我们感谢夫人卡罗琳·罗伯逊的语音和夫人埃莱娜皮罗瓦诺的视频制作，对她的帮助。这项研究是支持的米兰比可卡大学，"2011年雅典耀丰多二"。

1。如何使探头架（单站点还是多站点） <ol><li>所需的材料是天然橡胶，小塑料管和金属钢丝。探针架座可定制的动物的大小，激光多普勒探针的数量和大小，以及脑血管领土需要以被监视。 </li><li>切所需要的尺寸（约10毫米×10毫米的300克大鼠）的天然橡胶。 </li><li>标记探针的位置（s）和前囟门上的天然橡胶，根据所需的立体定位坐标;大脑中动脉闭塞，缺血核心典型的坐标在囟-1毫米，5毫米横向于预期中线外围设备的缺血性borderzone领土的，预期的坐标可以在囟+2毫米，2毫米正中线旁开。 </li><li>标记很清楚的X前囟门的位置，这是具有里程碑意义的安装探头持有人的头骨。 &lt;/ P&gt; <li>钢丝插入到一个小塑料管（其大小需要的大小相匹配的探针）。 </li><li>钢丝插入到探针具有被定位在点的天然橡胶;钢丝推入橡胶，直到塑料管已被插入到橡胶太。 </li><li>抽出钢丝。 </li><li>如果需要多个探头，重复步骤1.5-1.7其他探头位置。 </li><li>可选的多个探头:包装带，塑料管周围的探头，以确保更好的稳定。 </li><li>可选:进行化学消毒的几个探头支架以备日后使用。 </li></ol> 2。术前准备<ol><li>缺血中风模型通常是作为一种生存手术。在这种情况下，如在我们的视频所示（存活时间24小时后的手术），外科医生用灭菌的仪器及用品使用无菌技术。 </li><li>异氟醚麻醉大鼠（3％的归纳法N相，1.5％的维护）。 </li><li>将大鼠俯卧在手术台上。 </li><li>轻轻地剃了头的影响。 </li><li>应用纱布和消毒液消毒皮肤。 </li><li>管理2％利多卡因5毫克/公斤区皮下，颅。 </li></ol> 3。探测定位和固定在完整的头骨<ol><li>做一个正确的正中皮肤切口（右MCAO）和解剖皮下组织，，以达到颅筋膜， 帽状腱膜 。 </li><li>作出正确的的正中切口在颅筋膜和执行钝性分离，以达到颅骨，准备一个颅骨区域的探头支架的应用是足够大的。 </li><li>请注意:无需钻孔或骨质疏松。 </li><li>应用红药水消毒和干燥的颅骨表面的解决方案。 </li><li>使用吹风机（冷空气），以加速干燥颅骨表面。 </li&gt; <li>在这一点上，颅缝线和囟都清晰可见。 </li><li>自定义探针支架用无菌剪刀切割边缘。 </li><li>施加一个小的探针架座的表面下方的手术胶量（氰基丙烯酸酯，兽医批准），小心地避免的塑料管的下开口（请注意:如果一个显着量的胶之间仍然存在的光学表面的探头和头骨，这可能会产生一个低信号，并在多次使用后，可能会损坏探头）。 </li><li>应用探头持有人的颅骨表面，精心选配的X标志性建筑前囟门。轻轻压在探头支架。 </li><li>使用吹风机（冷空气设定），以加速干燥手术胶水。 </li><li>固定探头支架，通过把探头周围的人的动物头一个手术线;要小心定位的手术线，下颌骨，避免颌下区和日Ë的脖子上。 </li><li>可选的探头支架，填补了塑料管（S）的的光学凝胶（ 如常见的超声心电图凝胶），这将增加LD信号的质量。 </li><li>将探头（s）在探头支架和验证实际的LD流量计的读数。根据制造商的说明使用LD流量计。 </li><li>安全探针（S）捆绑各处的动物的头，要小心，避免颌下区和颈部定位下颌骨的手术线。 </li></ol> 4。在缺血的脑灌注监测<ol><li>将大鼠仰卧的姿势，小心地避免牵拉的力量（S）或上探探头支架。 </li><li>在MCAO脑灌注监测。 </li></ol> 5。拆除探头（S）和探头架<ol><li>切缝线探针（），探头支架的动物头周围。 </li><li>轻轻钝性解剖颅周围的软组织和皮肤的天然橡胶基地的探头支架。 </li><li>拆下探头支架。 </li><li>应用的颅骨表面的消毒液。 </li><li>颅皮肤缝合。 </li></ol> 6。手术后的护理<ol><li>管理2.5毫升的生理盐水皮下，以防止脱水，并停止气体麻醉后，使用加热垫，保持动物的体温。 </li><li>为了生存术:提供超前镇痛与酮洛芬4 mg / kg皮下注射，并在12小时手术后，重复相同剂量</li><li>在我们的实验条件下，安乐死在手术后24小时吸入CO 2。 </li></ol>

