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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6/0 suture&#160;</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&#38;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&#160;</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。

Stroke is a devastating disease that affects approximately 15 million people worldwide annually, according to the WHO. Around one-third of patients succumb to the condition, while another third experience permanent disability. Stroke is a complex pathology involving various cell types, such as neural and peripheral immune cells, vasculature, and systemic responses1. The intricate network of reactions triggered by stroke at the systems level cannot be currently replicated using in vitro models. Thus, experimental animal models are essential to delve into the disease&#39;s mechanisms and to develop and test new therapies. Currently, early tissue reperfusion is the only approved intervention, either through thrombolysis with tissue-type plasminogen activator (tPA) or endovascular thrombectomy1.
Occlusions of the middle cerebral artery (MCA) are frequent in stroke patients. Consequently, rodent models of transient MCA occlusion (tMCAo) were initially developed in rats2,3,4. Nowadays, genetically modified mice are the most commonly used animals in experimental stroke models. In this study, we describe a minimally invasive model of intraluminal tMCAo in mice. The approach is performed via the carotid artery at the neck level, without craniectomy.
The duration of the occlusion period is a critical factor that determines the extent of the ischemic lesion. Even short occlusions of 10 min can cause selective neuronal death without an apparent infarction, while longer occlusions, typically lasting 30 to 60 min, result in some degree of cerebral infarction. Unlike the proximal and distal branches of the MCA that supply the cortex and have collaterals, the lenticulo-striatal arteries providing blood to the striatum lack collaterals5. As a consequence, there is a greater reduction of blood flow in the striatum than in the cortex after tMCAo. Thus, occlusions of 30 min or less generally affect the striatum but not the cortex, whereas longer occlusions, from 45 min onwards, often generate an ischemic lesion in the entire MCA territory, including the striatum and dorsolateral cortex.
To ensure the well-being of the mice, we administer analgesics prior to the procedure and use anesthesia during surgery. Nevertheless, anesthesia can potentially introduce artificial alterations in the physiology of the mouse and affect some outcome measures6. The surgical intervention, when performed by experienced personnel, usually lasts about 15 min for inducing MCAo. Subsequently, the total time under anesthesia depends on the occlusion period. For experiments where minimizing anesthesia is crucial, an alternative step in the procedure involves discontinuing anesthesia during the occlusion period and limiting it only to the surgical steps for inserting and withdrawing the filament occluding the MCA. This approach reduces the duration of anesthesia and minimizes its potential artifactual effects on the experimental model7,8. Therefore, the method of inducing transient focal ischemia is presented by intraluminal occlusion of the MCA with two variants: with the mouse anesthetized during the entire occlusion period or with the mouse awake during this period. In either case, a sham surgery should be performed in parallel with the intervention carried out on the ischemic mice. Additionally, data on outcome assessment is provided as measured by behavioral tests and MRI at various time points after reperfusion. Finally, the main factors to consider when implementing the experimental procedure are discussed.

作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

脑卒中短暂性中动脉闭塞模型

The middle cerebral artery occlusion model is widely used to simulate ischemic stroke in mice. This model allows to investigate a temporal progression of ischemic damage, evaluate effectiveness of neuroprotective agents, and explore the role of specific genes and pathways in stroke pathology with a final aim of finding new potential treatments. The implementation of neuroimaging techniques such as MRI allows to make better diagnosis, improve the assessment of outcomes, and reduce the number of animals needed in each study.This protocol offers a clear and detailed explanation of the most commonly used technique for inducing transient middle cerebral artery occlusion. Moreover, it implements the technique by introducing the precision modification of anesthesia withdrawal during occlusion time, reducing the detrimental effect of prolonged anesthesia time.

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...

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

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。

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

Research

JoVE Journal

Neuroscience

Transient Middle Cerebral Artery Occlusion Model of Stroke

1.2K 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 

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

科研

神经科学

Surgical Procedure for Transient Middle Cerebral Artery Occlusion

To begin, position the anesthetized mouse on the operating table. Maintain body temperature using a rectal probe connected to a heating pad. Make an incision on the skin of the head in the direction of the sagittal suture, starting from the ears to the area between the eyes.Retract the skin and remove the periosteum on the right side of the skull. Then find the coordinates, 2.5 millimeters lateral from bregma. Apply cyanoacrylate to the doppler holder and attach it for laser-doppler flowmetry.After the glue has dried, connect the doppler probe. Then, check for the correct signal readout for assessing the cerebral blood flow. To perform artery occlusion, turn the mouse over to the supine position and fix it to the surgical table with medical tape.Then make a midline incision on the neck. Pull the skin laterally, followed by pulling away the salivary glands using retractors. This exposes the carotid territory.Identify the vascular anatomy of the common carotid artery, or CCA, the ICA, and the ECA, as well as the different arteries derived from them. For easier handling, detach CCA, followed by ECA from the connective tissue. Then cauterize the superior thyroid artery and detach ICA from the connective tissue.Check again to make sure the main arteries are fully detached from the connective tissue. Next, wrap 6-0 silk sutures around the ECA at the maxillary lingual bifurcation. Then suture around the CCA.Place a vascular clip interrupting blood circulation from the ICA. Make a small incision in the ECA close to the area where the tight knot is located. Insert the monofilament until the thick coating has completely entered the arterial lumen.Tighten the second knot to hold the monofilament inside the artery and to prevent the pressure exerted by the blood from pushing it out. Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA. Advance the monofilament via the ICA until the point where the MCA branches out.The occlusion is reflected in an abrupt blood flow drop in the LDF readout. After 40 minutes, anesthetize the mouse again and place it back on the surgical table. Remove the stitches from the neck.After 45 minutes of occlusion, loosen the knot holding the monofilament in place. Pull slowly and gently on the filament and check that tissue recanalization occurs. Then pull out the filament and tighten the knot to prevent blood loss, clean the blood and untie the CCA knot.Ensure that there is no arterial wall damage. Remove the retractors and reposition the muscles, glands, and skin. Suture the skin using a 6-0 suture and apply disinfectant.Disconnect the Doppler probe and detach the holder. Finally, suture and disinfect the skin of the head. The outcome of the transient middle cerebral artery occlusion procedure was evaluated using in-vivo MRI neuroimaging.Lesion evolution assessed at different time points after reperfusion showed that progression of the lesion volume takes approximately two days to complete. Behavioral tests, such as grip strength test, showed a loss of strength in mice after 24 hours of transient middle cerebral artery occlusion. Also, upon stimulation in the corner test, the mice showed a preference to turn to the right side, which was ipsilateral to the lesion, indicating a successful middle cerebral artery occlusion on the right side.