Funding was provided by the NIH K08 NS064094 and UCSF REAC grants. The authors would like to acknowledge Nikita Derguin, Zinalda Vexler, and Joel Faustino for their assistance in the development of this technique.

所有的动物研究得到批准由美国加州旧金山委员会大学动物研究，并按照指南实验动物的护理和使用 （卫生和人类服务，公开号85-23，1985年的美国能源部进行）。动物是由加州大学旧金山分校机构动物护理和使用委员会（IACUC），通过AAALAC认证的兽医密切监测。得到一个雌性Sprague-Dawley大鼠用8天龄的垫料（每窝10只幼仔）。母亲和她的幼崽被给予食物和水自由采食 ，并装在一个温度和光控动物护理设施，每天充实，每IACUC协议，直到幼崽为10天。在此过程中使用的所有外科手术器械高压灭菌，确保无菌。仪器提示的不育是保持整个手术。 1.大脑中动脉闭塞<ol><li>称量小狗和ensurË它是适当的重量（19-21克）。麻醉在100％O 2 3％异氟醚的小狗，并确保有一个脚捏没有反应。维持35.5℃和37℃之间的身体表面温度与手术阶段下使用加热垫。 </li><li>确保动物在跨越肩区域带的仰卧位置。使用无菌棉签，擦拭用聚维酮碘溶液，然后用70％乙醇在双蒸水拭子颈前区域，四个拭子总每种溶液之间交替。 </li><li>局部浸润0.25％布比卡因到计划切口部位。使用立体镜，使一个中线5至7mm颈前切口以暴露颈总动脉（CCA）。将2-4牵引器以保持腔开放和暴露的动脉。 </li><li>定位颈内动脉（ICA），枕动脉（OA），和颈外动脉（ECA）。新郎动脉得到一个明确的说法。是车eful不打扰迷走神经。 注:对于假手术组幼鼠，在inicision保持开放和动脉被曝光，之后，将切口缝合封闭。总的麻醉时间等同于闭塞手术。 </li><li>切6-0丝线编织缝合线为1.5厘米。 Unbraid缝合，拉出单链。确保单链整洁，不磨损。 注:如果有必要，通过浸渍在无菌水单链和修饰与镊子提示链smoothe磨破结束。 </li><li>按住45度镊子的缝合线，将镊子的清扫弧形运动的ICA去下，这样的镊子尖端的ICA和OA之间出现。 注:如果剥离做得好，这一步将是比较容易的。如果ICA和OA是感人的，要小心，不要使用镊子到ICA分离时破裂的动脉。如果发生出血，将压力施加到动脉镊子，直到出血停止。吸收血液用无菌棉签。 </li><li>抓住由镊子举行的缝合链的末端，并把它使得最终很容易访问。释放从钳子的链和从出背钳ICA下，逆转在步骤1.5（ 图1A）的动作。 </li><li>在基地，最近它从CCA分开绑围绕ICA临时结扎。 注意:这是重要的，以配合结，使得将被拉到取出结股线的端部足够长（大于1毫米，小于3mm），以容易地与钳子抓住，同时确保有一个在结退回的另一侧缝合线的合适的量。 </li><li>仔细缩回ICA横向，并使用夹子固定在切口相反侧的腋窝区域附近的股线多余的皮肤。确保该退避链是足够拉紧在进行下一步骤之前停止血液流动，以尽量减少无法控制的出血的风险。观察到动脉是平坦的和苍白。 注意:不要过度收缩，因为它可以导致ICA的部分或全部撕裂。回缩只需通过拉动上相反于夹子的一侧的链使动脉切口之前进行调整。 </li><li>使用45°镊子抓住另一未编织的缝合线和循环其下方和周围的ICA，如在步骤1.5。位置这股横向于回缩链（ 图1B）。 注:此步骤也可以之前回缩完成。如果不确定回缩的质量，这个心结，可以非常宽松的下一步骤前追平。 </li><li>切断捆绑和解开结扎线之间0.2mm的动脉切口中途，犯错靠近并列结扎。 注:血液在并列断动脉剩余可通过动脉切开空，但不得超过5微升。如果出血持续，小心地将回缩链增加回缩，小心的空隙从损伤过大的张力的动脉。 </li><li>用米尺，测量闭塞缝线和切开缝合用2-3毫米的再灌注过程中去除封堵器的额外津贴。握住45度钳封堵器，并使用直钳在适当的长度，以创建一个弯曲达到MCA，标志着一个升迁的停车点。 注意:从硅酮前端到弯曲将10mm遮挡长度用于P10的Sprague Dawley或的Long Evans大鼠幼仔在这个重量范围内。 </li><li>使用45°镊子，喂硅氧烷涂覆尼龙闭塞缝合到动脉切口并推进缝合到标志着预定距离到MCA（ 图1C）的折弯。确保推进手感光滑;如果感觉到阻力立即停止进步。