We would like to thank Prof. I Mhairi Macrae and Dr. Debbie Dewar, University of Glasgow, who kindly hosted CW and DD for training in this procedure. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 309731 to LM. This work was also supported by a &#8220;Serendipity Grant&#8221; from The Dunhill Medical Trust to LM [grant number: SA21/0512].

该协议被批准列明由伦敦国王学院的机构的指导方针，并按照英国内政部的指导方针和动物（科学程序）1986年指南者，可以机构之间的变化进行;请确保遵守机构指引尝试此过程之前。为了触摸设备时，要保持无菌技术，高压灭菌器一大块铝箔并使用该包装的显微镜和麻醉机上设备周围处理此类。也可以使用无菌的保鲜膜（塑料膜）。 1.准备<ol><li>熟悉凝胶流体包（兽医恢复胶）和软州城大鼠中风手术前至少48小时。由于动物在手术后经常发现与进食和饮水困难，手术前引进的动物这些项目，尽量减少恐新症。众议院老年人鼠木咀嚼一个BLO中正减少长满牙齿的发病率（见第6章）。 </li><li>通过高压开始的外科手术（最小121℃，15 PSI，15分钟）前消毒所有外科手术工具。消毒在70％乙醇，用1％的氯己定所有工作表面，并使用手术单和维持无菌技术的程序的持续时间。 </li><li>引起的对侧半球的首选爪子病变。分配病变的左侧或取决于各大鼠的优选前爪右半球，由手术前基线的蒙托亚楼梯测试15（ 参见图4）来确定。为简单起见，在该手稿描述此过程时，左侧爪子是优选爪子，和手术都在右半球进行。使用李斯特连帽长或Evans大鼠，这些行为测试，因为他们学得很快。 注:楼梯测试旨在衡量两个精细和粗大熟练米变化下列电机系统损坏ovements。楼梯包括在一个中央平台的每一侧七个步骤。如果设备的楼梯测试是不可用的，钢瓶测试是测试霸道一个合适的选择，但注意，在这个模型中风在这个测试只是一个短暂的赤字，因此将不适合衡量以下长期复苏治疗。 <ol><li>放置在每个步骤的井3糖粒料（每侧21粒料）。放置大鼠在楼梯装置10分钟，并记录检索粒料的数量和位移每侧粒料的数量。 注意:要被包括在任务术后，大鼠必须在基线检索的最低75％的粒料。 </li></ol></li></ol> 2.手术<ol><li>在16-18个月（250〜400 g）利用女性老年人利斯特-连帽大鼠和诱导用5％异氟烷麻醉在1.5升/分钟Ø2.继麻醉诱导，减少异氟醚的水平，并在手术充足而最小化深度（例如，1.5-2％）维持它。通过面罩提供麻醉动物，并使用清除系统，以限制医生接触到异氟醚。 <ol><li>执行所有的程序为止，但不包括在深水动物开颅手术，因为这个程序可以产生行为缺陷16。缺血设计实验时，考虑的实验目标，并决定是否不假手术组来控制开颅手术应包括在内。 </li></ol></li><li>辖疼痛缓解前或围操作性（Carprieve，0.25毫克/千克，S。切）。 <ol><li> 1毫升加至19毫升无菌0.9％盐水，以使该储液，并注入每300克鼠0.6毫升。使用Carprieve的优于卡洛芬，因为前者是稳定在室温。 </li></ol></li><li>剃须的右半球腹侧颈部和颞部的皮毛，露出皮肤。消毒手术部位ü唱乙醇棉签。一定要按照脱毛当地IACUC准则。 <ol><li>执行此步骤的操作区域，以在切口部位毛发的数量减少。 </li></ol></li><li>将大鼠上覆盖有一个加热垫仰卧位无菌的悬垂性软木板。应用利多卡因霜剂的头部和颈部的剃的区域。插入直肠探头，以监测和带有恒温系统保持36.5-37.5℃之间动物的温度。 <ol><li>戴上眼睛lacrilube膏，防止干燥。给予大鼠的硫酸阿托品溶液注射（0.05毫升600微克/毫升的溶液，皮下），以减少气管分泌物。 注意:研究人员应该考虑测量生理变量，如血气和压力。 </li></ol></li><li>手术开始前，检查后爪掐撤离，眨眼反射，以确认全身麻醉。 </li><li>根据解剖微范围，就使用解剖刀露出颈部2厘米中央中线切口。移动至唾液腺轻轻外侧两侧的气管。 </li><li>环不可吸收缝合丝线绕其覆盖在一侧上的颈动脉的皮肤上。轻轻地用丝线拉远离部位的皮肤，并坚持下来，这些用外科胶带透露颈总动脉的公告板。仔细解剖钝周围筋膜和使用细镊子迷走神经自由的动脉。 <ol><li>要小心，不要在此步骤中损坏肌肉或迷走神经，因为这会影响摄食，吞咽和呼吸。 </li></ol></li><li>一旦颈总动脉被曝光，使用非吸收性丝线缝合（5/0）分离颈动脉，先用细镊子反向剖析，并小心不要让与神经接触与迷走神经分离颈动脉。一旦容器已被环（但不并列）与缝线，叔猿使用医用胶带，以防止这一步得而复缝合在一起的目的。拆下固定回覆皮肤缝线。 </li><li>重复另一侧（第2.7-2.8） </li><li>放置一个无菌生理盐水浸泡的纱布在伤口保持湿润的组织在手术剩余时间。松散的缝合皮肤，并把额外的生理盐水浸湿的纱布在区域，以防止进一步的脱水。 </li><li>放置大鼠的横向位置，并在皮肤上的切口在正确轨道和外耳道之间的中点。收回使用最多钉在公告板5弹性3毫米挂钩拉钩皮肤，然后生硬的解剖颞肌，露出头骨。 </li><li>放置在开放场地上方的拇指轮调整重力驱动盐水滴注（流量约2毫升/分钟），并设置一个抽吸系统以去除骨碎片，并在整个手术的这一阶段从曝光部位清除小出血（教派离子2.13-2.16）。 <ol><li>放置在耳朵附近的头盖骨的最高点的盐水输送喷嘴，和在最低点的抽吸喷嘴。出血期间调整轮提供更多盐水的区域，以便更好地可视化的任何渗色的源容器的烧灼，降低了动物麻醉下的时间量。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/53106/53106fig1.jpg" /> 图1.手术一套永久远端大脑中动脉闭塞模型的。在建立了大鼠开颅手术使用的设备是显示了右半球和插图，围绕骨瓣部位抽吸及吊盐水的定位。还示出的脉管系统的主要功能;大脑中动脉（红色）和下脑静脉（蓝色）被示出，并且阴影区域表示，其中所述动脉的凝固发生。闭塞的确认是通过切割MCA下大脑大静脉下方进行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53106/53106fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="13"><li>在暴露的区域进行开颅使用牙钻用粗1.6毫米金刚石涂层钻毛刺在大约8000转，确保圆形和侧压而不是向下的压力的应用，而钻的露出面积（大约5毫米×5毫米）的。一旦骨是足够薄，它看起来完全透明，取出使用镊子。 </li><li>使用自制的硬脑膜钩，通过弯曲细镊子之一梢约180°，形成一个弧形提出，要小心地打开硬脑膜，谨慎，以避免大的体表血管，因为它们非常脆弱，容易破裂。 注:大脑的露出面积将揭示大脑中动脉（MCA）;所需的段措施约2毫米长（见图1，小图）。 </li><li>凝结MCA从那里下脑静脉穿过，到动脉分叉处的点，然后沿着使用一对透热镊子，直到完全闭塞17 MCA的尾分支机构。使用长为0.25 mm尖头角度的珠宝商透热钳。 <ol><li>当闭塞动脉，改变盐水流作为必要保持此区域凉，以防止凝固钳附着到血管。当闭塞，血管呈黑色，无血流的迹象应该存在;血液流动可以在部分地阻塞血管中看到。 </li><li>切马华在这一点上，确认完全闭塞。使用微血管剪刀，剪哪里下大脑大静脉穿过MCA下方。 </li><li>覆盖继续下一步骤之前的盐水浸泡的纱布垫的暴露面积。 </li></ol></li><li>转鼠回仰卧POSI重刑和重新脖子上的松散的缝合，再暴露颈动脉。永久被遮挡瞬时使用具有60125克压力13毫米的不锈钢动脉夹在周围的颈动脉中丝线打结，而左颈动脉结扎上的同一侧的闭塞MCA（右）的颈动脉分钟。松散缝合起来在颈部切开这段时间并放置一个无菌盐水浸泡的纱布在上面，以防止脱水。 </li></ol><ol start="1" style="margin-left: 40px;"><li>需要注意的是该动物的右手侧将在外科医生的左手侧，给出的仰卧定位。 </li><li>对于假手术动物中，打开颈和时间区域的腹侧部分和分离的肌肉来定位颈总动脉，但是不堵塞。有深水动物经过的程序，但不包括开颅手术，因为这个程序可以产生行为缺陷16。为了维持其他牛逼致盲EAM 成员 （例如，后行为测试期间），切口进行缝合。 </li></ol><img alt="图2" src="/files/ftp_upload/53106/53106fig2.jpg" /> 图2.下列中脑动脉闭塞串联颈动脉阻塞，右颈总动脉（CCA）被永久地通过捆扎丝缝合（5/0）周围血管（在图像的左手侧）封闭。左CCA（右侧）被闭塞用微血管钳1小时。进行这些手术注意不要使每边（白色）迷走神经联系。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53106/53106fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="17"><li>缝合左颈闭塞1小时期间在时间区域中的切口。 </li><li>给大鼠20毫升皮下注射生理盐水（5毫升两个站点上的每个侧面），以维持水合作用（见6.2）。 </li><li>当60分钟已经过去了，取下固定夹。局部应用生理盐水周围的肌肉，并关闭切口采用可吸收缝合线（4/0）皮下连续缝合颈部。 </li></ol> 3.术后护理<ol start="1"><li>放置老鼠在31℃培养箱从麻醉中恢复长达4小时。 注意:研究人员可能更愿意使用根据当地惯例其他温度和持续时间。 </li><li>在24小时的重复carprieve（0.25毫克/千克），用于缓解疼痛。因为所有的动物接受Carprieve的相同剂量，这个因素被控制系统。 Carprieve的任何神经保护作用可能是微不足道的。 </li><li>给予盐水注射每日以防止脱水为至少第一三天。在该大鼠需要每公斤体重（每24小时）65毫升流体的假设，确保每个300克大鼠每天接收〜22毫升流体。 </li></ol> 4.刀豆梗死firmation <ol start="1"><li>为了测量梗塞体积使用结构性MRI检查。 注意:另一种方法可以是使用组织染色，如四氮唑氯（TTC），先前已经相关于结构MRI数据18。然而，这只能在研究的结束点使用，而不是纵向地。 <ol><li> MCAO的诱导二十四小时后，在0.9升/分钟医用空气和1升/分钟的 O 2，在正交鸟笼磁共振线圈安全麻醉用异氟烷（诱导5％，为维护1-1.5％）的大鼠（43毫米直径的），并发生在7特斯拉水平的孔扫描仪。 </li><li>获得使用快速自旋回波序列T2加权扫描:回波时间（TE）（TR）4000毫秒（FOV）40×40毫米，采集矩阵128×128，获得40×0.5毫米视场60毫秒，重复时间厚的片约8分钟。随后，转换40片，放入20×1毫米厚的片，使用resamplING功能在医学成像软件。 </li></ol></li><li>通过测量在20体积梗塞的截面积得到的医用图像显示包病变卷。由厚度（1毫米），这些面积的总和乘以获得的总体积。计算的组平均体积和标准偏差。对于个病灶体积计算，也获得ipsilesional和对侧半球的体积。 </li><li>为了校正因脑水肿肿胀，采用Gerriets&#39;公式18调整这些值。仅包括含有皮质和根据标准鼠图谱19，以避免过度矫正不包含小脑或嗅球，因为这可能产生负病变卷片。 <ol><li>再次中风手术8周后获得T2加权像。 </li></ol></li></ol> 5.计算样本量为未来研究评估神经保护和行为恢复&lt;/ P&gt; <ol start="1"><li>执行样本量计算，估计会用两组（控制组，处理），以确定的三个不同大小的治疗效果（减少25％，减少50％，降低75％），假设的未来神经保护实验需要使用最小样本量先验算法在电力分析软件20实现。 注意:为使读者做类似的计算与自己的数据，还有来自可免​​费提供的功率分析软件截图（见代表性的结果）。使用下面的参数:上可接受的假阳性率（即，I型误差阈值;α）≤0.05，和功率（等于1-β）≥0.80（即，大于80％的功率）。参见下面的解释和讨论21。 </li></ol> 6.老年动物福利卒中后手术<ol start="1"><li>提供额外的软州城一第二补液凝胶包，该大鼠在手术前习惯于，除了水瓶更容易达到延长的提示。此外，在笼子里，而不是松散的床上用品可吸收恢复垫（ 即避免木屑）作为第一个24小时，并提供额外的嵌套的床上用品。不要用泥食物 （如婴儿食品）的大鼠吞咽困难可能会窒息。 </li><li>每日称重动物7天并监控恢复。体重减轻是脱水和应力的主要指示。在手术后的最初几天体重减轻主要反映脱水（而非体重的损失由于减少摄食）。 </li><li>老龄化大鼠显示减肥无关手术，与流体的当量重量替换丢失体重和检查的顶部和底部齿。凡牙齿杂草丛生，麻醉异氟烷大鼠（如第2.1节），并在仰卧位的地方<ol><li>放置一个1ml注射器筒的德后面ETH保护软组织。切以高的旋转速度，但速度慢，结实的手的动作（例如，3秒切口），使用手持式圆锯（例如，大约3厘米直径的）。让老鼠的牙齿切割之间冷却下来。确保旋转锯安装在心轴，使得该锯的齿面对旋转方向。这是值得保留独立包装，灭菌的旋转锯准备这种可能性。 注意:即使在硬颗粒饲料保持过度生长在老年大鼠发生。我们建议，牙齿老年大鼠定期检查。 </li></ol></li><li>其中，一对老年大鼠显示减肥和毛发直立无关的手术，讨论与兽医的治疗方案。考虑人道杀害动物。这种动物的MRI可显示垂体瘤（通常在老年雌性大鼠），它是不可操作和致命的。 </li></ol>

