Name: transient middle cerebral artery occlusion model of stroke
Uploaded: 2023-08-11T14:40:00.000+00:00
Duration: 5 min 32 s
Description: Watch this Scientific Journal Video about Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model at JoVE.com

该研究由 科学与创新部 （MCIN）/Agencia Estatal de Investigación （AEI）、 Gobierno de España/10.13039/501100011033 和“欧洲区域发展基金 （ERDF）”资助的赠款 PID2020-113202RB-I00 支持。一种创造欧洲的方式“。NCC 和 MAR 拥有由 MCIN/AEI/10.13039/501100011033 和“欧洲社会基金 （ESF） 投资您的未来”资助的博士前奖学金（分别为 PRE2021-099481 和 PRE2018-085737）。我们感谢弗朗西斯卡·鲁伊斯-哈恩和莱昂纳多·马尔克斯-基西努斯基的技术支持。我们感谢 Institut d'Investigacions Biomèdiques August Pi i Sunyer （IDIBAPS） 的 MRI 成像设施的支持。加泰罗尼亚政府加泰罗尼亚研究中心（CERCA）计划支持IDIBAPS。

动物工作是按照加泰罗尼亚和西班牙法律（Real Decreto 53/2013）和欧洲指令进行的，并得到了巴塞罗那大学伦理委员会（Comité Ètic d'Experimentació Animal，CEEA）和加泰罗尼亚政府当地监管机构的批准。研究报告符合ARRIVE指南。该程序旨在在成年小鼠中进行，从 8 周龄开始，没有年龄限制。这里提供了在10-12周龄的C57BL / 6小鼠中开发的外科手术的示例。应考虑取决于小鼠品系的解剖学差异。
1.动物准备
<ol><li>在开始外科手术之前，请收集并消毒所有必需的材料和工具。用所有必要的手术材料（在 材料表中列出）设置手术台。</li>
	<li>使用异氟醚吸入氧气和一氧化二氮（30%/70%）的混合物麻醉动物。</li>
	<li>皮下注射丁丙诺啡（见 材料表），剂量为0.05mg / kg BW，以提供镇痛并减轻任何疼痛和不适。 注意：镇痛是强制性的，但接受不同的方案。在MCAo后的第一天，疼痛和不适的体征也必须得到控制（见步骤4）。必要时应用纠正解决方案。</li>
	<li>将动物置于含有5%异氟烷的麻醉诱导箱（见 材料表）中，直至达到深度麻醉状态（爪子穿刺和眼反射反射丧失）。</li>
	<li>将鼠标放在手术台上，将异氟烷的含量降低到1.5%，通过面罩给药。涂抹兽医软膏以避免在手术过程中眼睛干涩。</li>
	<li>将体温保持在37±0.5°C，由连接到加热垫的直肠探头控制（参见 材料表）。</li>
	<li>用电动剃须刀剃掉颈部和头部的腹侧部分（颅骨）。小心地清除毛皮碎屑，并用碘基消毒剂和 70% 酒精以打圈的方式对皮肤区域进行三次消毒。 .</li>
</ol>2. 使用激光多普勒血流法 （LDF） 进行脑血流 （CBF） 评估
<ol><li>用剪刀在头部皮肤上切开一个切口，沿着矢状缝线的方向，从耳朵到眼睛之间的区域。</li>
	<li>缩回皮肤并去除颅骨右侧的骨膜。</li>
	<li>找到坐标（距 Bregma 侧 2.5 毫米）并使用氰基丙烯酸酯连接多普勒支架（参见 材料表）。胶水干燥后，连接多普勒探头并检查信号读数是否正确。</li>
</ol>3. 短暂性大脑中动脉闭塞 （tMCAo）
<ol><li>将鼠标翻转到仰卧位，并用医用胶带将其固定在手术台上。</li>
