这项工作是由欧洲共同体的第七框架计划（FP7/2007-2013）根据赠款协议N ° 201024和N ° 202213（欧洲中风网络）。 Bundesministerium献给教化和Forschung（柏林中风研究中心）和德意志研究联合会（Exzellenzcluster NEUROCURE）​​的额外资金。 作者想感谢Mareike Thielke（Dep.实验神经病学，柏林Charité）在操作过程中和埃尔克路德维希（Charité视频服务）制作动画的支持。

为了保证高质量和可重复性，我们建议使用的标准操作程序（SOP）。在这个视频，发表在我们的实验室开发和使用的SOP应用1 1。大脑中动脉闭塞<ol><li>小鼠与咨询兽医人员适当的麻醉制度麻醉。 （如诱导1.5 - 2％异氟醚和维护1.0 - 1.5％异氟醚2 / 3的N2O和1 / 3的氧气使用喷雾器）。 <ol><li>小鼠的体温保持在36.5 ° C ± 0.5 ° C在一个加热板手术。强烈建议，根据鼠标的直肠温度变暖的反馈控制的加热垫。 </li><li>一个合适的代理（如70％酒精）消毒的皮肤和周围的皮毛和事后干燥。 </li></ol></li><li>中线颈部切口及软组织拉开。 </li><li>左颈总动脉（LCCA）仔细解剖周围神经（不伤及迷走神经）和结扎是使用6.0/7.0字符串。也可用于5.0字符串。 </li><li>然后分离左侧颈外动脉（莱萨）和第二个结。 </li><li>接下来是孤立的，左侧颈内动脉（LICA）和6.0长丝准备了一个结。 </li><li>左侧颈内动脉（LICA）和左侧翼腭动脉（LPA）获得良好的查看后，两动脉被裁剪，使用微血管剪辑。 </li><li>之前，分叉的莱萨和LICA在LCCA切开一个小孔。然后引入一个单丝涂有硅固化剂混合物（见下文）的8.0尼龙LICA，直到它停在剪辑。要注意支付不进入枕动脉。 （图1） </li><li>剪切的动脉被打开，而灯丝插入到LICA闭塞Willis环的原产地的LMCA。 </li><li>第三个结对LICA封闭，固定位置的灯丝。 </li><li>小鼠接受生理盐水0.5毫升皮下注射量补货。缓解疼痛，利多卡因凝胶是在伤口局部应用。 </li><li>如果再灌注的目的是，老鼠留30 - 90分钟在激烈的笼子闭塞，可以封闭伤口，用一个小的缝合片段。之后，进行第二次麻醉，结对ICA第三是瞬间打开灯丝撤回。 </li><li>剩余的缝线缩短，适应与手术缝合皮肤。 </li><li>所有的动物都收到如上所述，第二卷补货。 </li><li>动物都放在了两个小时，体温控制在激烈的笼子。 <ol><li>动物必须每天检查，手术后不适的迹象。小鼠可能会​​表现出一定的手术减肥后。他们收到捣碎食物，在培养皿，培养皿放置在地板上，鼓励进食。七天每天食物取代。 </li></ol></li></ol> 2。假手术<ol><li>深水作业的灯丝是插入到闭塞LMCA立即取消允许即时再灌注（1.8）。随后的操作是相同的，发生脑缺血（1.9 - 1.14）的动物，包括第二次麻醉。 </li></ol> 3。灯丝的制备<ol><li>应考虑灯丝不育。事后使用消毒设备，以及适当处理灯丝是无菌手术的先决条件。消毒的灯丝是困难的，因为许多常见的灭菌方法可能会恶化长丝的质量。然而，如辐射的方法，例如紫外线或γ射线，或化学灭菌，例如高活性气体，如环氧乙烷，都适用。 </li><li> 8.0锦纶长丝被切成长度为11毫米，在显微镜下</li><li>灯丝尖端必须超过8毫米的长度，完全并均匀地涂固化剂混合物的Xantopren中号黏膜和激活的NF Optosil </li></ol> 4。代表性的成果血流量限制的期限，根据不同的电机和行为缺陷的结果。两个后30和脑缺血60分钟，在大多数情况下，动物抵抗力下降，横向推盘旋在运动障碍。温和的病变表现为一个在前面的李屈位置MBS。这些很容易观察到的迹象，可以用来作为手术成功的基本得分。 形态病变可使用或者组织学或磁共振成像（MRI）进行评估。六十分钟大脑中动脉闭塞，生产面积包括纹状体和大脑皮层组织pannecrosis，而30分钟，缺血引起主要限于纹状体的神经细胞死亡。梗死体积3（图2） ，我们预期标准偏差低于30％的一组操作。死亡率取决于阻断时间约5％后，30分钟缺血和10 - 60分钟后的20％。 另一种微创的可能性是使用激光多普勒血流计（LDF）的操作过程中，允许其成功的直接控制。在一个单个的动物，减少到10 - 20％preocclusion值明确表示局灶性脑缺血的成功归纳4然而，自卫队可以不被作为个体间的比较的方法使用，因为自卫队可以只测量定量的变化血（百分比）。流内的一个小而有限的组织样本量。它不给的血流量减少的空间范围的信息。 有几种测试，以评估中风后的行为方面，包括步态分析6,7，Rotarod 8，极测试9,10，不干胶去除测试11,12，13,14楼梯测试，梯级测试15,16和Morris水迷宫17。在所有这些测试中，受到缺血的小鼠比对照组动物执行较为成功。 <img src="/files/ftp_upload/2423/2423fig1.jpg" alt="图1" /> 图1。船只提供在小鼠大脑的架构（背景描绘）的计划。不同菌株可能会出现变化，例如的枕动脉，有时叶片颈内动脉。 <img src="/files/ftp_upload/2423/2423fig2.jpg" alt="图2" /> 图2。近端MCAO模型再灌注后不同时间点的典型病变大小的示意图。在中间，功能活动和脑血流量MCAO后的典型当然是描绘。 （MCAO:大脑中动脉闭塞，LDF激光多普勒血流测量）

