这项工作得到了国家自然科学基金（82173781和82373835）、博士后科研项目（BKS212055）、佛山市科学技术局科技创新项目（2320001007331）、广东省基础与应用基础研究基金（2019A1515010806）、广东省通用高校重点领域项目（智能制造）（2020ZDZX2057）和通用科研项目（特色创新）的支持。广东省大学排名 （2019KTSCX195）.

实验方案是按照成都中医药大学实验动物使用和机构动物护理和使用委员会指南（记录编号：2019-DL-002）进行的。所有动物研究数据均按照 ARRIVE（动物研究 ：体内实验 报告）指南进行记录。本研究使用体重 250 g ± 20 g 且年龄为 6-8 周龄的雄性 Sprague Dawley （SD） 大鼠。有关所用动物、试剂和设备的详细信息列在 材料表中。
1. 动物准备
<ol><li>使用 Zoletil 50 （50mg/kg， IM） 和盐酸甲苯噻嗪 （40mg/kg， IP） 诱导并维持大鼠的深度麻醉。确保在整个手术过程中使用连接到加热垫的直肠探针将体温保持在 37 ± 0.5 °C。在大鼠的眼睛上涂抹兽药膏，以防止它们干燥。</li>
	<li>剃掉大鼠头部和颈部的毛发。使用脱毛膏去除头部和颈部的皮毛，然后用生理盐水洗掉乳霜。使用无菌棉球涂抹乙醇和聚维酮碘 3 次，对手术部位的皮肤进行消毒。</li>
	<li>用手术刀沿矢状缝合线的方向在大鼠头部中间切开 2 厘米，小心地去除覆盖颅骨的肌肉。</li>
	<li>使用颅骨钻头将大鼠缺血侧的颅骨变薄。在研磨过程中使用生理盐水冷却并清除碎屑。使用激光散斑对比成像 （LSCI） 记录大鼠的基线血流。</li>
	<li>使用无菌棉球将乙醇和聚维酮碘涂抹在颈部皮肤上 3 次。使用手术刀片沿颈部中线切开 2 厘米。使用牵开器横向拉回皮肤和唾液腺，露出胸锁乳突肌和颈椎肌肉。</li>
	<li>将胸锁乳突肌和颈椎肌分开，露出颈动脉区域。确定颈总动脉 （CCA）、颈内动脉 （ICA） 和颈外动脉 （ECA） 的血管解剖结构。根据血管解剖结构，将 CCA 以及 CCA 衍生的 ECA 和 ICA15 分开。 注：大鼠的解剖结构表明 CCA 区域主要被胸锁乳突肌和颈肌覆盖，其中 ICA 和 ECA 来源于 CCA。分离后露出 CCA，沿着 CCA 可以看到一个 Y 形叉子，代表 ICA 和 ECA。小心不要损伤与 CCA 平行的迷走神经。</li>
</ol>2. MCA 的闭塞
<ol><li>使用 3-0 丝线在颈外动脉 （ECA） 和颈总动脉 （CCA） 上打一个容易解开的结，以暂时阻止血流。将容器夹放在 CCA 上，距离第一个结约 0.5 厘米。<ol><li>将 5 mL 注射器的针头染成黑色。使用黑色染色的针头在 CCA 中打一个小孔，并用黑色标记针孔。</li>
	</ol></li>
	<li>通过黑色标记将单丝尼龙缝合线插入 CCA。打开血管夹，将尼龙丝引导至颈内动脉 （ICA），直到它因轻微阻力而停止。牢固地拧紧第二个结，将单丝尼龙缝合线保持在动脉内，防止从阻塞位置移位。</li>
	<li>记下此时的缺血时间，并使用 LSCI 测量缺血侧的血流值。将布比卡因滴剂滴到颈部伤口上，以镇痛和闭合伤口。取下麻醉面罩，让大鼠恢复。 注意：如果单丝尼龙缝合线难以进入 ICA，请稍微缩回并再次尝试插入。</li>
</ol>3. MCAO 的再灌注
<ol><li>缺血 90 分钟后，用异氟醚重新麻醉大鼠。使用 LSCI 测量来自缺血侧的脑血流，并确保单丝尼龙缝合线没有移动。</li>
	<li>解开颈外动脉 （ECA） 上的结，以便血液再灌注。松开颈总动脉 （CCA） 上的结，固定单丝缝合线，然后撤回缝合线。在固定单丝缝合线之前应用血管夹以防止出血。</li>
	<li>用血管夹替换第一个 CCA 结。使用镊子将血管切口侧向旋转（图 1）。用镊子夹住切口，并使用 6-0 线结扎以修复切口。取下血管夹，检查是否有渗漏，并确认完全再灌注（视频 1）。 注意：进行结扎以修复切口，以避免过度夹住血管，这可能导致 CCA 狭窄。</li>
	<li>切口结扎后使用 LSCI 记录脑皮层血流值，以确认再灌注成功。用缝合线闭合颈部切口，并给予 1,00,000 单位青霉素和 2 mL 生理盐水，以防止感染和脱水。</li>
	<li>保持温暖，直到大鼠恢复意识。术后提供软食并监测动物的生命体征。</li>
</ol>4. 神经功能与脑缺血性损伤的评估
<ol><li>一旦大鼠完全醒来，研究人员将评估它们的神经功能，而不了解动物分组。利用 Longa 评分量表（表 1）和改良的神经严重程度评分 （mNSS） 量表（表 2）来评估所有大鼠的神经功能。</li>
	<li>手术后 24 小时后，在异氟醚诱导的麻醉下（4% 异氟醚，4 L/min 氧气） 通过 颈椎脱位16 （遵循机构批准的方案）对大鼠实施安乐死。用冰水冲洗大脑以去除任何残留的血液。</li>
	<li>将脑组织分成 5 个部分并用 TTC 染色。捕获脑组织切片两侧的图像，使用 Image J 软件测量前后两侧的缺血面积，并使用以下公式计算梗死率： <img alt="Equation 1" src="/files/ftp_upload/67060/67060eq01.jpg" style="margin: auto;"> 注：S1 和 S2 分别代表脑切片近端前脑和近端脑干侧的梗死区域。使用 Swanson 公式校正每个脑切片的梗塞区域，其中 h 表示脑切片的厚度。该公式由作者先前在先前的研究17 中概述，允许更准确地计算梗死病变的大小。</li>
	<li>使用统计和绘图软件分析数据，以评估模型的成功率和稳定性。</li>
	<li>采用免疫荧光法标记大鼠的皮层神经元，进一步验证该方法引起的神经元损伤。大鼠缺血再灌注 24 小时后，使用预冷 PBS 进行心脏灌注以获得脑组织。</li>
	<li>将脑组织嵌入并冷冻成切片。使用神经元抗体，例如未成熟神经元标志物双皮质素 （DCX）18、神经元核标志物神经元核 （NeuN）18 和神经元树突状标志物微管相关蛋白-2 （MAP-2）19。使用激光共聚焦显微镜观察缺血神经元的表达。</li>
</ol>

