作者感谢 Kathy Gage 的编辑支持。方案数字是用 BioRender.com 创建的。这项研究得到了麻醉学系（杜克大学医学中心）和 NIH 资助（NS099590、HL157354、NS117973 和 NS127163）的资金支持。

这项工作中描述的所有程序均按照美国国立卫生研究院关于研究动物护理和使用指南进行，该协议已获得杜克研究所动物护理和使用委员会 （IACUC） 的批准。本研究使用年轻（8-10周龄）和年龄（22个月大）雄性C57Bl/6小鼠。该协议的概述如图 1所示。
1. 手术准备
<ol><li>检查小鼠的严重异常和行为缺陷。 注意:手术前，外科医生必须穿戴适当的 PPE（个人防护设备），包括外科口罩、帽子、手套和防护服。</li>
	<li>称量鼠标;根据体重对呼吸机进行编程（参见 材料表）。</li>
	<li>将鼠标放入 4 英寸 x 4 英寸 x 7 英寸的麻醉诱导盒中。打开设置为 30 的氧气流量计（参见 材料表）和设置为 70 的一氧化二氮流量计。用 5% 异氟烷打开蒸发器。</li>
	<li>将导丝插入 20 G 静脉内 （IV） 导管。</li>
	<li>当鼠标的呼吸频率降低到每分钟 30-40 次呼吸时，将鼠标从感应箱中取出。</li>
	<li>将鼠标以仰卧位放在手术台上。将鼠标的舌头拉出并用左手的手指握住它。将喉镜（见 材料表）插入动物的嘴里，以观察声带。</li>
	<li>用右手中指稳定喉镜上的鼠标下巴。释放左手握住 20 G IV 导管。</li>
	<li>将导丝轻轻插入声带，然后将 20 G IV 导管缓慢推入气管，直到导管的翼部与鼻尖齐平。 注意: 如果鼠标正在移动，请勿插入电线。这可能会导致气管创伤和出血。</li>
	<li>打开呼吸机（见 材料表），并将其与插入鼠标的 20 G IV 导管连接。将异氟醚降至1.5%，并确保双肺机械通气。 注意:不要忘记降低异氟烷浓度。否则，小鼠将接受过量的麻醉。</li>
	<li>将眼膏涂抹在双眼上，皮下注射 5 mg/kg 卡洛芬。</li>
	<li>将鼠标保持在横向位置，右侧颞区朝上。使用加热垫（35°C）和由温度调节器控制的加热灯将直肠温度保持在37°C（参见 材料表）。</li>
	<li>剃掉右眼和耳朵之间的表面积，并用碘和酒精棉签对手术区域进行至少三次消毒。</li>
</ol>2. MCAO手术
<ol><li>打开用于MCAO手术的无菌器械包。戴上无菌手套，用手术剪刀在右眼和右耳之间做一个1厘米的皮肤切口。 注意: 每 15 分钟监测一次肤色、体温和对脚趾捏的反应。</li>
	<li>用镊子解剖下面的筋膜，露出颞肌和咬肌。 注意:注意不要损坏腮腺。</li>
	<li>用镊子触摸颞肌下部，检测颧弓的位置。小心地拉开面神经的分支。</li>
	<li>使用高温烧灼环的尖端（参见 材料表）在颞肌上切开 5 毫米的横向切口。</li>
	<li>使用两把镊子解剖下面的颧弓，露出上颌骨和颧骨之间的关节。</li>
	<li>用剪刀剪下颧弓的 3 毫米部分并将其移除。将咬肌与颅底分开。 注意:注意不要使眶后窦和颞浅静脉骨折。</li>