	<li>在脖子上做一个中线切口。使用牵开器侧向拉回皮肤和唾液腺（见 材料表），以暴露颈动脉区域。</li>
	<li>识别颈总动脉 （CCA）、ICA 和 ECA 的血管解剖结构，以及由它们衍生的不同动脉（上颌动脉和舌动脉、甲状腺上动脉、枕动脉和翼腭动脉）（图 1A）。</li>
	<li>将主动脉与相邻的结缔组织分离，以便处理它们。 注意：特别注意不要损伤神经，尤其是与 CCA 平行的迷走神经。</li>
	<li>在上颌/舌分叉处将 6-0 真丝缝合线（见 材料表）缠绕在 ECA 周围。紧紧打结以永久中断循环。</li>
	<li>在同一条动脉周围，在第一个结和 CCA 分叉之间通过第二条缝合线，并保持这个结松动。</li>
	<li>在 CCA 周围放置第三根线并打一个可以轻松解开的滑结。 注意： 这也可以使用血管夹进行，但螺纹允许更多的运动和灵活性。在这个阶段，可以观察到LDF信号中CBF的首次降低。</li>
	<li>放置一个血管夹（见 材料表），中断ICA的血液循环。</li>
	<li>在 ECA 上做一个小切口，靠近紧结所在的区域。</li>
	<li>插入单丝，直到厚涂层完全进入动脉腔。</li>
	<li>拧紧第二个结，将单丝固定在动脉内，防止血液施加的压力将其推出（图1B）。</li>
	<li>从ICA上取下血管夹。</li>
	<li>将ECA切到第一个结下方，并旋转树桩以使其朝向ICA的方向（图1C）。</li>
	<li>通过 ICA 推进单丝，直到 MCA 分支点。 注意：闭塞反映在 LDF 读数中的血流量突然下降。当 CBF 下降大于基础值的 70% 时，我们认为闭塞成功。如果没有CBF测量系统，则可以通过前进阻力来记录闭塞点，在成年小鼠中，该阻力通常距离CCA的分叉约11毫米。<ol><li>如果在闭塞期间继续麻醉，请监视小鼠并持续观察45分钟。</li>
		<li>如果老鼠在闭塞期间被唤醒，请缝合几针的颈部皮肤。在不断开LDF探头的情况下，将鼠标放入温控盒中，以便从麻醉中恢复。 注意：在此期间，鼠标表现出自发的盘旋行为是很常见的，表明成功闭塞。</li>
		<li>40分钟后，按照与1.4,1.5和1.7点相同的麻醉和消毒程序再次麻醉小鼠。将其放回手术台上，然后从颈部拆下缝线。</li>
	</ol></li>
	<li>闭塞 45 分钟后，松开将单丝固定到位的结。缓慢而轻柔地拉动细丝，检查是否发生了组织再通。</li>
	<li>拉出灯丝并拧紧结以防止失血。</li>
	<li>解开 CCA 结。确保动脉壁没有损伤。</li>
	<li>取下牵开器并重新定位肌肉、腺体和皮肤。缝合皮肤（6-0）并涂抹消毒剂。</li>
	<li>断开多普勒探头，然后拆下支架。缝合和消毒头部皮肤。</li>
	<li>在麻醉恢复期间，将小鼠留在装有加热器的笼子中以保持温度。持续观察，直到它从麻醉中完全恢复。恢复后，老鼠可以放回笼子里。 注意：强烈建议提供具有社会丰富的住房。但是，切勿将操作的小鼠与未操作的小鼠混合在同一个笼子中，没有任何物理隔离，以防止攻击性。</li>
</ol>4. 术后护理
<ol><li>按照当地法规制定的程序和规定定期监督动物。按适当的时间表提供镇痛治疗，以尽量减少手术后的疼痛。 注意：在本研究中，在手术后 6 小时和 24 小时施用与干预开始时相同的镇痛剂（丁丙诺啡 0.05mg/kg BW）。</li>
	<li>当监督参数表明安乐死时，按照机构批准的协议进行安乐死。</li>
	<li>每天监测动物的体重。在手术后的最初几天为动物提供软食。此外，在手术后立即通过皮下注射生理盐水（200μL）来水化它们，如果观察到小鼠不自行水化，则定期水化。以动物容易获得的方式安排食物和水。</li>
	<li>体内研究完成后，麻醉小鼠，对它们实施安乐死，并取出脑组织进行进一步的组织病理学分析（如果需要）。</li>
</ol>