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M., Prass, K., Meisel, A., 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=Central+nervous+system+injury-induced+immune+deficiency+syndrome.">Central nervous system injury-induced immune deficiency syndrome.</a> Nat Rev Neurosci. 6, 775-786 (2005).</li><li>Engel, O., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Infection+Before+and+After+Stroke:+Investigating+New+Targets.">Models of Infection Before and After Stroke: Investigating New Targets.</a> Infect Disord Drug Targets. 9, (2010).</li><li>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=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> J Cereb Blood Flow Metab. 26, 1465-1478 (2006).</li><li>Macleod, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+laboratory+practice:+preventing+introduction+of+bias+at+the+bench.">Good laboratory practice: preventing introduction of bias at the bench.</a> Stroke. 40, 50-52 (2009).</li><li>Group, S. T. A. I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+standards+regarding+preclinical+neuroprotective+and+restorative+drug+development.">Recommendations for standards regarding preclinical neuroprotective and restorative drug development.</a> Stroke. 30, 2752-2758 (1999).</li></ol>

动物实验Landesamt附耳Gesundheit，柏林，德国和Soziales规定的准则和法规的规定执行。

短暂的，18,19近端MCA闭塞模型，介绍了这里的模拟缺血性中风患者最常见的类型之一 。20变量灌注时间的基础上，该模型提供了不同档次的损害，从短暂性脑缺血发作（ TIA）到大不等包括主要部件的缺血半球梗死。这使研究人员研究中风后不同的病理生理机制。 20,21 手术可以在很短的时间内，生产高度重复性病变。不过，这需要彻底控制了混杂因素。22在操作技术的细微差别可能占不同的效果梗死。23,24此外，由于脑血管解剖的差异，不同的小鼠品系表现出不同的结果 。 25,26机构温度影响，低温导致较小的病灶27和热疗，更严重的赤字的神经损伤 ，28因此，温度控制和维护在这个模型是高度相关的 。29此外，血压和血气结果的重要混杂因素和需要30,31快速，微创方法的使用（非侵入性血压测量，适合和容易访问的采血网站）进行监测。建议。麻醉剂的选择也是非常重要的，因为有些人可能有保护作用，和/或血管扩张剂，例如异氟醚 32因此，接触麻醉应尽可能和规范化。我们排除经历了手术时间超过15分钟的动物。 剃须手术部位产生microabrasions和炎症，并释放头发片段。这可能会进一步加重炎症和促进当地的感染，这可能会影响中风的病理生理。住房条件，特别是使用浓缩，可能会影响中风的结果，并应标准化，并在研究报告中描述的。6,33,34使用环境的丰富和重复性其效果是一个讨论的问题。35另一个重要混杂因素是中风引起的感染的风险，特别是经过较长时间的缺血36的，这会导致额外的发病率和死亡率增加。37,38感染症状在3至5天左右，这已长期在这种模型的后续行动的重要后果。 产生结果，为发展新的治疗中风，标准化和质量控制策略有关，是非常重要的翻译中风在临床前研究39"良好实验室规范 "40,41任务。 <ol><li>使用动物的适当和详细的描述; </li><li>样本量的计算和报告预期的效果大小; </li><li>纳入和排除标准，之前设置的研究; </li><li>随机的分配到组; </li><li>分配隐蔽性调查; </li><li>报告从分析中排除的动物; </li><li>盲法评估的结果; </li><li>报告潜在的利益冲突和研究经费。 </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical scissors (skin cut)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>14028-10</td>
<td>&nbsp;</td>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors (according to Vannas)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Applying forceps for micro clamps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00072-14</td>
<td>&nbsp;</td>
<tr>
<td>Micro vascular clamp (e.g. S&amp;G B1-V)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00396-01</td>
<td>Also Serrefine possible</td>
</tr>
<tr>
<td>2 Hemostats according to Hartmann</td>
<td>&nbsp;</td>
<td>FST</td>
<td>13002-10</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder (according Olsen-Hegar or other)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>12002-14</td>
<td>&nbsp;</td>
<tr>
<td>Standard anatomical forceps (for wound closure)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>5/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for vascular ligatures</td>
</tr>
<tr>
<td>6/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for skin suture, 5/0 also possible</td>
</tr>
<tr>
<td>Lidocaine (e.g. Xylocain Gel 2%)</td>
<td>&nbsp;</td>
<td>Astra Zeneca</td>
<td>&nbsp;</td>
<td>or other local pain relief</td>
</tr>
<tr>
<td>Dissecting microscope (stereo microscope), magnification 6x to max. 40x</td>
<td>&nbsp;</td>
<td>Leica Zeiss</td>
<td>MZ6 Stemi2000C</td>
<td>&nbsp;</td>
<tr>
<td>Cold light source</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>KL1500</td>
<td>&nbsp;</td>
<tr>
<td>Temperature feedback controlled heating pad system</td>
<td>&nbsp;</td>
<td>FST</td>
<td>21052-00</td>
<td>With mouse pad and small probe</td>
</tr>
<tr>
<td>Anaethesia system for isoflurane</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>Abott</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Veterinary Recovery Chamber</td>
<td>&nbsp;</td>
<td>Peco Services</td>
<td>V1200</td>
<td>Heated cage</td>
</tr>
<tr>
<td>8.0 nylon filament</td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>TEL181005</td>
<td>for coating</td>
</tr>
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cutting filament in pieces</td>
</tr>
<tr>
<td>Ruler</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To cut correct length of filament</td>
</tr>
<tr>
<td>Xantopren M Mucosa</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Activator Universal Plus</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>Optosil - Xantopren</td>
</tr>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>A soft underlay for storing and grasping the uncoated filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. swabs, foam, &#x2026;</td>
</tr>
<tr>
<td>Receptacle for new build filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. petri dish, flexible foam,&#x2026;</td>
</tr>		</tbody>
		</table>
		</div>