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O., 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=Ischemic+Lesion+Volumes+in+Acute+Stroke+by+Diffusion-Weighted+Magnetic+Resonance+Imaging+Correlate+with+Clinical+Outcome.">Ischemic Lesion Volumes in Acute Stroke by Diffusion-Weighted Magnetic Resonance Imaging Correlate with Clinical Outcome.</a> Ann Neurol. 42, 164-170 (1997).</li><li>Norrving, B., Brainin, M., Heiss, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Textbook of Stroke Medicine. , 28-39 (2009).</li><li>Truelsen, T., 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=Stroke+incidence+and+prevalence+in+Europe:+a+review+of+available+data.">Stroke incidence and prevalence in Europe: a review of available data.</a> Eur J Neurol. 13, 581-598 (2006).</li><li>Ord, E. N., 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=Positive+impact+of+pre-stroke+surgery+on+survival+following+transient+focal+ischemia+in+hypertensive+rats.">Positive impact of pre-stroke surgery on survival following transient focal ischemia in hypertensive rats.</a> J Neurosci Methods. 211, 305-308 (2012).</li><li>Chen, S. T., Hsu, C. Y., Hogan, E. L., Maricq, H., Balentine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+focal+ischemic+stroke+in+the+rat:+reproducible+extensive+cortical+infarction.">A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction.</a> Stroke. 17, 738-743 (1986).</li><li>Montoya, C. P., Campbell-Hope, L. J., Pemberton, K. D., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'staircase+test':+a+measure+of+independent+forelimb+reaching+and+grasping+abilities+in+rats.">The 'staircase test': a measure of independent forelimb reaching and grasping abilities in rats.</a> J Neurosci Methods. 36, 219-228 (1991).</li><li>Adams, F. S., Schwarting, R. K., Huston, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+neurochemical+asymmetries+following+unilateral+trephination+of+the+rat+skull:+is+this+control+operation+always+appropriate?.">Behavioral and neurochemical asymmetries following unilateral trephination of the rat skull: is this control operation always appropriate?.</a> Physiol Behav. 55, 947-952 (1994).</li><li>Macrae, I. M., U, D. i. r. n. a. g. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Rodent Models of Stroke, Neuromethods. 47, 41-53 (2010).</li><li>Gerriets, T., 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=Noninvasive+Quantification+of+Brain+Edema+and+the+Space-Occupying+Effect+in+Rat+Stroke+Models+Using+Magnetic+Resonance+Imaging.">Noninvasive Quantification of Brain Edema and the Space-Occupying Effect in Rat Stroke Models Using Magnetic Resonance Imaging.</a> Stroke. 35, 566-571 (2004).</li><li>Paxinos, G., Watson, C. R., Emson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AChE-stained+horizontal+sections+of+the+rat+brain+in+stereotaxic+coordinates.">AChE-stained horizontal sections of the rat brain in stereotaxic coordinates.</a> J Neurosci Methods. 3, 129-149 (1980).</li><li>Faul, F., Erdfelder, E., Buchner, A., Lang, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+power+analyses+using+G*Power+3.1:+tests+for+correlation+and+regression+analyses.">Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses.</a> Behav Res Methods. 41, 1149-1160 (2009).</li><li>Button, K. S., 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=Power+failure:+why+small+sample+size+undermines+the+reliability+of+neuroscience.">Power failure: why small sample size undermines the reliability of neuroscience.</a> Nature Reviews Neuroscience. 14, 365-376 (2013).</li><li>Fox, G., Gallacher, D., Shevde, S., Loftus, J., Swayne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomic+variation+of+the+middle+cerebral+artery+in+the+Sprague-Dawley+rat.">Anatomic variation of the middle cerebral artery in the Sprague-Dawley rat.</a> Stroke. 24, 2087-2093 (1993).</li><li>Tamura, A., Graham, D. I., McColloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J. Cereb. Blood Flow Metab. 1, 53-60 (1981).</li></ol>