大脑中动脉闭塞的短暂性脑缺血模型

<ol>
	<li>Siddiqi, A. Z., Wadhwa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+acute+stroke:+current+practices+and+future+horizons.">Treatment of acute stroke: current practices and future horizons.</a> Cardiovascular Revascularization Medicine. 49, 56-65 (2023).</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+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> Journal of Cerebral Blood Flow &amp; Metabolism. 1, 53-60 (1981).</li><li>Koizumi, J., 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.+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. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Japanese Journal of Stroke. 8, 1-8 (1986).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, R., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+ischemia:+Models,+methods,+and+outcomes.">Cerebral ischemia: Models, methods, and outcomes.</a> Neuropharmacology. 55, 257-270 (2008).</li><li>Seto, A., 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=Induction+of+ischemic+stroke+in+awake+freely+moving+mice+reveals+that+isoflurane+anesthesia+can+mask+the+benefits+of+a+neuroprotection+therapy.">Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.</a> Frontiers in Neuroenergetics. 6, 1 (2014).</li><li>D&#237;az-Marugan, L., 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=Poststroke+lung+infection+by+opportunistic+commensal+bacteria+is+not+mediated+by+their+expansion+in+the+gut+microbiota.">Poststroke lung infection by opportunistic commensal bacteria is not mediated by their expansion in the gut microbiota.</a> Stroke. 54 (7), 1875-1887 (2023).</li><li>Xie, L., Kang, H., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+transient+occlusion+of+the+middle+cerebral+artery+in+awake+mice.">A novel model of transient occlusion of the middle cerebral artery in awake mice.</a> Journal of Natural Sciences. 2 (2), e176 (2016).</li><li>Arbaizar-Rovirosa, M., 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=Aged+lipid-laden+microglia+display+impaired+responses+to+stroke.">Aged lipid-laden microglia display impaired responses to stroke.</a> EMBO Molecular Medicine. 15 (2), e17175 (2023).</li><li>Orsini, F., 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=Targeting+mannose-binding+lectin+confers+long-lasting+protection+with+a+surprisingly+wide+therapeutic+window+in+cerebral+ischemia.">Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.</a> Circulation. 126 (12), 1484-1494 (2012).</li><li>Majid, A., 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=Differences+in+vulnerability+to+permanent+focal+cerebral+ischemia+among+3+common+mouse+strains.">Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains.</a> Stroke. 31, 2707-2714 (2000).</li><li>Rogers, D. C., Campbell, C. A., Stretton, J. L., Mackay, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+motor+impairment+and+infarct+volume+after+permanent+and+transient+middle+cerebral+artery+occlusion+in+the+rat.">Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat.</a> Stroke. 28, 2060-2065 (1997).</li><li>Hedna, V. 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=Validity+of+Laser+Doppler+flowmetry+in+predicting+outcome+in+murine+intraluminal+middle+cerebral+artery+occlusion+stroke.">Validity of Laser Doppler flowmetry in predicting outcome in murine intraluminal middle cerebral artery occlusion stroke.</a> Journal of Vascular and Interventional Neurology. 8 (3), 74-82 (2015).</li><li>Yin, L., 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=Laser+speckle+contrast+imaging+for+blood+flow+monitoring+in+predicting+outcomes+after+cerebral+ischemia-reperfusion+injury+in+mice.">Laser speckle contrast imaging for blood flow monitoring in predicting outcomes after cerebral ischemia-reperfusion injury in mice.</a> BMC Neuroscience. 23, 80 (2022).</li><li>Thakkar, P. C., 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=Therapeutic+relevance+of+elevated+blood+pressure+after+ischemic+stroke+in+the+hypertensive+rats.">Therapeutic relevance of elevated blood pressure after ischemic stroke in the hypertensive rats.</a> Hypertension. 75 (3), 740-747 (2020).</li><li>Yu, X., Feng, Y., Liu, R., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+protects+mice+against+ischemic+stroke+by+modulating+macrophage+polarization+through+upregulation+of+interferon+regulatory+factor-4.">Hypothermia protects mice against ischemic stroke by modulating macrophage polarization through upregulation of interferon regulatory factor-4.</a> Journal of Inflammation Research. 14, 1271-1281 (2021).</li><li>Denorme, F., Portier, I., Kosaka, Y., Campbell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+exacerbates+ischemic+stroke+outcome+independent+of+platelet+glucose+uptake.">Hyperglycemia exacerbates ischemic stroke outcome independent of platelet glucose uptake.</a> Journal of Thrombosis and Haemostasis. 19, 536-546 (2021).</li></ol>

