这项研究是由82946一个CIHR队授予＃CTP，CIHR授予的＃MOP 102741，以及一个加拿大的椅子＃216684和施特劳斯基金会的支持。 ER得到了从加拿大心脏及中风基金会的奖学金。

短暂性大脑中动脉闭塞（MCAO） 1。外科手术（ 图1） 短暂性大脑中动脉的阻塞（tMCAO）上进行2 - 3个月大的雄性C57BL / 6小鼠（22-28G）。 IRCM生物伦理委员会动物护理批准该协议。外科工具被灭菌，高压灭菌（121℃60分钟，在15 psi）。每只动物之间，他们使用热珠灭菌器（15秒）消毒。用70％的乙醇消毒的外科手术和其他设备。 手术前两小时，的小鼠，analgesized与丁丙诺啡（0.03毫克/公斤BWIP的）。 图1A中。 <ol><li>深麻醉小鼠异氟醚5％，然后维持在2.5％异氟醚麻醉。的体温的小鼠的是，用加热垫的外科手术过程中保持恒定。 </li><li>消毒的皮毛和sk在用70％乙醇或聚维酮碘。由于剃须产生头发片段释放，微打磨和炎症，可能对脑卒中病理生理的影响，我们，不刮胡子鼠标毛皮。 </li><li>中线颈部切口，然后轻轻的拉开的软组织。 </li><li>在立体显微镜（STEMI 2000年，蔡司），钝性分离暴露气管和收回的肌肉来定位的颈动脉。 </li><li>仔细解剖的左颈总动脉（CCA）从周围组织和CCA的暂时闭塞的一个临时的缝合线（1），用5-0丝线缝合切成20毫米分部。大应当谨慎行事，不伤害迷走神经。 </li><li>分离分叉内部的左侧颈总动脉（ICA）和外部颈总动脉（ECA）。被放置的永久缝合线（2）周围的ECA，为尽可能向远侧，和稍紧另一个临时缝合线（3）被放置在远端的ECA分叉。 </li><li>剪辑的左ICA（4）是使用反向动作镊子避免出血。大应当谨慎行事，不伤害迷走神经。 </li><li>到ECA（5）之间永久（2）和临时（3）缝合切开一个小口。 </li></ol>图1B。 <ol start="9"><li>介绍一个12毫米长的6-0硅涂层（约9-10mm的涂硅）的单丝的缝合（Doccol公司）的ECA（6），完全切断远端永久缝合的非洲经委会和反转的封堵器到ICA 。紧缚缝合线的单丝，以防止出血和周围的反向动作镊子除去。 </li><li>封堵器，Willis环的MCA闭塞的起源。停止其插入超出ECA和CCA分叉处约9-10毫米。封堵器被堵塞，不能动了。必须小心渗透到翼腭动脉。 （3）在ECA的缝合是TIGhtly绑在固定的单丝的位置。 </li><li>关闭的皮肤的autoclip伤口闭合系统。 </li><li>注入1毫升生理盐水溶液皮下和放置在红外加热灯的小鼠在所有的时期（60分钟）后闭塞。 </li></ol> 2。大脑中动脉血流的恢复在reanesthesia之前，neuroscore可以进行检查，以评估成功的手术。 <ol><li>麻醉小鼠如前所述，，并移除autoclips。 </li><li>稍微打开缝合到ECA（3），允许单丝撤销和血液灌注。 </li><li>永久的的ECA，以防止失血临时缝合结扎。的单丝保持重用。 </li><li>删除临时缝合（1）到CCA，让血液循环。 </li><li>关闭伤口。这些老鼠应该接受另1 mL生理盐水皮下注射。 </li><li>将小鼠1小时下的红外加热灯。检查的动物重新获得流动性后，鼠标将返回到它的笼子，遗留下72小时的加热垫和毛巾。请注意，只有一半的笼放置在加热垫，以允许选择他们的环境的小鼠。由于手术后的重量损失一般观察，捣碎食物放在培养皿中，鼓励进食。 12小时的手术后，小鼠接受了另一种剂量丁丙诺啡（0.03毫克/公斤BWIP的）。 </li></ol> 3。假手术假手术，所有的程序都是相同的，只是，封堵器未插入。 4。 Neuroscore 神经功能缺损允许再灌注后不同程度的伤害程度后，估计成功的tMCAO的评价。神经功能缺损评分如前所述5 &lt;/ SUP&gt; 1，24，48和72小时再灌注后执行。扩大的六分： <ul><li> 0：正常。 </li><li> 1：轻度或没有不一致旋转时捡到的尾巴转圈行为，&lt;50％到对侧旋转。 </li><li> 2：轻度一致盘旋，&gt; 50％到对侧旋转。 </li><li> 3：持续强劲，立即盘旋，鼠标拥有超过1-2秒的旋转位置，用鼻子几乎达到了它的尾巴。 </li><li> 4：严重的旋转推进到装桶，步行或翻正反射的损失。 </li><li> 5：昏迷或奄奄一息。 </li></ul> 5。甲酚紫染色（ 图2） 的PBS溶液灌注之后，大脑快速冷冻在异戊烷中，并贮存于-80℃C.小鼠大脑也可以灌注，用4％多聚甲醛（PFA）取决于计划的免疫组化研究。冠状脑切片（17微米）的低温恒温器的快捷方式进行。一节收集了每30同一张幻灯片上有代表性的脑损伤。量量化，2张幻灯片被染成甲酚紫染色和图像分析系统（Scion的公司，冯检基，MD）是用来评估病变。以任意单位（像素）计算损伤体积，并作为每个区段对侧非损伤的面积的百分比表示。其他的幻灯片保持在-80°C免疫组化研究。另外，PFA固定的大脑可以减少在30-50微米，，使用vibratome和脑梗死体积测量的建议，Han 等人（2009）6。