	<li>应用四个位于不同方向的小牵开器，露出颅骨底部，三叉神经分支由一个牵开器横向拉动。 注意:颅底外表面的沟标志着额叶和颞叶之间侧裂的位置。MCA位于此处（图2A），通过薄而透明的头骨可以看到其躯干和树枝（图2B）。该动脉与其他主要脑动脉的关系如图 2A所示。</li>
	<li>在MCA躯干上方和鼻皮质分支近端的头骨上滴0.9%生理盐水。使用电动研磨机使颅骨变薄，直到可见小骨折。 注意: 请勿将研磨机推到颅骨上，因为它可能会穿透颅骨并伤害下面的动脉。</li>
	<li>使用镊子的尖端提起变薄的头骨并将其取出。对于假老鼠，请停在这里，不要结扎动脉。 注意: 在 MCA 干线上形成一个小矩形窗口。</li>
	<li>在MCA顶部放置一圈黑色编织丝的单股环（图2C）。插入 8-0显微手术针提起MCA躯干，并在针头下方系紧缝线（见 材料表），将针头的两端留在丝线环结的顶部（图2D）。</li>
	<li>对于短暂的MCAO，将针头下方的丝线结稍微收紧以阻断动脉血流（图2E），代表MCAO发作。</li>
	<li>使用镊子固定缝合线，并在缺血结束时（例如，60 分钟或更长时间）慢慢取出针头。 注意:当针头被移除时，丝线结从MCA上滑落，大脑被重新灌注（图2F）。</li>
	<li>对于永久性 MCAO，用力拧紧动脉周围的丝线环，然后取出针头。切割并去除多余的缝合材料。</li>
	<li>在皮肤切口处滴一滴0.25%布比卡因，间歇性地使用6-0尼龙缝合线将肌肉和皮肤分开缝合（见 材料表）。将抗生素软膏涂抹在皮肤切口表面。 注意:皮肤切口也可以用无菌订书钉或胶水闭合。</li>
</ol>3. 术后护理
<ol><li>关闭异氟醚以唤醒鼠标。恢复自主呼吸后断开呼吸机。</li>
	<li>将鼠标转移到温度受控的恢复室（参见 材料表）。</li>
	<li>当鼠标的矫正反射恢复或开始移动时拔管。</li>
	<li>在温度和湿度受控的室中密切监视鼠标。在小鼠获得完全意识后（恢复期~2小时），将小鼠放回家笼中。每天皮下注射 5 mg/kg 卡洛芬，持续 3 天。</li>
</ol>4. 激光散斑对比成像（LSCI）
<ol><li>MCAO后6小时和24小时，将麻醉的小鼠安装在立体定位框架上。剃掉头顶，并用三支交替的碘和酒精棉签清洁。 注意:麻醉是按照步骤1.3所述进行的。LSCI 也在 MCAO 之前进行。</li>
	<li>做一个 3 厘米的中线皮肤切口，并从颅骨上解剖皮肤。使用四个小针牵开器暴露颅骨顶部。</li>
	<li>将激光散斑相机（参见 材料表）移动到头顶上方，然后调整相机的焦距。对脑血流进行成像。</li>
</ol>5. 2,3,5-三苯基氯化四氮唑（TTC）染色
<ol><li>在实验结束时用5%异氟醚深度麻醉小鼠，通常在中风后第1天，第3天或第28天。捏住尾巴以确保没有疼痛反应。</li>
	<li>用手术剪刀将小鼠斩首并收获大脑。将大脑在冰冷的盐水中孵育20分钟。</li>
	<li>将大脑放入冰上的脑切片机基质中，然后将冷盐水滴在大脑上。使用薄剃须刀片将大脑切成 1 毫米的薄片。</li>
	<li>将相同方向的脑切片浸入2%TTC溶液的培养皿中（参见 材料表）。将盘子在室温下避光15分钟。 注意:正常脑组织变红，缺血组织保持白色。</li>
	<li>将脑切片转移到10%福尔马林中固定24小时。对脑切片进行成像并测量梗死区域。</li>
</ol>