The authors have nothing to disclose.

MCAO在啮齿类动物是经常用于人类中风模型的技术。这种模式确实有一些细节在协议中要注意。首先，它是保持动物的体温整个实验，因为这会影响梗塞的尺寸和死亡率的研究的数量是必不可少的。它可能会正确的CCA的瞬态闭塞期间停止异氟醚，并保持大鼠温热的，安静的环境，减少暴露于异氟醚提高存活率。研究人员应该考虑是否缩短麻醉时间远远超过额外感应的压力。大鼠血管（如MCA分支）内的动物22同伙之间变化。它开始一项新的研究时，牢记这一点是非常重要的。不同CCA闭塞时间可以进行评估（ 例如 ，30，45，60和90分钟）。在这项研究中使用的60分钟的阻断时间。在其它研究中，我们已发现，45分闭塞导致类似大小的皮质梗死但提高生存率的传闻证据。因此，有外科医生开始使用短闭塞时间 （例如，30分钟），以查看是否获得充分的病灶体积（和/或要求的行为缺陷），然后才以增加闭塞倍必要。行为赤字不会在成年大鼠相比持续老年大鼠相同的闭塞倍。 MRI可以被用来判断是否（后一特定阻塞时间）病变卷适合的研究目标。一个小的病灶将跨越不到十0.5毫米冠状切片（满分40）。一个中等大小的病灶将10到20冠状切片之间的跨度。大病灶将二三十个冠状切片之间的跨度。一个非常大的病灶将跨越三十多四十片。根据我们的经验，老鼠具有非常大的病变（超过三十片）和/或脑疝ACROS的证据S中的中线通常有预后差:更短的时间闭塞可以考虑。 MRI对评估梗死位置有用:一些更位于尾端，有些更吻侧位置。 来自颈总动脉分离迷走神经时要格外小心。罗音（锉）中风手术后可能会出现，这可能是由于在某些动物的神经损害，虽然病因目前尚不清楚:根据我们的经验，预后这些动物很可怜，通常建议人道杀死他们。 永久透热缺血模型结果可复制的皮质梗死和老年大鼠接受手术后的存活率。该技术确实，但是，需要在立体显微镜下侵入性手术。保持无菌技术操作，如果动物是从手术恢复的不错是很重要的。必须小心，不要损坏MCA同时揭露和混凝动脉，和皮质表面损伤应该最小化否则皮层的露出面积可以形成梗塞区的一部分。因此建议以获得尽可能多的经验，可能建立的程序和之前进行的一项研究，以测试候选疗法，例如用于实现一致梗死和确定闭塞次。实验者应随机手术阶段中的任何处理（"块随机"），在可能的情况。值得注意的是，这种模式不涉及马华再灌注（除非瞬态MCA结扎代替透热）。死亡率可以在老年大鼠高与这些大皮质梗塞但应该可以通过使用更短的闭塞倍并通过在可能的情况（例如，闭塞期间）减少暴露于全身麻醉降低死亡率。作为载体使用的70％的N 2 O和 30％的O 2的可允许的异氟烷较低水平被使用:这降低了暴露在isofluRANE可能会导致更高的存活率。 另一个需要考虑的一点是，动脉粥样硬化是一个渐进的过程，而在这个协议中，我们与急性闭塞CCA模拟它。然而，在血流量和持续的财政赤字大幅减少模拟发生在许多中风患者串联闭塞。永久远端MCAO无汇接CCA闭塞大鼠不能诱导笔画重复地14:此外，没有串联CCA闭塞，我们已经发现相当大的自发恢复发生时排除中风疗法长期行为评价超过8周。与此相反，我们表明，远端MCAO与串联CCA闭塞诱导长期赤字老年大鼠。 总之，在大鼠此过程会导致在大小和位置，以那些在人类状况见过类似的笔触，用持续的残疾，人们可以用它来实现的新的治疗方法和重新阐发测试缺血性中风后对机制。