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	<li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema,+I:+a+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Jpn. J. Stroke. 8, 1-8 (1986).</li><li>Chan, P. H., Kamii, H., Yang, G., Gafni, J., Epstein, C. J., Carlson, E., Reola, L. B. r. a. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=infarction+is+not+reduced+in+SOD-1+transgenic+mice+after+a+permanent+focal+cerebral+ischemia.">infarction is not reduced in SOD-1 transgenic mice after a permanent focal cerebral ischemia.</a> Neuroreport. 5, 293-296 (1993).</li><li>Yang, G., Chan, P. H., Chen, J., Carlson, E., Chen, S. F., Weinstein, P., Epstein, C. J., Kamii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+copper-zinc+superoxide+dismutase+transgenic+mice+are+highly+resistant+to+reperfusion+injury+after+focal+cerebral+ischemia.">Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia.</a> Stroke. 25, 165-170 (1994).</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>Jiang, S. X., Lertvorachon, J., Hou, S. T., Konishi, Y., Webster, J., Mealing, G., Brunette, E., Tauskela, J., Preston, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlortetracycline+and+demeclocycline+inhibit+calpains+and+protect+mouse+neurons+against+glutamate+toxicity+and+cerebral+ischemia.">Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia.</a> J. Biol. Chem. 280, 33811-33888 (2005).</li><li>Han, R. Q., Ouyang, Y. B., Xu, L., Agrawal, R., Patterson, A. J., Giffard, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postischemic+brain+injury+is+attenuated+in+mice+lacking+the+beta2-adrenergic+receptor.">Postischemic brain injury is attenuated in mice lacking the beta2-adrenergic receptor.</a> Anesth. Analg. 108, 280-287 (2009).</li><li>Rousselet, E., Marcinkiewicz, J., Kriz, J., Zhou, A., Hatten, M. E., Prat, A., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCSK9+reduces+the+protein+levels+of+the+LDL+receptor+in+mouse+brain+during+development+and+after+ischemic+stroke.">PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke.</a> J. Lipid Res. 52, 1383-1391 (2011).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J. Cereb. Blood Flow Metab. 1, 53-60 (1986).</li><li>Chen, Y., Ito, A., Takai, K., Saito, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+pterygopalatine+arterial+blood+flow+decreases+infarct+volume+variability+in+a+mouse+model+of+intraluminal+suture+middle+cerebral+artery+occlusion.">Blocking pterygopalatine arterial blood flow decreases infarct volume variability in a mouse model of intraluminal suture middle cerebral artery occlusion.</a> J. Neurosci. Methods. 174, 18-24 (2008).</li><li>Tsuchiya, D., Hong, S., Kayama, T., Panter, S. S., Weinstein, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+suture+size+and+carotid+clip+application+upon+blood+flow+and+infarct+volume+after+permanent+and+temporary+middle+cerebral+artery+occlusion+in+mice.">Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice.</a> Brain Res. 970, 131-192 (2003).</li><li>Tureyen, K., Vemuganti, R., Sailor, K. A., Dempsey, R. 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作者有没有利益冲突披露。 生产和免费使用这篇文章是由蔡司公司

