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。

<table><tbody><tr><td>Isoflourane</td><td>Henry Schein</td><td>50033</td><td>anesthetic, at 3%&amp;nbsp;</td></tr><tr><td>Trinocular Surgioscope</td><td>World Precision Instruments</td><td>PSMT5N</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>000731-500-000</td><td>low to medium setting</td></tr><tr><td>IR Thermometer</td><td>Extech Instruments</td><td>72-5270</td><td></td></tr><tr><td>Retraction kit for small animals&amp;nbsp;</td><td>Fine Science Tools</td><td>18200-20</td><td></td></tr><tr><td>CermaCut Scissors</td><td>Fine Science Tools</td><td>14958-09</td><td></td></tr><tr><td>Dumont #5SF Forceps</td><td>Fine Science Tools</td><td>112522-00</td><td>2x</td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>2x</td></tr><tr><td>B-2 Micro Clamp</td><td>Fine Science Tools</td><td>00398-02</td><td></td></tr><tr><td>Forcepts for Clamp Application</td><td>Fine Science Tools</td><td>00072-14</td><td></td></tr><tr><td>Micro Vannas Scissors</td><td>Fine Science Tools</td><td>15000-03</td><td>2mm cutting edge</td></tr><tr><td>Occlusion Sutures</td><td>Doccol</td><td>602123PK10</td><td>701712PK5Re</td></tr><tr><td>Ruler</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Hemostatic Agent&amp;nbsp;</td><td>Avitene</td><td>DVL1010590</td><td></td></tr><tr><td>6-0 Perma-Hand Silk Reverse CuttingSuture</td><td>Ethicon</td><td>769G</td><td></td></tr><tr><td>Euthasol</td><td>Virbac</td><td>710101</td><td>0.22 mL/kg</td></tr><tr><td>Cotton Tipped Applicators</td><td>Henry Schein</td><td>100-9249</td><td></td></tr><tr><td>Laboratory Tape</td><td>VWR</td><td>89097-990</td><td></td></tr></tbody></table>

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="http://ecsource.jove.com/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="http://ecsource.jove.com/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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

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 ...

医学

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 ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

电刺激在大鼠中建立急性庞廷梗死

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

模拟小鼠中风:通过颈外动脉的短暂性脑中动脉闭塞

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

建立经颅大脑中动脉闭塞小鼠模型

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

作者聚焦:中风研究的创新技术和未来方向 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

大脑中动脉闭塞的短暂性脑缺血模型

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

脑卒中短暂性中动脉闭塞模型

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

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脑卒中短暂性中动脉闭塞模型