在进步，针对在平行于CC / ECA，朝向头部的方向上的缝合线。 注意:如果缝合先进背部，朝动物的脊椎，它可能会碰到翼腭动脉（PTA）。如果阻力后3-5毫米进步的感觉，缝合一直打到PTA结。回封堵了动脉，直到硅头部动脉切口附近调整前进方向之前。这是没有必要从动脉切口完全删除封堵。 </li><li>通过把一个临时结扎，使用链从步骤1.10（ 图1D）固定封堵器。 </li><li>取下牵引器夹。修剪两个临时结扎的股，使得被拉到去除结结的链很容易与直钳把握和比拉收紧打结链长。 注:链必须足够短，使他们不会在空洞闭合后纠结。 </li><li>取出拉钩，关闭使用6-0编织丝线建立三到四个间断缝合腔。 </li><li>取下小狗麻醉，并将其放置在室内空气的加热垫。监视小狗，直到它已重新获得足够的意识，以保持胸骨斜卧，并确保它已经将其返回到坝前完全恢复。确保小狗维持35.5℃和37℃之间的身体表面温度。 </li><li>在闭塞，异氟烷麻醉下扩散加权磁共振成像（DW-MRI）可以用来验证损伤的适当诱导。 图2演示了tMCAO 9期间由DW-MRI检测正在进行的缺血性损伤。 注:扩散加权自旋回波平面成像再灌注之前进行10-15分钟。全脑进行成像使用以下脉冲序列设置串行2毫米厚的冠状切片:TR / TE = 5000/60毫秒，4个平均值，视场= 35毫米，数据矩阵= 128×128，扩散梯度时间= 20毫秒，分离= 29.7毫秒，幅度=出70mT / m和b因子= 1045秒/毫米2。表现出缺乏皮质参与或非典型缺血性损伤，如在脑干的动物，被排除在外。 </li></ol> 2.再灌注注:闭塞3小时进行造成伤害的，涉及的纹状体和皮质中度至重度量。 <ol><li>在大约2小时和50分钟之后的MCA闭塞，麻醉在100％O 2 3％异氟烷的小狗。维持35.5℃和37℃之间的身体表面温度与手术阶段下使用加热垫。 </li><li>从步骤1.16中取出间断缝合和定位的交界处，这是由两个连字和闭塞缝合线的尾端标记。 注:假手术动物诱导和维持麻醉下等效时间段闭塞的动物。切口再次打开和闭合缝合。对于假手术的动物，继续执行步骤2.11。 </li><li>仔细解开最横向结先前通过拉动长链在步骤1.14并列。 注:如果在解开结电阻，停下来确保结的正确链被拉动。如果阻力仍然存在，增加更好的视野放大倍率。解开的时候，因为有可能在此步骤中损伤动脉谨慎。 </li><li>除去从空腔中的缝合线。 </li><li>在下面的MCA闭塞正好3小时，慢慢走回栓塞缝合了动脉。不会有任何阻力。 注:在大多数情况下，也不会有出血。如果有少量的出血发生时，将压力施加到动脉的动脉切开部位。 </li><li>使用镊子将压力施加到动脉切开术，为下一步恢复再灌注ICA血流。 </li><li>仔细解开使用相同的方法内侧结如在步骤2.3。 </li><li>取下身上缝合线并应用止血剂到动脉切口止血。 </LI&gt; <li>确保出血已停止。 注:该动脉的原始形状和红色的回归证实，ICA血流量已经恢复。 </li><li>取出拉钩，并用6-0编织丝线的三到四个间断缝合关闭腔。 </li><li>从麻醉中取出小狗，并将其放置在室内空气加热垫。监控它，直到它已经恢复了足够的意识，以保持胸骨斜卧，并确保其返回到坝前全面复苏。确保小狗维持35.5℃和37℃之间的身体表面温度。 </li><li>每天检查仔兔5天。 Recprd它们的权重，并仔细检查，将切口部位进行适当的愈合。后7-14天拆除缝线。后tMCAO损伤可以与MRI成像，如果需要的话9。 注:动物可能会失去多达1克重的首日，但通常会恢复的重量而不干预，并处于类似的权衡由天4-5吨范围内的控制。罕见地，小狗可能会失去体重超标或有喂养困难，需要口服灌胃2-3天源。 </li><li>在P21，损伤也能够可靠地与感觉运动行为测试，如转棒或气缸饲养评估。认知测试可以执行早4-6周的使用龄评估诸如新物体识别或Morris水迷宫13。 </li><li>通过Euthasol的腹膜内注射（50毫克/千克）对脑收获11,12安乐死大鼠。 </li><li>组织学分析，以评估损伤体积或响应于干预可以用甲酚紫或H＆E染色（ 图3）10来执行。 </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+ischemic+stroke.">Perinatal ischemic stroke.</a> Stroke. 38, 742-745 (2007).</li></ol>