作者声明没有利益冲突。

腔内 tMCAo 手术是基础研究中最常用的局灶性脑缺血再灌注模型。目前，由于转基因菌株的可用性，小鼠是首选的动物模型。然而，必须承认转基因小鼠及其遗传背景会影响脑血管形成。不同动脉区域之间侧支循环和吻合的存在可以显着影响实验程序的结果 11。
在执行此程序时，必须考虑某些关键点。损伤可能发生在 MCA 区域之外，累及海马体、丘脑或下丘脑等区域，通常是由于后交通动脉闭塞所致。此外，尽管外科手术看似成功，但一小部分小鼠可能不会表现出明显的梗塞。
在此过程中需要监控几个变量。脑部病变的发展直接取决于脑血流量 （CBF） 下降的严重程度和这种减少的持续时间 5,12。为了在手术过程中跟踪 CBF 并评估闭塞期间和再灌注后的流量变化，强烈建议使用 LDF（激光多普勒流量计）或激光散斑流量计13,14 等系统。闭塞的持续时间也会影响病变的程度，闭塞持续时间 30 分钟或更短，主要影响纹状体，闭塞时间超过 45 分钟，也影响 MCA 提供的皮质区域。考虑到多种变异性因素，在研究开始之前建立纳入/排除标准并报告它们至关重要。
此外，血压、体温和血糖等其他因素也会显着影响中风结果。在闭塞期间将小鼠保持在麻醉状态可能会影响血压、突触兴奋性或炎症等参数 6,15。另一种选择是在闭塞期间唤醒动物。
麻醉会影响血压，进而影响梗塞的大小 15.由于体温过低和体温过高对脑缺血的影响有据可查，因此保持适当的体温至关重要16。此外，高血糖已被证明会增加缺血性损伤17。此外，年龄和性别是设计实验和分析结果时必须考虑的因素。
不应将因素的多样性视为缺点，而应将其视为优势，但在计算样本量时记录变量并考虑可变性至关重要。将实验研究结果转化为临床实践的失败部分可归因于实验组动力不足以及使用不能充分代表人类病理状况的动物模型。通常，在实验模型中使用年轻，健康，主要是雄性小鼠，但这些可以增强以研究患有高血压，高血糖或高胆固醇血症等合并症的小鼠，以及不同年龄组和性别的小鼠。

根据世界卫生组织的数据，中风是一种毁灭性的疾病，每年影响全球约 1500 万人。大约三分之一的患者死于这种疾病，而另外三分之一的患者则经历永久性残疾。卒中是一种复杂的病理学，涉及各种细胞类型，例如神经和外周免疫细胞、脉管系统和全身反应1。目前无法使用 体外 模型复制由系统水平的中风引发的复杂反应网络。因此，实验动物模型对于深入研究疾病的机制以及开发和测试新疗法至关重要。目前，早期组织再灌注是唯一获批的干预措施，通过组织型纤溶酶原激活剂 （tPA） 溶栓或血管内血栓切除术1。
脑中动脉 （MCA） 闭塞在卒中患者中很常见。因此，最初在大鼠中开发了瞬时 MCA 闭塞 （tMCAo） 的啮齿动物模型 2,3,4。如今，转基因小鼠是实验性中风模型中最常用的动物。在这项研究中，我们描述了小鼠腔内 tMCAo 的微创模型。该方法通过颈部水平的颈动脉进行，无需开颅手术。
闭塞期的持续时间是决定缺血性病变程度的关键因素。即使是 10 分钟的短暂闭塞也会导致选择性神经元死亡，而不会出现明显的梗死，而较长时间的闭塞（通常持续 30 至 60 分钟）会导致一定程度的脑梗死。与供应皮层并具有侧支的 MCA 的近端和远端分支不同，向纹状体提供血液的透镜状体动脉缺乏侧支5。因此，在 tMCAo 之后，纹状体中的血流量比皮层中的血流量减少更大。因此，30 分钟或更短时间的闭塞通常影响纹状体但不影响皮质，而从 45 分钟开始的较长时间闭塞通常会在整个 MCA 区域（包括纹状体和背外侧皮层）产生缺血性病变。
为了确保小鼠的健康，我们在手术前使用镇痛药，并在手术过程中使用麻醉剂。然而，麻醉可能会在小鼠的生理机能中引入人为的改变，并影响一些结果测量6。当由有经验的人员进行手术干预时，通常持续约 15 分钟以诱导 MCAo。随后，麻醉下的总时间取决于闭塞期。对于最小化麻醉至关重要的实验，手术中的替代步骤是在闭塞期间停止麻醉，并将其限制在插入和拔出闭塞 MCA 的细丝的手术步骤中。这种方法缩短了麻醉的持续时间，并最大限度地减少了其对实验模型的潜在人工影响7,8。因此，诱导短暂性局灶性缺血的方法通过MCA的腔内闭塞呈现，具有两种变体：在整个闭塞期间小鼠麻醉或在此期间小鼠清醒。在任何一种情况下，假手术都应与对缺血小鼠进行的干预同时进行。此外，在再灌注后的不同时间点通过行为测试和 MRI 测量，提供有关结果评估的数据。最后，讨论了实施实验程序时要考虑的主要因素。

