。虽然人类卒中往往涉及到皮质下梗死，造型在啮齿类动物中，需要增加的侵袭（阻塞脑血管皮质branchi之前吴先生需要访问通过在脖子上的颈动脉的动脉，必须在技术和梗死面积的变化增加额外闭塞）。这里所描述的模型，不能执行更近侧的早期分支MCA 通过一个简单的开颅手术是不可能的。虽然它可能是手术可能诱发皮层下通过PMCAO梗死，闭塞将需要一个非常侵入性的程序，因此并不理想。 闭塞效果，可以确认通过激光多普勒激光散斑成像12,24,25（ 图3），或组织学宰后（ 图2）。应该指出的是，以往的研究表明，感官刺激可以起到主要作用于心肌梗死的演变和结果赋予保护免受损害时2小时内PMCAO，造成给药后3小时PMCAO上增加中风损伤时24,25,28。我们已经证实，在5小时后PMCAO，刺激不再有效果的结果（未发表资料）。因此，感官刺激的主题应该被最小化后5小时，获得PMCAO梗死体积最小变异。因此，本集团运行"未处理"这种类型的控制，保持麻醉大鼠5小时后PMCAO的，在黑暗中，用最少的感官刺激，并明确表示无晶须的刺激。 还应当指出的是，偶尔在MCA的结构变化，包括过度支化，多个主段，或没有交通动脉可以发生在频率为10〜30％的雄性成年Sprague Dawley大鼠29,30。如果马华异常的观察，最好是不使用特定的主题添加动物如血管异常，会增加心肌梗死的变异。 此外，还有一些实用方面的o你的程序，使这个闭塞的方法，便于中风调查。首先，缝合线可能被放置周围的动脉，但没有拧紧以收集基线评估，然后结扎横断后缺血后评估，。以这种方式，有效地控制了手术的准备所需的闭塞，在主体之内。由于主体可能会保持静止或整个闭塞的一个立体框架内，就可以进行各学科的实验评估之前，期间和之后，闭塞而移动主题或干扰任何实验设备在使用25,28。此外，这一过程的结果在一个非常低的死亡率，即使在老化啮齿动物受试者21-24个月的婴儿（相当于老年人类）31，并且因此可以用于评估的影响，中风的治疗方法更精确地模拟最常见的中风患者的年龄段25,28。船只transectio的N还提供一些实用的目的。横断后出血的情况下确认该船只都结扎站点完全闭塞。此外，横断确保一个永久的血流中断。最后，切断确保闭塞血管的前端部的任何检测到的血流量必须来自备用源。 最后，虽然具体描述此技术MCA闭塞在这个手稿和视频，相同的双重结扎横断技术可能适用于任何可通过开颅脑血管。例如，我们的实验室，利用PMCAO配合一些额外的永久闭塞远端MCA分支，以阻止小学和侧支血流24的方式，类似的技术设计选择性诱导缺血内的初级躯体感觉皮层32。 总之，T他的方法适用于马华永久闭塞密切模拟人类缺血性中风的三个主要方面:最常见的位置（MCA），类型（缺血），并与人类中风的临床文献相关程度的损伤（心肌梗死）。此外，闭塞该方法可被应用到整个大脑的单个或多个闭塞站点，也可以进行具有高的存活率老年受试者。鉴于动态的，永久性的，相对无创性闭塞，这种技术代表评价新方法，为保护和治疗中风的临床前研究人员一个额外的工具。

<table border="1">
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Extra Fine Graefe Forceps - 0.5 mm Tips Slight Curve (1)</td>
 <td>Fine Science Tools </td>
 <td>11151-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Ceramic Coated Dumont #5 Forceps (2)</td>
 <td>Fine Science Tools </td>
 <td>11252-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Extra Fine Bonn Scissors, straight (1)</td>
 <td>Fine Science Tools </td>
 <td>14084-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Round 3/8 (16 mm) Suture Needles</td>
 <td>Fine Science Tools</td>
 <td>12050-02</td>
 <td></td>
 </tr>
 <tr>
 <td>6-0 Braided Silk Suture</td>
 <td>Fine Science Tools</td>
 <td>NC9071061 
 Harvard Apparatus 
 No.:510461</td>
 <td></td>
 </tr>
 <tr>
 <td>30 gauge needle, &frac12;" length</td>
 <td>Fine Science Tools</td>
 <td>NC9867376 
 
 No.:ZT-5-030-5-L/COL</td>
 <td></td>
 </tr>
</table>

permanent cerebral vessel occlusion via double ligature and transection

中风是导致死亡，残疾和全球社会经济损失。大多数中风导致血流中断（缺血）1。大脑中动脉（MCA）提供了一个伟大的大部分血液皮层2的侧面，是人类中风的最常见的部位，并在其领土内的缺血会导致广泛的功能障碍或死亡1,4,5。缺血性中风的幸存者经常遭受损失或中断的机动能力，感觉障碍，心肌梗死。在努力捕捉到这些中风的关键特性，从而制定有效的治疗方法，一个很大的重点是放在MCA缺血动物模型。 在这里，我们提出了一个永久阻断皮质表面血管的方法。我们将这种方法使用一个例子，相关的血管闭塞，模拟最常见的类型，位置和OUTCOM辰人力中风，永久性大脑中动脉闭塞（PMCAO）。在这个模型中，我们手术暴露马华在成年大鼠，通过双重结扎和切断船只随后闭塞。这PMCAO阻止皮质支近端马华MCA皮质境内，大部分的皮质，造成缺血。 ，闭塞此方法也可用于皮质血管闭塞更远侧的部分，以实现局灶性脑缺血，针对一个较小的皮层区域。的PMCAO的主要缺点是，有些侵入性外科手术作为一个小的开颅手术需要访问MCA，虽然这样的结果在最小的组织损伤。然而，这种模式的主要优点是:定义，血流量减少的程度是一致的，功能和神经功能缺损发生迅速闭塞部位，梗死面积是一致的，并且可以长期生存率高的长期慢性评估。

可以确认使用的激光散斑成像（LSI），其中包括血流成像技术成功闭塞的船只。 MCA主要的皮质分支的血流量下降到〜25％的基准以下的闭塞，根据在记录系统中的噪声和灵敏度的技术水平。见如图3段的皮质支马华的代表LSI形象前后的大脑中动脉闭塞。当所描述的闭塞技术应用到MCA M1段，阻止所有皮质马华分支机构，并防止〜5小时以下闭塞的感官刺激，结果是28.4±2.4毫米?...

Watch this Scientific Journal Video about Permanent Cerebral Vessel Occlusion via Double Ligature and Transection at JoVE.com

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

我们描述了一个高度可重复性的方法永久啮齿类动物的主要脑血管闭塞。这种技术可以完成与周损伤非常少，最小的失血，长期生存的一种高速率，和一致的梗死体积与人类的临床人口。

永久性脑血管闭塞<em&gt;通过</em&gt;双人结扎横断

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

permanent-cerebral-vessel-occlusion-via-double-ligature

Research

JoVE Journal

Medicine

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

14.8K 次观看.  University of California, Irvine. 我们描述了一个高度可重复性的方法永久啮齿类动物的主要脑血管闭塞。这种技术可以完成与周损伤非常少，最小的失血，长期生存的一种高速率，和一致的梗死体积与人类的临床人口。

视频: Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

大脑中动脉闭塞的小鼠模型

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

在小鼠永久性大脑中动脉结扎术中的应用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压:全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.