The authors have nothing to disclose.

在协议中的关键步骤首先，要保持从麻醉开始正常体温，直到完全恢复是非常重要的，因为已知在未成熟和成熟的动物都低温17热疗18脑损伤的进展的效果。其次，在确保动物和回缩切口，最佳定位监测呼吸，以确保气管是免费的压缩是必不可少的。三，避免挤压或拉伸的迷走神经，因为这可能会导致心脏率与迷走神经刺激的变化。第四，由于ICA的回缩是必要的动脉切开术中控制出血，必须注意回缩期间支付给紧张程度，以避免损坏动脉。如果动脉并从回缩撕裂，或者如果有一个贫穷的动脉切开切口，动物应该从分析中排除由于风险出血和再灌注不良。 修改和故障排除使用MRI作为指导，缝线长度可被优化以确保该硅酮尖端正确地闭塞MCA创建局灶性缺血。如果MRI是不可用，幼崽可能会再灌注前解剖以观察缝合的位置安乐死。根据需要调整所述缝合线段。小狗重量闭塞缝线长度要求高度相关。闭塞时间可以被修改以调节损伤严重程度。 此外，缝合线的形状和长度是至关重要的。对于P10的Sprague-Dawley和Long Evans大鼠体重19-21克，10毫米插入在我们的经验的最佳长度。闭塞缝合的进一步插入可能导致MCA的穿孔。此外，在每个手术闭塞灯丝的形状的一致性会导致伤害p的增加的一致性attern 19,20。出于这个原因，我们建议使用专业制造的缝合线为这一特定目的。同样重要的是要注意的是，受伤的图案可以从业者之间由于技术看似微小的差异有所不同。 该技术的局限性表演在一个小本技术，开发啮齿动物需要显著的经验。如果正确执行，外科医生能够使不同规模的动物一个非常一致的损伤模式，达到一个存活率大于95％。此外，适当的手术工具是必不可少的。手术器械必须严格控制，以确保所有的仪器尖端接近正常。 相对于现有的或可供选择的方法这种技术的显着性而缺氧缺血，或稻万努奇模型2 </SUP&gt;，最常用来研究发育中的大脑缺氧缺血性损伤，需要注意的是tMCAO的这种模式是不同于HI中，有局灶性缺血没有全球缺氧，随后再灌注阶段是很重要的当阻塞被移除且血流恢复。这将导致更一致的和可重复的损伤，是临床上多由翻译引起类似于足月新生儿中风看到受伤的图案。这使的局灶性损伤模式和未受伤的组织代偿反应的研究。 掌握这一技术后，未来的应用这个模型类似于行程在人类新生儿的最常见原因，即在围产期11,21发生了瞬态闭塞性血栓。其病因尚不完全清楚，最可能是多因素，但在大多数情况下，假定ŧø从胎盘11穿过栓塞引起。此外，假定围产期中风通常出现后癫痫发作或细微的局灶性神经功能检查许多新生儿异常22。这使得使用一个一致的，平移损伤模型，以确定损伤的进展和可能的治疗策略的关键机制。