不同的行程模型已经发展到模仿患者中风的后果。中风模型的选择取决于对生物的问题。管腔内的MCAO模型模仿患者缺血性脑卒中的最常见的类型之一是微创和更一致的比田村模型4,7。这是一个非常有趣的神经保护作用，neurorepair和细胞死亡的分析模型。腔内模型的成功取决于许多因素，如动物的性别，年龄和体重，温度，麻醉和手术的时间，以进行控制的。封堵器（尖端直径，长度，形状和fexibility）的物理礼模型4的一致性的关键。在此，我们使用12毫米长的6-0单丝上涂有硅Doccol公司从9-10毫米。这种单丝的巨大优势是减少蛛网膜下腔出血，因为它的长度覆盖了所有的ICA长，残留的血流量，其中来自威利斯和PPA圈的前，后交通动脉，防止梗死体积减小的变化（在我们手中15％的变异）。 CCA的缝合线也降低了变异的心肌梗死卷9。 可以通过不同的方式评估脑损伤的程度tMCAO。如前面提到的，可以测量神经功能缺损。然而，这是难以通过这种方式，有一个有效的措施。 TTC染色程度的心肌梗死是常见的表现。最近，磁共振技术也用于神经保护治疗的一个特别有趣的技术。要了解参与行程，如neurorepair，细胞死亡或细胞增殖的细胞机制的基础，不同的标志物免疫组化的研究是至关重要的，内容非常丰富。因此，我们的协议传输醇的脑切片的准备工作，让这些分析具有很大的优势，并最大限度地减少了所用的小鼠。此外，Tureyen 等 10报道，有良好的相关性，间甲酚紫染色法和TTC。