<ol>
	<li>Sutherland, B. A., Papadakis, M., Chen, R. L., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+blood+flow+alteration+in+neuroprotection+following+cerebral+ischemia.">Cerebral blood flow alteration in neuroprotection following cerebral ischemia.</a> J. Physiol. 589, 4105-4114 (2011).</li><li>Prinz, V., Endres, M., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Modeling+focal+cerebral+ischemia+in+rodents:+Introduction+and+overview.">Chapter 3 Modeling focal cerebral ischemia in rodents: Introduction and overview.</a> Rodent models of stroke. , (2010).</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><li>Riva, M., Pappad&#224;, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+monitoring+of+intracranial+collateral+flow+predicts+tissue+and+functional+outcome+in+experimental+ischemic+stroke.">Hemodynamic monitoring of intracranial collateral flow predicts tissue and functional outcome in experimental ischemic stroke.</a> Exp. Neurol. 233, 815-820 (2012).</li><li>Menon, B. K., Smith, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+leptomeningeal+score+on+CT+angiography+predicts+clinical+and+imaging+outcomes+in+patients+with+acute+anterior+circulation+occlusions.">Regional leptomeningeal score on CT angiography predicts clinical and imaging outcomes in patients with acute anterior circulation occlusions.</a> Am. J. Neuroradiol. 32, 1640-1645 (2011).</li><li>Bang, O. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+flow+predicts+response+to+endovascular+therapy+for+acute+ischemic+stroke.">Collateral flow predicts response to endovascular therapy for acute ischemic stroke.</a> Stroke. 42, 693-699 (2011).</li><li>Shuaib, A., Butcher, K., Mohammad, A. A., Saqqur, M., Liebeskind, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+blood+vessels+in+acute+ischaemic+stroke:+a+potential+therapeutic+target.">Collateral blood vessels in acute ischaemic stroke: a potential therapeutic target.</a> Lancet Neurol. 10, 909-921 (2011).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Stroke+Model+Guidelines+for+preclinical+stroke+trials+(1st+edition).">Rodent Stroke Model Guidelines for preclinical stroke trials (1st edition).</a> J. Exp. Stroke Transl. Med. 2, 2-27 (2009).</li><li>Shepherd, A. P., &#214;berg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser-Doppler Blood Flowmetry. , (1990).</li><li>Ulu&#231;, K., Miranpuri, A., Kujoth, G. C., Akt&#252;re, E., Ba&#351;kaya, M. K. <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+Ischemia+Model+by+Endovascular+Suture+Occlusion+of+the+Middle+Cerebral+Artery+in+the+Rat.">Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat.</a> J. Vis. Exp. (48), e1978 (2011).</li><li>Garcia, J. H., Wagner, S., Liu, K. F., Hu, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+deficit+and+extent+of+neuronal+necrosis+attributable+to+middle+cerebral+artery+occlusion+in+rats.+Statistical+validation.">Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation.</a> Stroke. 26, 627-634 (1995).</li></ol>

摩尔仪器有限公司（阿克明斯特，英国德文郡），它产生的激光多普勒仪器显示在我们的实验研究提供了这个视频文章的资金用于生产。 米兰比可卡大学动物保护委员会的批准，在使用的实验动物（DL 116/1992）的国家指导方针，按照欧盟指令对动物实验实验方案（2010/63/EU ）下的项目许可证（N. 219/2011-B）从意大利卫生部。