<table><tbody><tr><td>6/0 suture&amp;nbsp;</td><td>Arago</td><td></td><td>Vascular ligatures</td></tr><tr><td>6/0 suture with curved needle</td><td>Arago</td><td></td><td>Skin sutures</td></tr><tr><td>9 mg/mL Saline</td><td>Fresenius Kabi</td><td>CN616003 EC</td><td>For hydration</td></tr><tr><td>Anaesthesia system</td><td>SurgiVet</td><td></td><td></td></tr><tr><td>Blunt retractors, 1 mm wide</td><td>Fine Science Tools</td><td>18200-09</td><td></td></tr><tr><td>Buprenorfine</td><td>Buprex</td><td></td><td>For pain relief</td></tr><tr><td>Clamp applying forceps</td><td>Fine Science Tools</td><td>S&amp;amp;T CAF4</td><td></td></tr><tr><td>Dumont mini forceps</td><td>Fine Science Tools</td><td>M3S 11200-10</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>91106-12</td><td></td></tr><tr><td>Glue</td><td>Loctite</td><td></td><td>To stick LDF probe to the skull</td></tr><tr><td>Grip Strength Meter</td><td>IITC Life Science Inc.</td><td>#2200</td><td></td></tr><tr><td>Isoflurane</td><td>B-Braun</td><td>CN571105.8</td><td></td></tr><tr><td>LDF Perimed</td><td>Perimed</td><td>Periflux System 5000</td><td></td></tr><tr><td>LDF Probe Holders</td><td>Perimed</td><td>PH 07-4</td><td></td></tr><tr><td>Medical tape</td><td></td><td></td><td></td></tr><tr><td>MRI magnet</td><td>Bruker BioSpin, Ettlingen, Germany</td><td></td><td>BioSpec 70/30 horizontal animal scanner&amp;nbsp;</td></tr><tr><td>Needle Holder with Suture Cutter</td><td>Fine Science Tools</td><td>12002-14</td><td></td></tr><tr><td>Nylon filament</td><td>Doccol</td><td>701912PK5Re</td><td></td></tr><tr><td>Recovery cage with heating pad</td><td></td><td></td><td></td></tr><tr><td>Sirgical scissors</td><td>Fine Science Tools</td><td>91401-12</td><td></td></tr><tr><td>Small vessel cauterizer kit</td><td>Fine Science Tools</td><td>18000-00</td><td></td></tr><tr><td>Stereomicroscope and cold light</td><td>Leica</td><td>M60</td><td></td></tr><tr><td>Suture tying forceps</td><td>Fine Science Tools</td><td>18025-10</td><td></td></tr><tr><td>Thermostat, rectal probe and mouse pad</td><td>Letica Science Instruments</td><td>LE 13206</td><td></td></tr><tr><td>Vannas spring scissors (4mm cutting edge)</td><td>Fine Science Tools</td><td>15019-10</td><td></td></tr><tr><td>Vascular clamps</td><td>Fine Science Tools</td><td>00396-01</td><td></td></tr></tbody></table>