建立经颅大脑中动脉闭塞小鼠模型

<ol>
	<li>Tsao, C. W., 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=Heart+disease+and+stroke+statistics-2022+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2022 update: a report from the American Heart Association.</a> Circulation. 145 (8), e153 (2022).</li><li>Nogueira, R. G., 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=Thrombectomy+6+to+24+hours+after+stroke+with+a+mismatch+between+deficit+and+infarct.">Thrombectomy 6 to 24 hours after stroke with a mismatch between deficit and infarct.</a> The New England Journal of Medicine. 378 (1), 11-21 (2018).</li><li>Fisher, M., Savitz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+brain+cytoprotection+in+acute+ischaemic+stroke-renewed+hope+in+the+reperfusion+era.">Pharmacological brain cytoprotection in acute ischaemic stroke-renewed hope in the reperfusion era.</a> Nature Reviews Neurology. 18 (4), 193-202 (2022).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., 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>Knauss, 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=A+semiquantitative+non-invasive+measurement+of+PcomA+patency+in+C57BL/6+mice+explains+variance+in+ischemic+brain+damage+in+filament+MCAo.">A semiquantitative non-invasive measurement of PcomA patency in C57BL/6 mice explains variance in ischemic brain damage in filament MCAo.</a> Frontiers in Neuroscience. 14, 576741 (2020).</li><li>Yang, Z., 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=Post-ischemia+common+carotid+artery+occlusion+worsens+memory+loss,+but+not+sensorimotor+deficits,+in+long-term+survived+stroke+mice.">Post-ischemia common carotid artery occlusion worsens memory loss, but not sensorimotor deficits, in long-term survived stroke mice.</a> Brain Research Bulletin. 183, 153-161 (2022).</li><li>Wang, Z., 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=Increasing+O-GlcNAcylation+is+neuroprotective+in+young+and+aged+brains+after+ischemic+stroke.">Increasing O-GlcNAcylation is neuroprotective in young and aged brains after ischemic stroke.</a> Experimental Neurology. 339, 113646 (2021).</li><li>Jiang, 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=XBP1+(X-box-binding+protein-1)-dependent+O-GlcNAcylation+Is+neuroprotective+in+ischemic+stroke+in+young+mice+and+its+impairment+in+aged+mice+is+rescued+by+thiamet-G.">XBP1 (X-box-binding protein-1)-dependent O-GlcNAcylation Is neuroprotective in ischemic stroke in young mice and its impairment in aged mice is rescued by thiamet-G.</a> Stroke. 48 (6), 1646-1654 (2017).</li><li>Li, X., 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=Single-cell+transcriptomic+analysis+of+the+immune+cell+landscape+in+the+aged+mouse+brain+after+ischemic+stroke.">Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.</a> Journal of Neuroinflammation. 19 (1), 83 (2022).</li><li>Li, X., 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=Beneficial+effects+of+neuronal+ATF6+activation+in+permanent+ischemic+stroke.">Beneficial effects of neuronal ATF6 activation in permanent ischemic stroke.</a> Frontiers in Cellular Neuroscience. 16, 1016391 (2022).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+2.+Regional+cerebral+blood+flow+determined+by+[14C]iodoantipyrine+autoradiography+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 1 (1), 61-69 (1981).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 1 (1), 53-60 (1981).</li></ol>

所有作者均无利益冲突。

1981 年在大鼠中建立了第一个经颅 MCA 闭塞模型11,12，并于 1989 年被无颅切除术 MCAO 模型取代4.最初的经颅 MCA 闭塞具有较宽的手术区域，因此整个颧弓被切除，肌肉被横向拉扯。手术后局部组织肿胀，导致动物压力和食物摄入量减少。在我们改良的经颅MCAO模型中，切口的侵入性较小，并且只切除了一小部分颧弓。使用四个小针牵开器暴...