我们内部开发的一个简单的，低成本的系统的安全附件的一个或多个双光纤（深层渗透）LD探头的大鼠在MCAO程序的完整的头骨。虽然获得可靠的连接的LD探测到的头骨显然是一个微不足道的问题，实际上是这个实验中的一个主要问题，因为它是一个平滑的信号检测和监测脑灌注成功的前提。 侵入性操作，如钻孔和骨螺钉，通常延长手术时间，引进更多的实验变量相关的开颅手术，而这可能会阻碍研究人员从使用LD监控的，不要。另一方面，在使用单纤维（低渗透）探针，它是较薄的和相对容易到被粘直接颅骨表面，给出了低质量的信号，可以不被用于可靠地与成年大鼠无需钻或减薄的头骨。 我们使用简单和成本低的材料，如天然橡胶，塑料管和金属钢丝。甲量身定制的探头支架可以在几分钟和生产适于本实验条件下。这些探头支架可以容纳一个或多个深穿透LD探头，传统的单一的现场监控缺血核心或动脉在不同的地区，在同一个半球或跨越两个半球的多个现场监控。可以生产许多探头支架，化学灭菌，并存储，以备将来使用。兽医批准手术的胶水（cyanoacrilate），加速由冷空气，是用来安装探针架座的完整表面的大鼠颅骨，根据所需的颅坐标。最后，探头设置，进一步巩固了在常见的缝合线。 这个LD探针的整体时间设置，掌握这种技术后，约10分钟。 如所示n这个视频中，我们定期监测脑灌注在中央MCA领土（LD头1:缺血性核心）和MCA领土的外围设备（LD探头2:主要是半影区）。在我们最近的研究中，我们发现，在LD中的探针2（​​平均52％±16％的SD，与基准相比）的血流量的变化的可变性是高于LD探头1（平均3​​1％±6％的SD，相比基线）和可用于，预测中风结果4。 我们的研究人员想利用我们在内部开发的系统可能会提供一些​​故障排除建议。在实验开始时，要小心，干的很不错的的颅骨表面（与红药水和冷空气）前安装探头支架，以防止过早支队。此外，可以肯定申请天然橡胶上的胶水，避免接触的塑料管中，并与开口端的LD探针的光学表面，以防止不良的信号和潜在的损坏探头。周围的动物头，追平了缝合时要小心，以避免气道阻塞（这是防止通过定位在下颚骨缝合）。定位和固定的探针后，转弯时不小心牵引探头电缆的动物在仰卧位颈椎手术，这一步通常需要两个人，一个人的动物和第二个人的探头电缆，轻轻地定位到所需的位置。最后，血液污染的双纤维LD探头易于管理的最终制造商所提供的清洁说明。 我们已优化的脑灌注监测系统，在这个视频中，可以提供一个更简单，更快速，更可靠的替代的探头设置系统，这是目前在售的商业公司在这一领域的。此外，我们认为，由其他研究人员使用该系统可以提高螺柱y的脑血流动力学的的试验行程中，导致脑抵押品的新一代疗法的发展。

