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. 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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。

Nearly 800,000 people suffer a stroke in the US every year, and most of these strokes are ischemic in nature1. Timely restoration of the cerebral blood flow with tissue plasminogen activator (tPA) and/or thrombectomy is currently the most effective treatment for stroke patients; however, the full recovery of neurologic functions in the long term is rare2,3. Thus, searching for novel stroke therapy that targets functional improvement is an intense area of research that requires clinically relevant animal models of stroke. 
The most common ischemic stroke model in rodents uses intraluminal middle cerebral artery occlusion (MCAO) to induce stroke. In this model, initially developed by Zea Longa in 1989, a nylon filament is introduced into the internal carotid artery (ICA) to block the blood flow to the middle cerebral artery (MCA)4. However, this model has limitations. First, when the filament is inserted into the ICA, the blood flow to the posterior cerebral artery (PCA) could be partially blocked as well, especially in mice. Critically, the posterior communicating artery (PcomA), a small artery that connects anterior and posterior cerebral circulation, is frequently underdeveloped in some mouse strains, such as C57Bl/6, the strain predominantly used in experimental stroke research. This patency of the PcomA is believed to contribute to the variability in lesion size in mice after stroke5. Indeed, when blood flow to the PCA drops precipitously during MCAO, and the PcomA is unable to provide sufficient collateral blood flow, the stroke infarct can expand into the territory of the PCA. Moreover, in this model, a long duration of ischemia leads to a higher chance of mortality in mice. Consequently, a short MCAO duration of 30-60 min is typically used in mice. However, most stroke patients experience a few hours of ischemia before reperfusion treatment. Thus, a mouse stroke model with an extended duration of ischemia is of high clinical relevance. 
The overall goal of this procedure is to model ischemic stroke in mice that have a medium-sized infarct and an excellent survival rate. This transcranial MCAO model addresses critical attributes of clinical stroke, as prolonged ischemia can be performed, and aged mice tolerate this model well, allowing for the long-term assessment of functional recovery.

作者聚焦：中风研究的创新技术和未来方向 

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

Stroke affects nearly 800, 000 people annually in the US.Around 87%of this, ischemic, presenting as a severe medical condition. However, there are limited treatment options available especially during the chronic stroke phase. Our research aims to develop a recovery enhancing therapy to reduce disability and improve the quality of life for stroke patients.They widely use the filament, the middle cerebral artery occlusion model, as a drawback where the inserted filament can block a blood flow to the posterior cerebral artery. The C57BL/6 in mice often have an undisturbed posterior communicating artery leading to a high mortality in the later chronic stroke recovery phase. Our modified transcranial middle cerebral artery occlusion model partially across the MCA at the trunk generating brain damage in the cortex and the striatum via a small cranial window.This model achieves a high long-term survival rate, detectable neurological deficits, and works well even in aged mice. Our transcranial MCAO model can incorporate the critical attributes of clinical stroke, such as delayed reperfusion, neurological deficits, and aging factor. This model can be used to evaluate the therapeutical potential of new stroke treatments on long-term function recovery.

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

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。

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

1.9K 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 

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.

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.

科研

神经科学

Middle Cerebral Artery Occlusion (MCAO) to Induce Stroke in a Murine Model

To begin, lay an eight to 10 weeks anesthetized C57 black six male mouse on the surgical bench. Using blunt forceps, pull out the tongue and hold it with fingers. Insert a laryngoscope into the mouth and visualize the vocal cord.Stabilize the mouse chin on the laryngoscope and hold the 20 gauge intravenous, or IV catheter with a guide wire inserted in it. Then slightly insert the guide wire into the vocal cord and slowly push the 20 gauge IV catheter into the trachea until its wing part is even with the nose tip. Once the mouse is connected to the ventilator, keep it in a lateral position with the right temporal area facing up.Maintain the rectal temperature at 37 degrees Celsius using a heating pad and a heat lamp. After locating the zygomatic arch and incising the temporal muscle, use two forceps to dissect the underlying zygomatic arch and expose the joint between the maxilla and zygomatic bones. Then using scissors, cut a three millimeter portion of the zygomatic arch and remove it.Finally, separate the masseter muscle from the skull base. Position four small retractors in different directions and expose the cranial skull base with one retractor pulling the trigeminal nerve branches laterally. Apply a saline drop to the skull above the middle cerebral artery, or MCA trunk and proximal to the rhinal cortex branch.Use an electrical grinder to thin the skull until a small fracture is visible. And with the tip of the forceps, remove the thin skull. Place a single-strand loop of black braided silk on top of the MCA.Then insert an 8-O microsurgical needle to lift the MCA trunk and tie the suture under the needle, leaving the needle's two ends on top of the silk thread loop knot. For transient middle cerebral artery occlusion, or MCAO, slightly tighten the silk thread knot under the needle to block arterial blood flow, representing MCAO onset. Use the forceps to hold the suture and slowly remove the needle at the end of the ischemia.The laser speckle contrast imaging confirmed the reduced blood supply in the right MCA. For transient MCAO after suture removal, the cerebral blood flow reperfusion was evident, and further improved after 24 hours. TTC staining of the brain after 24 hours of stroke demonstrated the generation of infarcted tissue in both the cortical and lateral striatum areas in young and aged mice.The infarct size was moderate compared to filament MCAO.