在美国，每年有近 800,000 人患有中风，其中大多数是缺血性的1。及时使用组织纤溶酶原激活剂（tPA）和/或血栓切除术恢复脑血流是目前对脑卒中患者最有效的治疗方法;然而，从长远来看，神经功能的完全恢复是罕见的2,3。因此，寻找以功能改善为目标的新型卒中疗法是一个紧张的研究领域，需要临床相关的卒中动物模型。
啮齿动物中最常见的缺血性卒中模型使用腔内大脑中动脉闭塞 （MCAO） 来诱发卒中。在这个模型中，最初由 Zea Longa 于 1989 年开发，将尼龙丝引入颈内动脉 （ICA） 以阻止流向大脑中动脉 （MCA）4。但是，这种模式有局限性。首先，当细丝插入ICA时，流向大脑后动脉（PCA）的血流也可能被部分阻塞，特别是在小鼠中。至关重要的是，后交通动脉 （PcomA） 是连接大脑前循环和后脑循环的小动脉，在某些小鼠品系中经常发育不全，例如 C57Bl/6，该菌株主要用于实验性卒中研究。PcomA 的这种通畅性被认为有助于卒中后小鼠病变大小的变异性5.事实上，当 MCAO 期间流向 PCA 的血流量急剧下降，并且 PcomA 无法提供足够的侧支血流时，中风梗死可以扩展到 PCA 的领域。此外，在该模型中，长时间的缺血导致小鼠死亡的几率更高。因此，通常在小鼠中使用30-60分钟的短MCAO持续时间。然而，大多数卒中患者在再灌注治疗前会经历几个小时的缺血。因此，具有较长缺血持续时间的小鼠卒中模型具有高度的临床相关性。 
该程序的总体目标是模拟具有中等大小梗死和出色存活率的小鼠的缺血性中风。这种经颅MCAO模型解决了临床中风的关键属性，因为可以进行长时间的缺血，并且老年小鼠对这种模型的耐受性很好，可以对功能恢复进行长期评估。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% bupivacaine</td>
			<td>Hospira</td>
			<td>NDC 0409-1159-18</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% sodium chloride</td>
			<td>ICU Medical</td>
			<td>NDC 0990-7983-03</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium Chloride (TTC)&#160;</td>
			<td>Sigma or any available vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 G IV catheter</td>
			<td>BD</td>
			<td>381534</td>
			<td>20 GA 1.6 IN</td>
		</tr>
		<tr>
			<td>30 G needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 silk suture</td>
			<td>Look</td>
			<td>SP116</td>
			<td>Black braided silk</td>
		</tr>
		<tr>
			<td>8-0 suture with needle&#160;</td>
			<td>Ethilon</td>
			<td>2822G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>BD</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia induction box</td>
			<td>Any suitable vendor</td>
			<td></td>
			<td>Pexiglass make&#160;</td>
		</tr>
		<tr>
			<td>Electrical grinder</td>
			<td>JSDA</td>
			<td>JD 700</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature cautery loop tip</td>
			<td>Bovie</td>
			<td>AA03</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus</td>
			<td>NDC 11695-6777-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser doppler perfusion monitor</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant eye ointment</td>
			<td>Bausch + Lomb</td>
			<td>339081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rectal probe</td>
			<td>Physitemp</td>
			<td>RET-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrous Oxide</td>
			<td>Airgas</td>
			<td>UN1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Otoscope</td>
			<td>Welchallyn</td>
			<td>728</td>
			<td>2.5 mm Speculum</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Airgas</td>
			<td>UN1072</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>CVS</td>
			<td>955338</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery box</td>
			<td>Brinsea&#160;</td>
			<td>TLC eco</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl (carprofen)</td>
			<td>Zoetis</td>
			<td>6100701</td>
			<td>Injectable 50 mg/mL</td>
		</tr>
		<tr>
			<td>Rodent ventilator</td>
			<td>Harvard</td>
			<td>Model 683</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Physitemp</td>
			<td>TCAT-2DF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple antibioric &#38; pain relief</td>
			<td>CVS</td>
			<td>NDC 59770-823-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>RWD</td>
			<td>R583S</td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified transcranial middle cerebral artery occlusion model to study stroke outcomes in aged mice