transient middle cerebral artery occlusion model of stroke

中风是全球死亡或慢性残疾的主要原因。然而，现有的最佳治疗方法仅限于缺血性卒中急性期的再灌注治疗。为了深入了解中风生理学并开发创新的治疗方法，中风 的体内 啮齿动物模型起着重要作用。转基因动物的可用性特别推动了小鼠作为实验性中风模型的使用。
在卒中患者中，大脑中动脉 （MCA） 闭塞很常见。因此，最普遍的实验模型涉及 MCA 的腔内闭塞，这是一种不需要开颅手术的微创技术。该过程包括通过颈外动脉 （ECA） 插入单丝并将其推进通过颈内动脉 （ICA），直到它到达 MCA 的分支点。动脉闭塞 45 分钟后，移除单丝以允许再灌注。在整个过程中，监测脑血流以确认闭塞期间的减少和随后的再灌注恢复。使用行为测试和磁共振成像 （MRI） 研究评估神经和组织结果。

有不同的方法可以评估 tMCAo 程序的结果。这里使用 体内 神经成像方法 （MRI） 和行为测试。
小鼠在大脑中出现缺血性病变，主要影响MCA同侧提供给闭塞的区域，如纹状体和背外侧皮层。有几种方法可以确定病变范围，包括 2,3,5-三苯基氯化四氮唑 （TTC） 组织染色、组织学染色（苏木精/伊红素、乙酸亚硫氨酸）和 MRI 等 体内 神经影像学检查。这里之所以选择 MRI，是因为它具有非侵入性，并且能够将相同的组织用于其他研究，从而对每只小鼠的病变进行全面评估。此外，MRI允许在同一只动物中重复测量，提高了结果的可重复性，并且通常减少了研究所需的动物数量。
在 MRI 会议中使用异氟醚的相同麻醉方案（诱导 5%，维持 1.5%）。对于病变体积评估，使用快速 T2 加权序列（T2w turbo RARE 快速自旋回波）9 来最大限度地减少动物被麻醉的时间，这对于在同一只小鼠中进行不同时间的 MRI 采集的纵向研究很重要。该程序允许评估同一动物的病变随时间的变化，并且在用于神经保护研究或测试药物疗效等时非常有用。在7T卧式动物扫描仪上进行图像实验。解剖序列的技术规格（可能因磁场强度而异）：T2_TurboRARE;22个冠状切片;0.5 毫米厚;回波时间 （TE） = 33 ms;重复时间 （TR） = 2336.39 ms. 2 个平均值。翻转角度，90°;视场 （FOV） = 20 mm x 20mm，矩阵尺寸为 256 x 256。 图2A 显示了在同一只小鼠中病变演变的MR图像的代表性示例，在再灌注后40分钟，6小时，24小时和48小时进行评估。病变体积的进展需要数小时至大约两天才能完成。病变体积的量化显示了这种随时间的演变（图2B）。
已经描述了各种神经学量表来评估缺血性损伤引起的神经损伤。我们建议使用在以前的手稿中广泛描述的神经评分测试。例如，推荐使用Orsini等人（2012）10 详细报告的测试。
有各种各样的行为测试可供选择，主要是为了检测运动和感觉功能障碍的差异。为此，使用了握力测试和拐角测试。握力测试用于评估运动功能。前肢力量是用连接到数字力传感器的握力计测量的（见 材料表）。老鼠用两只前爪抓住单杠，同时轻轻地将其从尾巴向后拉。注意前爪松开前握力的最大强度。每只动物进行五次试验，在排除最大值和最小值后计算主要值。角试验用于检测感觉和运动功能的单侧异常。该装置由一个角组成，角有两块板（30 厘米× 20 厘米× 1 厘米），角度为 30°，末端有一个小开口。鼠标朝向角落放置一半。当鼠标深入角落时，振动的两侧一起受到刺激。然后鼠标转回面向开口端。每只动物总共进行 10 次试验，并注明所选的方面。在生理条件下，预计有 50% 的左转和右转，而在具有右 MCAo 的小鼠中，预计会有右转偏好。当完成完全转圈或鼠标将头转≥ 90º 时，试验被认为是有效的。结果显示为右转（同侧）转弯的百分比。
显示了通过握力测试测量的tMCAo后24小时小鼠表现出的力量损失的代表性结果（图3A），以及它们在角落测试中受到刺激时转向病变同侧的偏好（图3B）。在手术的同一天进行行为测试可能不太精确，因为某些参数可能会因麻醉和术后时间的接近而改变。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65857/65857fig01.jpg"> 图1：颈部维管树示意图（右侧）。 （A） 图像显示了主要动脉（颈总动脉-CCA、颈外动脉-ECA、颈内动脉-ICA）和不同的分支（翼腭动脉 Pt;枕动脉 Occ;甲状腺上动脉 St;上颌动脉和舌动脉Max/Lin）。（B） 外科手术的第一步，通过缝合线结扎 CCA，血管夹中断 ICA 循环， 并通过 ECA 引入单丝。（C） 重新定向 ECA 以将单丝推到闭塞区。 <a href="https://www.jove.com/files/ftp_upload/65857/65857fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65857/65857fig02.jpg"> 图 2：代表性的 MR 图像。 （A）再灌注后同一小鼠在不同时间点的T2-w图像显示急性期病变的演变。受梗死影响的区域在最初的几个小时内迅速增加，此后几乎没有变化。（B） MCAo 后急性期病变体积的演变。每个条形代表病变体积百分比 （%） 的平均值± SD。在再灌注后的前 24 小时内病变体积显着增加（*p = 0.0182;**p = 0.0088;单因素方差分析/Kruskal-Wallis 检验）。 <a href="https://www.jove.com/files/ftp_upload/65857/65857fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65857/65857fig03v2.jpg"> 图 3：tMCAo 之前（基础）和 24 小时后的行为测试（n = 16 只小鼠）。 （A） 握力测试显示每只小鼠的最大（最大）力量。（B） 弯道测试显示右转的百分比 （%）。图表显示每组的方框和胡须（最小值到最大值），点对应于单个小鼠（****p < 0.0001;Wilcoxon matched-pairs signed rank test）。 <a href="https://www.jove.com/files/ftp_upload/65857/65857fig03largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model at JoVE.com