血流动力学因素影响中风的病理生理和治疗的需要实施的转化研究，重要的问题，因为这往往是矛盾的所忽视的基本研究。 脑灌注的监测是重要的，但未被充分利用的，准确的缺血性中风的建模工具，用于2。除了 ​​动脉血管闭塞的确认和排除蛛网膜下腔出血3，持续脑灌注监测灌注赤字，颅内侧支血管的功能状态和血流动力学的影响的新的治疗方法的程度和一致性，可以提供有用的数据。 本集团最近的一项研究表明，多部位的血流动力学监测可用于评价颅内侧支循环，并能预测梗死面积和功能障碍4。这些实验结果的注意事项，支架的临床研究表明，脑侧支循环的功能性预测的临床结果在缺血性中风患者的5，6。出于这个原因，脑抵押品一直主张在急性期缺血性中风的7作为一个潜在的治疗目标。 激光多普勒（LD）的工具是最常用的工具，用于测量脑灌注缺血性中风的实验，其使用推荐最近关于这一主题的指导方针8。 LD仪器测量微血管灌注在一个小的皮质体积，所记录的信号的深度依赖于纤维分离的宽度，与双纤维LD探针允许更深的渗透相比，单纤维LD探针9。血流量值表示为相对的而不是绝对脑血流量的任意灌注单位（PU）表示。的PU的校准通常是进行全光照克的蠕动标准，根据制造商的说明。 LD血流仪可以连续动态监测和定量数据的生成在同一个会话。 其中的技术问题，目前限制使用的LD，一个主要问题是获得一个安全的和长期的深穿透激光多普勒探头附件的动物头骨。长期监测，这是非常重要的，如果有多个探头用于不同的脑动脉领土，我们执行的是在我们的实验室。 特别是，如果需要探针使用钻孔或颅骨螺钉连接到颅骨延长手术时间，而会发生不良的信号和不安全附件，如果单纤维（低渗透性）LD探针被连接到通过简单的手术胶水颅骨。双床纤维（深穿透）LD探头提供了一个更高，更一致的信号，但它们是大于单一的光纤探头，并不能在tached的头骨仅采用手术胶。 在这段视频中，我们将展示我们已优化的线栓在大鼠大脑中动脉闭塞的脑灌注监测系统。我们描述了一个简单的方法来获得一个高效，产品订制，成本低持有人单个或多个双光纤（深穿透）LD探头，用于长期监测脑灌注较完整的头骨。 瞬态MCAO在大鼠的手术程序中可以看出在视频文章由Uluç和同事10中未示出该播放器。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> MoorVMS-LDF 2通道激光多普勒监测</td><td>穆尔工具</td><td></td><td></td></tr><tr><td> VP12探头</td><td>穆尔工具</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 试剂/材料 </td></tr><tr><td> Doccol涂硅纤度4-0，直径涂层0.39毫米</td><td> Doccol公司</td><td> 403956PK10 </td><td></td></tr><tr><td>天然橡胶， 例如常见的奶嘴新生儿</td><td></td><td></td><td>多种供应商</td></tr><tr><td>金属管芯， 如脊髓针18 GA×90毫米</td><td></td><td></td><td>多供应商</td></tr><tr><td> 例如，从静脉输液25 GA×20毫米的塑料管， </td><td></td><td></td><td>多供应商</td></tr><tr><td>非吸收性缝合，涂布，编织丝</td><td></td><td></td><td>多供应商</td></tr><tr><td>氰基丙烯酸酯手术胶</td><td></td><td></td><td>多供应商</td></tr><tr><td>兽医用异氟醚100％（V / V） </td><td></td><td></td><td>多供应商</td></tr></tbody></table>

optimized system for cerebral perfusion monitoring in the rat stroke model of intraluminal middle cerebral artery occlusion

临床前中风研究的平移潜力取决于实验模拟的准确性。在动物模型中的急性缺血性中风的脑灌注监测，可以确认成功的动脉闭塞和排除蛛网膜下腔出血。脑灌注监测，也可用于研究颅内侧支循环，这正在成为一个强大的中风结果的决定因素和可能的治疗靶点。尽管认可的准则进行实验性脑缺血作用的激光多普勒血流监测，存在一些技术上的困难，限制了其广泛使用。的主要问题之一是获得一个安全的和长期的深穿透激光多普勒探头附件的动物头骨。在这段视频中，我们将展示我们已优化的线栓在大鼠大脑中动脉闭塞的脑灌注监测系统。我们开发了我N-房子一个简单的方法来获得一个定制的双光纤（深穿透）激光多普勒探头，如果需要的话，允许多站点监控的持有人。 A连续和长期的监测脑灌注可以很容易地获得较完整的头骨。