在新生儿期中风是死亡和残疾的显著原因，在多达100 2300个活产1的发生。这导致改变中枢神经系统发育，提高长期的发病率，包括增加癫痫的发生，脑瘫，智力低下，以及其他类型的运动或认知功能障碍。早期中风的终身影响使平移动物模型研究在这一人群中损伤修复的机制，包括战略，以保护脑损伤或提高维修是必不可少的。 不同的缺血模型已被用于研究成年动物的脑损伤，并且在2过程通常用来研究在发育中的大脑缺氧缺血性损伤稻万努奇（修改莱文），局灶性脑缺血再灌注损伤的独特的机制引起局灶性损伤，具有损伤的核心和penuMBRA和未受伤的远程组织。小泉3和龙格4模型，在成年大鼠分别开发经颈总动脉（CCA）和颈外动脉（ECA）来实现短暂大脑中动脉闭塞。在这两种模式，永久结扎动脉分支的烧灼是很重要的，尽量减少出血和简化手术过程，这也导致对动物的能力，饲料和发胖损伤后的不良影响。此外，存在于未成熟的脑损伤不同机制和视为结果损伤的特定图案。 最近，光化学中风（玫瑰孟加拉法）5和永久性MCA结扎6已经被用于研究新生儿和成人中风。无论光化学中风和MCA结扎创建脑血流量导致缺乏reperfus的永久性变化离子。再灌注是病灶性损伤的发生和发展的一个重要组成部分，具有增加的兴奋性中毒，自由基形成，和一氧化氮的产生，导致其涉及的信令是从缺血阶段7不同级联延迟细胞死亡。缺氧缺血包括永久性单侧颈总动脉结扎后跟全球缺氧，这也不同于缺氧缺血性损伤的原因在人类和不引起一致的局灶性损伤模式，使受伤的核心和半影更具挑战性的研究。 使用短暂大脑中动脉闭塞（MCAO）8，9，10我们先前已经描述的非出血性缺血再灌注性中风模型中的未成熟的大鼠。这是访问和通过没有永久ligati颈内动脉闭塞MCA较小侵入性方法或烧灼。这提供了损伤的类似行程的在围产期11,12的最常见原因的模型。在同侧纹状体和parieto，颞叶皮层损伤损伤导致的这种缺血再灌注模型。 tMCAO的该模型还允许通过改变闭塞的持续时间而造成的伤害的严重程度的控制。信号通路，并在受伤的核心和半影和未受伤的同侧和对侧组织学变化的检查可以进一步阐明在未成熟脑损伤和修复的机制。这项研究将展示发育中的大脑这个重要的损伤模型。

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transient middle cerebral artery occlusion model of neonatal stroke in p10 rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