中风是目前世界范围内死亡的第三最常见的原因和残疾1的首要原因。缺血性中风，其包括所有笔划的80％，经常导致梗塞皮层引起感觉损失（例如，本体）中，运动功能和在关注患侧2-4。大脑中动脉（MCA）是最大的，从Willis环彩票供给，并从颈内动脉5茎船只。马华是脑血管缺血性脑卒中最常见的影响，在这片领土中风占所有缺血性中风6,7的65％。在MCA提供既皮层和皮层下区域和由MCA冲程神经异常取决于闭塞7的精确位置。近端闭塞MCA影响通过lenticulostriatal动脉深领土，并引起大梗死既包括科蒂CAL和皮质下区域。相反，剥夺血流完全皮层区域更远端闭塞往往会产生较小的皮层梗死。 在人口众多的研究中，人类中风病灶范围从同侧半球8,9的5-14％;恶性中风占笔划的10％，并引起更大的梗塞，需要骨瓣减压降低颅内压，和患者的较小的病灶更可能存活10。我们证明产生病变占据半球类似比例的人多招一个可重复的模型。 中风是一种异质性疾病;缺血性中风的75％（从颅内小血管阻塞）引起的任何腔隙性梗死;心源性卒中;或大型动脉粥样硬化，它占冲程的30％。症状动脉粥样硬化是在点的常见的C最常观察到的arotid动脉（CCA）分支为内部和外部颈动脉11。 脑卒中的临床前模型应尽可能接近人的条件尽可能地模拟其病理生理学和应纳入中风的危险因素。缺血性中风的92％在人的65岁以上的情况时，和其他危险因素包括肥胖，高血压，动脉粥样硬化，如前所述12。更好地代表这些危险因素，它是推荐使用可能共享一些的自然条件的病理生理特征的模型。在这个协议中，我们已包括通过颈动脉高龄和阻碍血液流动。 大脑中动脉闭塞的经典模型（MCAO）是近端MCA闭塞，这减少了在前血流量及大脑中动脉腔内灯丝模型。采用这种模式的短闭塞次集中第lesion到皮质下区域，而会导致大的病变招募既皮质和皮质下区域的区域，从而导致更高的死亡率在老年大鼠再闭塞倍。相比之下，我们小组使用的模型涉及实施开颅手术和硬脑膜接着使用双极烧灼镊子MCA的一小部分的血液和破坏凝血开口。此电疗模型是从1981年的论文由Tamura等适于23和使用骨瓣的可能限制颅内压升高，它是封闭的颅骨的一个特征，并在较高的再现性的结果和下部死亡率在我们的手术队列相比一些其他车型13。以生成可再现的梗死和持续残疾我们永久阻塞近端CCA和瞬时阻塞远端CCA按照Chen等人的14我们使用非侵入性的T2加权核磁共振成像（MRI）评价的程度和脑梗塞的位置，和大脑中的感觉运动皮层肿胀程度。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Carprofen</td>
			<td>Norbrook</td>
			<td>Vm No; 02000/4229</td>
			<td>give 0.25 mg/kg</td>
		</tr>
		<tr>
			<td>Atropine Sulfate</td>
			<td>AmTech</td>
			<td>RXATRINJ-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>UHS</td>
			<td align="right">20021</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine cream (Emla)</td>
			<td>AstraZeneca</td>
			<td>0012901</td>
			<td>Apply a pea sized drop to the shaved neck and temporal regions</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Harvard Instruments</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11019-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk sutures</td>
			<td>Harvard Apparatus</td>
			<td align="right">723288</td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery system</td>
			<td>Eschmann</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 mm Jeweler caurtery forceps</td>
			<td>Eschmann</td>
			<td align="right">8330349</td>
			<td></td>
		</tr>
		<tr>
			<td>fine Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thumb driven saline drip system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacusafe aspirator system</td>
			<td>INTEGRA BIOSCIENCES</td>
			<td align="right">158320</td>
			<td></td>
		</tr>
		<tr>
			<td>16 mm coarse diamond coated Steel burrs</td>
			<td></td>
			<td>K801 104 016</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld dental drill</td>
			<td>NSK</td>
			<td>NSKVMAXVRE (Handpiece NSKEX6B)</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular scissors</td>
			<td>World Precision Instruments</td>
			<td align="right">501790</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Vicryl sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clip and applicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Anaesthesia System Isoflurane K/F Single Gas</td>
			<td>VetTech Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel Scalpel blades No. 10</td>
			<td>Swann-Morton</td>
			<td align="right">201</td>
			<td></td>
		</tr>
		<tr>
			<td>25g needles</td>
			<td>Terumo</td>
			<td>NN-2525R</td>
			<td></td>
		</tr>
		<tr>
			<td>syringes (1 ml and 5 ml)</td>
			<td>Terumo</td>
			<td>SS+01T1 / SS*05SE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (Sodium Chloride 0.9%)</td>
			<td>Fresenius Kabi</td>
			<td>Pl 08828/0178</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton buds</td>
			<td>Johnson and Johnson</td>
			<td>5000207582502</td>
			<td>sterilize before use</td>
		</tr>
		<tr>
			<td>gauze</td>
			<td></td>
			<td></td>
			<td>sterilize before use</td>
		</tr>
		<tr>
			<td>Medical Imaging Package (Jim)</td>
			<td>Xinapse</td>
			<td></td>
			<td>Free software</td>
		</tr>
		<tr>
			<td>Statistical Parametric Mapping Software (SPM8)</td>
			<td>UCL</td>
			<td></td>
			<td>Free software</td>
		</tr>
		<tr>
			<td>Power Analysis Software (G*Power)</td>
			<td>Universit&#228;t D&#252;sseldorf</td>
			<td></td>
			<td>Free software</td>
		</tr>
	</tbody>
</table>