作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

该方案描述了通过大脑中动脉腔内闭塞在小鼠中短暂性局灶性脑缺血的模型。此外，还显示了使用磁共振成像和行为测试的结果评估示例。

脑卒中短暂性中动脉闭塞模型

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

大脑中动脉闭塞模型被广泛用于模拟小鼠缺血性脑卒中。该模型允许研究缺血性损伤的时间进展，评估神经保护剂的有效性，并探索特定基因和通路在中风病理学中的作用，最终目的是寻找新的潜在治疗方法。MRI等神经成像技术的实施可以更好地诊断，改善结果评估，并减少每项研究中所需的动物数量。该协议对诱导短暂性大脑中动脉闭塞的最常用技术进行了清晰而详细的解释。此外，它通过引入闭塞期间麻醉退出的精确修改来实现该技术，从而减少了长时间麻醉时间的不利影响。

transient-middle-cerebral-artery-occlusion-model-of-stroke

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Transient Middle Cerebral Artery Occlusion Model of Stroke

1.7K 次观看.  Spanish National Research Council (CSIC). 大脑中动脉闭塞模型被广泛用于模拟小鼠缺血性脑卒中。该模型允许研究缺血性损伤的时间进展，评估神经保护剂的有效性，并探索特定基因和通路在中风病理学中的作用，最终目的是寻找新的潜在治疗方法。MRI等神经成像技术的实施可以更好地诊断，改善结果评估，并减少每项研究中所需的动物数量。该协议对诱导短暂性大脑中动脉闭塞的最常用技术进行了清晰而详?...

视频: 作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

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

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 ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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

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

基于短暂性脑缺氧缺血性血栓性中风模型

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

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

对大脑中动脉闭塞再灌注损伤诱发中风小鼠的手术方法

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

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

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

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

模拟小鼠中风:通过颈外动脉的短暂性脑中动脉闭塞

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

神经科学

建立经颅大脑中动脉闭塞小鼠模型

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

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

作者聚焦:中风研究的创新技术和未来方向 

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

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

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

脑卒中短暂性中动脉闭塞模型

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在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

模拟小鼠中风:通过颈外动脉的短暂性脑中动脉闭塞

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局