造成tMCAO损伤的严重程度在很大程度上取决于闭塞时间和外科医生的经验双方。 A 90分钟闭塞常常产生轻度至中度损伤图案，而3小时产生中度至严重的伤害。损伤的严重性可以通过多种方法，包括MRI，组织学，或短期或长期行为的分析来评估。 图2演示了DW-MRI的一个例子进行75分钟到90分钟闭塞，证实累及同侧半球缺血性损伤。弥散加权成像演示期间急性缺血相同侧纹状体和广大同侧皮层的扩散增加，而无需改变对侧，。这关联到同时涉及皮质和深部灰质长期伤害的中等水平。 马华导致闭塞细胞死亡开始于纹状体不太严重伤害并开发了更长的时间闭塞，更严重的伤害加重皮层和海马损伤。在手术技术的优化，例如确定缝线插入长度，MRI强烈推荐，因为它允许进行适当的缝合线放置和水肿和损伤进展的可视化的确认闭塞14，15，16中。如果MRI是不可用，H＆E或甲酚紫染色是简单和可靠的组织学方法，以确定损伤形态，并可在tMCAO后早期和晚期的时间点被使用。 图3证明以下3小时闭塞上组织病理学检查中度至严重损伤图案，表明在同侧纹状体和皮质囊肿形成和体积。 <p class="jove_content" fo:keep-together.within-page="1"&gt;甲酚紫染色切片来计算的同侧半球对侧体积的比率的无偏体视学定量证实同侧局部缺血 - 再灌注损伤。具体来说，麻醉用Euthasol动物后，大脑被transcardiac灌注在0.1M PBS中收集细胞，用4％多聚甲醛。以下在30％蔗糖过夜后固定和平衡，整个大脑，在50周微米的间隔切片，并选择每第十二部，安装，并用甲酚紫染色10。 即使轻度损伤，运动的变化，如盘旋和偏瘫，在接合期间被指出。随着越来越多的严重伤害，这些变化会再灌注后持续存在。额外行为测试可用于评估损伤的严重程度，包括感觉运动功能转棒或气缸饲养测试和Morris水迷宫认知功能13。 <img alt="图1" src="/files/ftp_upload/54830/54830fig1.jpg" /> 图1:tMCAO 过程的现场手术照片。 （A）第一缝合线围绕ICA环，如在步骤1.6中详述。 （B）第一临时结扎被栓和ICA缩回。第二缝合线围绕ICA环，横向于第一缝合线，如在步骤1.9中详述。 （C）的有机硅涂覆的闭塞缝线被送入动脉切口位点，如在步骤1.12中详述。 （D）第二临时结扎被绑定到固定封堵器在适当位置，如在步骤1.14中详述。比例尺= 1mm左右。 <img alt="图2" src="/files/ftp_upload/54830/54830fig2.jpg" /> 图2:MRI OCCL期间演示延髓的适当的单边损伤。从前至后，在DW-MRI的冠状图像切片，一个90分钟闭塞期间执行，表明涉及同侧半球（箭头），这与在急性期持续缺血性损伤一致增加扩散。从行程11重印许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54830/54830fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54830/54830fig3.jpg" /> 图3: 单侧损伤累及纹状体和Cortex在以下tMCAO 4周。后到前，甲酚紫染色的冠状脑切片（各为50μm）从P38动物中收集证明相当严重损伤（箭头显示同侧囊肿形成和降低的皮层和以下在P10一个3小时tMCAO纹状体体积）。在左侧圆孔表示对侧半球标识符。比例尺= 5毫米。转载与疾病12神经生物学许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54830/54830fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats at JoVE.com

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

transient-middle-cerebral-artery-occlusion-model-neonatal-stroke-p10

Research

JoVE Journal

Medicine

10.5K 次观看.  University of California, San Francisco. Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

视频: Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

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 ...

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 ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

栓塞性脑梗塞（缺血）的大鼠缺血性中风与血同源凝块

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...