诱导瞬态MCAO（60分钟）通过插入一个聚硅氧烷涂覆的长丝在颈外动脉。然后将长丝通过颈内动脉的末端推压最多的MCA的起源，根据LD的监测。瞬时普通颈动脉和pterygopalatin的动脉闭塞期间灯丝手术插入。的外科手术过程中的示意性表示在图1A所示。 坐标定位LD探头的选择颅根据底层动脉领土。明胶墨汁灌注（ 图1B）的初步实验表明，缺血核心，预计在中央MCA领土（前囟门-1毫米，5毫米，中线探针1），而预计在borderzone领土之间的侧支循环皮层中，前脑动脉分支（前囟2毫米，从中线2毫米;探头2）。 脑血流动力学进行了研究，采用多点激光多普勒探头的整个期间的外科手术， 即之前，期间和之后MCAO（ 图2）。在MCAO脑灌注赤字较小，表现出较高程度的可变性探头2探头1，个体间差异的功能性颅内侧支循环缺血条件下。多点激光多普勒监测也可以研究脑血流动力学的变化闭塞的近端额外的颅内脑动脉（颈总动脉，颈内动脉，动脉pterygopalatin）的过程中。 中风结果评估再灌注24小时后的梗死体积，计算与甲酚紫（ 图3），和加西亚官能neuroscore 11染色的19个连续的部分。免疫组化特异性标志物ASSOciated与缺血性脑损伤的进行，为了获得地形分布的神经元丢失（微管相关蛋白2，MAP2）和缺血半影区（热休克蛋白70，热休克蛋白70）有关的多站点颅内循环血流动力学监测（ 图4）。 <img src="/files/ftp_upload/50214/50214fig1.jpg" alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50214/50214fig1highres.jpg"/> 图1。在管腔内MCAO在大鼠脑灌注的监测。 A.瞬态MCAO手术程序的示意图。使用甲硅氧烷包覆长丝的起源的MCA闭塞后，被引入在外部的颈动脉，并推动通过颈内动脉。近端宫颈动脉结扎（颈外动脉）或瞬时闭塞的（pterygopalatin动脉和颈总动脉）在多项式dure。一位代表大脑后明胶墨汁染色。 transcardiac灌注明胶油墨溶液进行缺血发作，60分钟后，没有再灌注。正常灌注的脑染色明胶墨和色为灰色与黑色染色血管，而出现缺血性（灌注）的区域依然是不染（粉红色）。颅坐标定位两个LD探针被示出。探头1 = 1毫米5毫米中线前囟门，探头2 = +2毫米，2毫米的中线前囟门。 <img src="/files/ftp_upload/50214/50214fig2.jpg" alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50214/50214fig2highres.jpg"/> 图2。使用多站点激光多普勒探头的脑血流动力学记录。一个典型的血流动力学模式，这表明缺血条件下的功能活性的颅内抵押品所示。在这种动物，LD描呈小呃灌注赤字探头2通道，探头1通道，在CCA闭塞及大脑中动脉闭塞。 MCA-O =大脑中动脉闭塞。 CCA-O =常见的颈内动脉闭塞。 PU =灌注单位。 <img src="/files/ftp_upload/50214/50214fig3.jpg" alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50214/50214fig3highres.jpg"/> 图3。代表的脑切片的梗塞灶体积的计算。组织学冠状切片（50μm的19例250μm的间隔;囟2.5毫米-3.0毫米）被固定在4％多聚甲醛和甲酚紫染色的0.1％。梗塞体积计算使用ImageJ图像处理软件，校正因脑水肿的半球间的不对称，并表示在3毫米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50214/50214fig3large.jpg" target="_blank">点击这里查看大图</a> <img srC ="所有/ files/ftp_upload/50214/50214fig4.jpg"ALT ="图4"FO:内容的宽度="6英寸的"佛:SRC ="/ files/ftp_upload/50214/50214fig4highres.jpg"/&gt; 图4。免疫组织化学染色的分子标记的神经元的损失和半影。代表连续脑切片示出，进行染色甲酚紫0.1％（A）或与标记的神经元丢失（微管相关蛋白2，MAP2，B）和缺血半影区（热免疫染色休克蛋白70，热休克蛋白70，C）。

Watch this Scientific Journal Video about Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion at JoVE.com

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

已被证明是改善脑灌注监测缺血性中风模型的准确性。技术困难往往限制了使用这个脑血管研究的重要工具。在这段视频中，一个优化的系统，以获得单个或多个网站在管腔内对大鼠大脑中动脉闭塞的血流动力学监测。

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 ...

optimized-system-for-cerebral-perfusion-monitoring-rat-stroke-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

23.0K 次观看.  University of Milano Bicocca. 已被证明是改善脑灌注监测缺血性中风模型的准确性。技术困难往往限制了使用这个脑血管研究的重要工具。在这段视频中，一个优化的系统，以获得单个或多个网站在管腔内对大鼠大脑中动脉闭塞的血流动力学监测。

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

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 ...

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 ...

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 ...

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 ...