performing permanent distal middle cerebral with common carotid artery occlusion in aged rats to study cortical ischemia with sustained disability

中风通常发生在老年人的范围合并症包括颈动脉（或其他动脉）粥样硬化，高血压，肥胖症和糖尿病。因此，在动物中风评估治疗时，选择具有优异的表面效度的模型是非常重要的。缺血性中风占所有中风的80％，其中大部分发生在大脑中动脉（MCA）的领土，往往诱发影响感觉运动皮层，在身体对侧引起持续plegia或轻瘫梗死。我们在这个视频中示范生产老年大鼠缺血性中风，从而导致持续的感觉残疾和大量的皮质梗死的方法。具体来说，我们用电疗钳闭塞该动脉的短段诱发老年雌性大鼠永久远端大脑中动脉闭塞（MCAO）。同侧的病变的颈动脉是则permanently闭塞和对侧颈动脉瞬时闭塞60分钟。我们在24小时和中风8周后使用结构T2加权磁共振成像（MRI）测量梗死面积。在这项研究中，平均梗死体积为4.5％±同侧半球的2.0％（标准偏差）在24小时（对于脑使用Gerriet方程肿胀校正中，n = 5）。这种模式是可行的和临床上相关的，因为它允许持续感觉运动缺损的诱导，这是病理生理机制和新的治疗方法的澄清重要。