<table border="1"&gt;<tr&gt;<td<strong&gt;描述</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;</tr&gt;<tr&gt;<td&gt;体视显微镜</td&gt;<td&gt;蔡司</td&gt;<td&gt; STEMI 2000</td&gt;</tr&gt;<tr&gt;<td&gt;光纤灯高强度照明灯</td&gt;<td多兰 - 詹纳工业公司</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;，异氟醚麻醉系统</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;异氟醚</td&gt;<td&gt; CDMV</td&gt;<td&gt; 108737</td&gt;</tr&gt;<tr&gt;<td&gt;加热毯</td&gt;<td&gt; Gaymar Medsearch</td&gt;<td&gt; TP22G</td&gt;</tr&gt;<tr&gt;<td红外线加热灯</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;超精细镊子，花柱5</td&gt;<td&gt;电子显微学</td&gt;<td78320-5TI</td&gt;</tr&gt;<tr&gt;<td&gt; Vannas剪刀3毫米切削刃，直</td&gt;<td&gt;电子显微学</td&gt;<td&gt; 72932-01</td&gt;</tr&gt;<tr&gt;<td&gt;样式LA-1和MPF-1所有的光滑叶片</td&gt;<td&gt;电子显微学</td&gt;<td&gt; 77926-5S</td&gt;</tr&gt;<tr&gt;<td&gt;负作用的镊子，风格KOPINC 5</td&gt;<td&gt;电子显微学</td&gt;<td&gt; 72850-F</td&gt;</tr&gt;<tr&gt;<td&gt;解剖剪5 1/2“直</td&gt;<td&gt; CEDARLANE</td&gt;<td&gt; 72960</td&gt;</tr&gt;<tr&gt;<td&gt; Autoclip入门套装</td&gt;<td&gt;哈佛设备</td&gt;<td&gt; 34-0557</td&gt;</tr&gt;<tr&gt;<td&gt; BD Autoclip结束，9毫米长，100/PK截止SYST。</td&gt;<td&gt;费舍尔</td&gt;<td&gt; 01-804-5</td&gt;</tr&gt;<tr&gt;<td&gt; 5-0缝合</td&gt;<td&gt;哈佛设备</td&gt;<td&gt; 517763</td&gt;</tr&gt;<tr&gt;<td缝合材料PDS II单丝紫，5-0，RB-1; Z303H</td&gt;<td&gt; CDMV</td&gt;<td&gt; 6167</td&gt;</tr&gt;<tr&gt;<td&gt; 12毫米长的6-0涂硅单丝缝合</td&gt;<td&gt; Doccol公司</td&gt;<td&gt; 60SPRePK5</td&gt;</tr&gt;<tr&gt;<td&gt; Seringes1毫升</td&gt;<td&gt;费舍尔</td&gt;<td&gt; 14-823-2F</td&gt;</tr&gt;<tr&gt;<td&gt;针26G1 / 2</td&gt;<td&gt;费舍尔</td&gt;<td&gt; BD-305111</td&gt;</tr&gt;<tr&gt;<td<em&gt; C57BL / 6</em&gt;鼠标</td&gt;<td&gt; IRCM</td&gt;<td</td&gt;</tr&gt;</table

mouse model of intraluminal mcao: cerebral infarct evaluation by cresyl violet staining

中风是第三大死亡原因和在世界上的残疾的首要原因。缺血性中风占所有中风的约80％。然而，溶栓组织型纤溶酶原激活剂（tPA）治疗急性缺血性中风的存在是唯一的。这使研究人员开发了几个缺血性中风模型，在不同的物种。两种主要类型的啮齿类动物模型已经开发全脑缺血或局灶性脑缺血模型。为了模拟缺血性中风的患者中，约80％的人在血栓形成或栓塞性中风发生在大脑中动脉（MCA），管腔内的大脑中动脉闭塞（MCAO）模型是非常相关的中风研究的领土。此模型的大鼠Koizumi 等人首先开发于1986年1。由于在小鼠体内的遗传操作的容易性，这些模型也被开发在这一物种2-3。 内容“&gt;在这里，我们提出在C57/BL6小鼠大脑中动脉闭塞的瞬态过程。以前的研究报告的封堵器的物理特性，如齿顶圆直径，长度，形状和灵活性是至关重要的重现性梗死体积的4。这里，一个商业硅涂覆的单丝（Doccol公司）已被使用。另一个很大的优点是，这种单丝诱导蛛网膜下腔出血的风险降低。使用蔡司立体显微镜STEMI 2000，硅涂布的单丝导入颈内动脉（ICA）的外颈动脉（ECA），直到单丝通过一个切割阻塞MCA的基础上恢复血流量，1小时后除去的单丝，以模仿裂解后的血栓栓塞血块中的血流恢复人类。脑梗塞的程度，可以首先计算由神经系统的得分和所测量的梗死体积。因此，分析他们的神经行为学评分在再灌注后不同时间缺血小鼠。为了评估梗死体积，2,3,5 - 氯化三苯基四氮唑（TTC）染色，通常执行。在此，我们用甲酚紫染色，因为它提供了一个机会来测试许多重要的标志物免疫组化。在这段视频中，我们报道了MCAO程序;神经甲酚紫染色的梗死体积分数和评价。