在实验性卒中研究中，带有腔内细丝的大脑中动脉闭塞 （MCAO） 被广泛用于模拟小鼠缺血性卒中。细丝MCAO模型通常在C57Bl / 6小鼠中表现出巨大的脑梗死，有时包括大脑后动脉供应区域的脑组织，这主要是由于后交通动脉闭锁的高发生率。这种现象被认为是在细丝MCAO后长期卒中恢复期间在C57Bl / 6小鼠中观察到的高死亡率的主要原因。因此，许多慢性卒中研究都利用了远端MCAO模型。然而，这些模型通常只在皮层区域产生梗死，因此，中风后神经功能缺损的评估可能是一个挑战。本研究建立了一种改良的经颅 MCAO 模型，其中躯干处的 MCA 通过 一个小颅窗永久或短暂地部分闭塞。由于闭塞位置相对靠近 MCA 的起源，因此该模型会在皮层和纹状体中产生脑损伤。该模型的广泛表征表明，即使在老年小鼠中也具有出色的长期存活率，并且易于检测到神经功能缺损。因此，这里描述的MCAO小鼠模型代表了实验性中风研究的宝贵工具。

在手术显微镜下直接观察，可以目视确认缺血期间MCA血流受阻。我们之前的研究表明，使用激光多普勒监测仪 6 >缺血区域的血流量减少了80%。为了确定MCAO后血流变化，LSCI可用于进一步确认缺血性损伤和再灌注（图1）。事实上，在图3A中，观察到右侧MCA区域的血液供应减少。对于瞬时MCAO，拆线后，脑血流的再灌注很明显（图<strong clas...

Watch this Scientific Journal Video about Innovative Techniques and Future Directions in Stroke Research at JoVE.com

作者聚焦:中风研究的创新技术和未来方向 

该协议展示了一种独特的小鼠卒中模型，具有中等大小的梗死和出色的生存率。该模型允许临床前卒中研究人员延长缺血持续时间，使用老年小鼠，并评估长期功能结果。

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. ...

在美国，每年有近 800， 000 人受到中风的影响。其中约87%为缺血性，表现为严重疾病。然而，可用的治疗选择有限，尤其是在慢性卒中阶段。我们的研究旨在开发一种促进康复的疗法，以减少中风患者的残疾并改善生活质量。他们广泛使用细丝，即大脑中动脉闭塞模型，作为插入的细丝可以阻止血液流向大脑后动脉的缺点。小鼠的 C57BL/6 通常具有未受干扰的后交通动脉，导致慢性卒中恢复后期的高死亡率。我们改良的经颅大脑中动脉阻塞模型部分穿过躯干处的 MCA，通过一个小颅窗在皮层和纹状体中产生脑损伤。该模型实现了高长期存活率，可检测到的神经功能缺损，即使在老年小鼠中也效果良好。我们的经颅MCAO模型可以纳入临床卒中的关键属性，例如延迟再灌注、神经功能缺损和衰老因素。该模型可用于评估新的中风治疗对长期功能恢复的治疗潜力。

a-modified-transcranial-middle-cerebral-artery-occlusion-model-to

Research

JoVE Journal

Neuroscience

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

3.0K 次观看.  Duke University Medical Center. 在美国，每年有近 800， 000 人受到中风的影响。其中约87%为缺血性，表现为严重疾病。然而，可用的治疗选择有限，尤其是在慢性卒中阶段。我们的研究旨在开发一种促进康复的疗法，以减少中风患者的残疾并改善生活质量。他们广泛使用细丝，即大脑中动脉闭塞模型，作为插入的细丝可以阻止血液流向大脑后动脉的缺点。小鼠的 C57BL/6 通常具有未受干扰的后交通动脉?...

视频: 作者聚焦:中风研究的创新技术和未来方向 

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

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 ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

小鼠脑中动脉闭塞诱导脑卒中后脑肌粘连血管病治疗模型

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

大脑中动脉闭塞的短暂性脑缺血模型

Transient Middle Cerebral Artery Occlusion Model of Stroke

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.

作者聚焦:通过大脑中动脉闭塞模型评估缺血性卒中损伤 

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.

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 ...

远端中动脉闭塞开发缺血性卒中小鼠模型

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

作者聚焦:在小鼠中建立可靠的远端 MCA 闭塞模型用于中风研究

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...