永久MCAO诱导通过进行开颅，接着通过电疗中脑动脉与ipsilesional颈总动脉的永久闭塞和对侧颈总动脉60分钟堵塞组合的凝结和破坏。设备的设置的示意图和闭塞MCA 示于图1，和图 2（上图）的颈动脉。 中风结果采用权益工具的地区在医疗图像显示包评估24小时，通过测量40×0.5 mm层厚梗死体积（从嗅球脊髓延髓的结束喙末端）中风后8周。代表性的T2加权结构MRI扫描示出了用于在24小时和8周（图3A）相同的动物。梗死体积被确定通过表示一个高信号的大鼠脑的区域;为T2重编辑图像显示水或血浆为明亮的白色区域。众所周知，有在中风后水肿和脑肿胀的增加，这可以从已经相关的梗塞体积18的组织学测量值的T2加权的扫描来测量。然而，水肿本早期中风（例如，在24小时）后可导致最终的损伤体积（例如，在8周）的过高估计，因此，我们也调整本平均梗死体积使用Gerriet的公式。 图3B示出了从平均数据原始（未经调整）病灶在24小时体积62.8毫米3（±25.4毫米3 SD，上图）;这个占据了受影响的半球（±4.2％SD，中图）的9.8％。当纠正脑使用Gerriets"公式将此值降低到4.5％（±2.0％SD，下图）肿胀。 中风严重程度用蒙托亚楼梯测试15还测量。简言之，一个nimals进行预训练的检索糖粒料MCAO之前4周中风手术和中风后（图4），以确认持续赤字测试8周。大鼠放置在楼梯装置10分钟，并记录检索粒料的数量（下21粒料）和百分比（基是指±标准误差）显示。进行回归分析，以适应该行的数据。 图5示出样本大小计算使用梗塞体积数据（候选疗法的潜在影响），分析了使用"两个独立的装置（两组）之间的差异"，并使用（未校正用电力分析软件的算法t-检验）的装置，并从图3B的标准偏差。在图 5和表 1显示的信息12只大鼠。将每组需要检测一个疗法降低的inf通过24小时50％ARCT量，而图6显示了权力的"XY图"使用的动物数目不等。 表1总结了样本大小计算所有时间点来实现的。 <img alt="图3" src="/files/ftp_upload/53106/53106fig3.jpg" /> 图3. T2加权结构的MRI被用于测量梗塞，脑中风后肿胀的大小。（A）中相同的大鼠脑24小时的T2加权的磁共振图像和中风诱导后8周。白色区域表示病变组织，而且还包含该八周后，解决了一些血管性水肿。使用兴趣工具包的医用图像显示包指定地区进行测定（B）的梗死体积，并在表示平均值±标准差为所使用的3个时间点（6）的曲线图绘制。原病灶体积（大脑得不到纠正由于ED肿胀EMA），受影响的半球病变的比例（未修正的肿胀），以及半球大脑使用Gerriets"公式肿胀纠正的百分比病变显示在这里。 SD被使用，而不是SEM以执行计算样本量（ 见图 5）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53106/53106fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53106/53106fig4.jpg" /> 图 4. 楼梯试验表明把握和检索颗粒损伤。在这种缺血模型有非常少的自然恢复了。中风老年大鼠持续损害灵巧，使用颗粒的"楼梯测试"每周达到测试中。插图:执行行为测试大鼠的照片。该图显示平均（±支架检索（选自21粒料ARD误差）数，表示为受影响的前爪每周的百分比）。 N = 5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53106/53106fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53106/53106fig5.jpg" /> 图5.样本大小计算来确定，以检测期望的治疗效果所需的大鼠的组号，此画面拍摄，从功率分析软件采取表明每组12只大鼠，将需要以检测疗法，通过减少50％的梗死体积在24小时左右。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53106/53106fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53106/53106fig6.jpg" /><br使用动物的各种总数给出的参数/&gt; 图6.电源来实现的。从电源分析软件的"XY绘图为一个值的范围"表示，将使用老年大鼠不同（总）号实验中获得的功率， 如图5所示。 表1总结了我们所有的结果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53106/53106fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 中风后时间: </td><td colspan="3"> 每次检测的病变体积减少了所需的组大鼠数量: </td></tr><tr><td align="right"> 75％ </td><td align="right"> 50％ </td><td align="right"> 25％ </td></tr><tr><td> 24小时</td><td align="right"> 6 </td><td align="right"> 12 </td><td align="right"> 42 </td></tr><tr><td> 8周</td><td align="right"> 4 </td><td align="right">五</td><td align="right"> 17 </td></tr></tbody></table> 表1每组样本大小计算为假想以后的实验。使用动力分析软件计算值（ 见图 5和6）。表显示每一个双组实验在每个在这项研究中的时间点的以检测病灶体积25％，降低了50％和75％所需组大鼠数。

Watch this Scientific Journal Video about Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability at JoVE.com

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

在这里，我们提出了一个协议，以生产中老年雌性大鼠永久远端大脑中动脉闭塞的颈动脉闭塞的同时产生大量的皮质梗死和持续的赤字。我们采用结构MRI 24小时，行程8周后显示病灶大小的确认。

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

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

performing-permanent-distal-middle-cerebral-with-common-carotid

Nanyang Technological University

Research

JoVE Journal

Medicine

21.4K 次观看.  King's College London, University of London. 在这里，我们提出了一个协议，以生产中老年雌性大鼠永久远端大脑中动脉闭塞的颈动脉闭塞的同时产生大量的皮质梗死和持续的赤字。我们采用结构MRI 24小时，行程8周后显示病灶大小的确认。 

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

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 ...

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 ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

永久性脑血管闭塞<em&gt;通过</em&gt;双人结扎横断

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

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 ...