modeling stroke in mice - middle cerebral artery occlusion with the filament model

中风是之间的死亡和残疾成人，尤其是在高度发达的国家，最常见的原因。然而，迄今为止的治疗方案是非常有限的的。为了满足新的治疗方法需要，中风的实验研究经常采用局灶性脑缺血啮齿动物模型。大多数研究人员使用的小鼠或大鼠大脑中动脉（MCA）的永久或暂时闭塞。 通过腔内缝合技术（所谓长丝或缝合模型）（MCA）的大脑中动脉近端闭塞可能是实验性中风的研究中最常用的模型。腔内MCAO模型提供了在一个相对非侵入性的方式重现短暂或永久MCA的领土缺血诱导的优势。腔内的方法中断的整个领土的这个动脉的血流量。因此，长丝闭塞逮捕流近端lenticulo纹动脉，供应基底节。长丝闭塞，在皮层和纹状体的重现性病变MCA的结果可以是永久或短暂。相比之下，MCA闭塞模型，诱导远端（lenticulo纹动脉分支）通常备用纹状体和主要涉及的大脑皮层。此外，这些模型需要去骨瓣。本文演示模型，硅涂层长丝引入左颈总动脉和先进的威利斯，它阻断大脑中动脉的起源圈沿颈内动脉。大脑中动脉闭塞的患者，是缺血性中风的最常见的原因。由于不同缺血时间间隔可自由选择在这个模型再灌注时间点上，可以产生不同程度的缺血性病变。再灌注去除咬合长丝至少部分模型自发或治疗（TPA）在人类的血栓栓塞血块溶解后恢复血液流动。 在这个视频中，我们将目前的基本技术以及主要缺陷和混杂因素，这可能会限制这个模型的预测值。

Watch this Scientific Journal Video about Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model at JoVE.com

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

丝状的大脑中动脉闭塞，是一个研究小鼠缺血性中风的常见模型。

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 ...

modeling-stroke-mice-middle-cerebral-artery-occlusion-with-filament

Research

JoVE Journal

Medicine

91.4K 次观看.  Center for Stroke Research Berlin, Charité Universitätsmedizin. 丝状的大脑中动脉闭塞，是一个研究小鼠缺血性中风的常见模型。

视频: Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

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 ...

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 ...

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

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

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

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

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

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

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

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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

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

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

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

栓塞性脑梗塞（缺血）的大鼠缺血性中风与血同源凝块

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

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

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

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

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

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

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

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

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, ...