的neuroscore评价证实的tMCAO脑卒中后的成功，并且确定的tMCAO再灌注后的效率。在我们的手中，所有的老鼠至少有一个温和的一致卷曲（neuroscore 2）和神经功能缺损一般是稳定的72小时腔内的过程。大多数小鼠表现出轻度一致的卷曲（neuroscore 2）。没有卷曲的小鼠被排除在研究之外。然而，neuroscore评估不允许我们能够区分小鼠海马受伤的，一个完整的海马。 没有观察到的死亡率在手术当天，表明蛛网膜下腔出血没有发生。当确定在小鼠蛛网膜下腔出血，这个系统从分析中排除。从Doccol公司，这是比家庭自制的单丝光滑，增加了使用的硅涂层单丝成功tMCAO，并降低蛛网膜下腔出血。在这个模型中，24小时和72小时后的死亡率是14％，一般报道为60分钟的阻塞。可能是由于一个大梗死体积的这个小鼠品系的死亡率。还观察到一个强大的病变可重复性（标准偏差为15％），这是非常有趣的研究神经保护作用的分子，可以隐藏他们的影响（S）的模型的变化。 的程度脑损伤tMCAO的评估，我们选择使用甲酚紫染色（ 图2），而不是TTC有大量的材料测试相关的标记6。病变的程度是相对一致的。然而，我们注意到，在一些小鼠，海马受伤（约30％的小鼠）。有趣的是，要注意，甲酚紫染色法可以应用至1周以后。在文献中，脑梗死的百分比从一个不同的研究的。这取决于选择的小鼠品系，麻醉，脑切片的厚度，所用的单丝，或使用6，7的染色。 <img src="/files/ftp_upload/4038/4038fig1.jpg" alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/4038/4038fig1highres.jpg" /> 图1。计划使用硅涂层的管腔内的单丝中脑动脉的闭塞。 A。简化方案的小鼠大脑和脑动脉连续缝合和剪辑的硅涂层，以准备采用单丝。B.通过Willis环的位置，单丝表示。单丝通过 ECA引入ICA，MCA，ACA，大脑前动脉闭塞的基础上，BA，基底动脉，颈总动脉CCA C.威利斯，Willis环; ECA，颈外ARTERY ICA，颈内动脉，MCA，大脑中动脉; PCA，后交通动脉; PPA，翼腭动脉。 <img src="/files/ftp_upload/4038/4038fig2.jpg" alt="图2" /> 图2。甲酚紫染色后72小时再灌注后梗死面积（主要是纹状体，大脑皮层相邻的脑区） 的小鼠脑组织的代表冠状面显示白色（不染甲酚紫）。的损伤体积（白色部分，大脑右半球）分隔和对侧非损伤区域（左侧大脑半球）的百分比表示。

Watch this Scientific Journal Video about Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining at JoVE.com

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

可植入大脑中闭塞小鼠模型中，本文提出的。脑梗塞的程度进行评估，神经行为学评分和甲酚紫染色，染色，TTC替代，提供了巨大的优势，以测试在并行的兴趣标记。

小鼠模型的腔内MCAO脑梗塞评价甲酚紫染色

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

mouse-model-intraluminal-mcao-cerebral-infarct-evaluation-cresyl

Research

JoVE Journal

Medicine

62.8K 次观看.  Clinical Research Institute of Montreal. 可植入大脑中闭塞小鼠模型中，本文提出的。脑梗塞的程度进行评估，神经行为学评分和甲酚紫染色，染色，TTC替代，提供了巨大的优势，以测试在并行的兴趣标记。

视频: Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

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 ...

科研

医学

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 ...

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 ...

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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

汽车测试的脑瘫新生儿小鼠模型电池

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

腔内药物输送到鼠标动静脉瘘内皮

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

小鼠脑海绵体畸形的诱导及显微 CT 成像

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

脑缺血再灌注的双血管闭塞小鼠模型

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...