保留 ECA 的改良 MCAO/R 模型，用于增强再灌注和稳定性

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E., Janus, J., Fern, R., Gibson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+surgical+approach+reduces+variability+following+filament+induction+of+experimental+stroke+in+mice.">An alternative surgical approach reduces variability following filament induction of experimental stroke in mice.</a> Dis Model Mech. 10 (7), 931-938 (2017).</li></ol>

由单丝尼龙缝合线诱导的大脑中动脉闭塞 （MCAO） 模型是制备 MCAO 模型的最常用方法。这种方法在临床前研究中被广泛采用，并因其简单、无需开颅手术、手术创伤最小以及能够实现再灌注而获得许多从业者的认可。
腔内细丝 MCAO 有两种经典的手术技术：小泉法8 和 Longa 法9。这些方法之间的主要区别在于如何引入缝合线以闭塞 MCA。在 Koizumi 方法中，缝合线通过颈总动脉 （CCA） 切口插入颈内动脉 （ICA）。缝合线的前端涂有硅胶，以阻挡 MCA 的起点。相反，在 Longa 方法中，缝合线通过切断的颈外动脉 （ECA） 插入 ICA。缝合线的前端展开成球形，以阻挡 MCA 的起点（图5）。在再灌注期间，轻轻抽出缝线以恢复血流。
Longa 方法包括通过切断的颈外动脉 （ECA） 将缝合线引入颈内动脉 （ICA）。这个过程需要分离和断开 ECA，以及分离和去除小树枝，然后使用电凝笔将其切断以防止出血。因此，这种方法需要仔细和精确的操作。此外，切断的 ECA 的短残余端会使缝合线在插入过程中很容易滑落，进一步增加了手术的复杂性。在准备脑缺血小鼠模型时，这一挑战尤为明显，因为它通常需要使用显微镜来确保准确性，从而增加了操作的整体复杂性。
小泉法的主要关注点在于颈总动脉 （CCA） 的结扎，这导致缺血侧脑组织仅通过 Willis 环进行代偿性再灌注。CCA 是主动脉弓从心脏开始的重要分支，是供应脑血流灌注的主要动脉。因此，由 Koizumi 方法创建的脑缺血再灌注模型经常经历灌注不足的状态。这种低灌注不仅限于大脑中动脉区域，而是延伸到同一侧的整个缺血半球。即使在建模 35 天后，血流值也没有恢复到基线水平，这表明小泉法应被视为慢性低灌注的缺血模型，而不是缺血再灌注模型10。
相比之下，Longa 方法可以在 MCAO/R 动物中实现 100% 的血流值。然而，重要的是要注意，实现完全再通并不一定意味着应该放弃小泉法。在考虑临床转化时，小泉法可能更适合动脉闭塞后自发性缓慢再通的患者。然而，这种现象在临床实践中相对较少。只有一小部分缺血性卒中患者符合溶栓标准并在治疗时间窗口内接受有效治疗20,21。此外，一些患者即使在溶栓治疗后仍未实现完全血运重建22。因此，Koizumi 方法可以用来模拟此类患者的病情。
另一方面，Longa 方法允许血管在闭塞后快速再通，使其更适合模拟临床实践中接受有效溶栓治疗或血栓切除术的患者。对于模拟未接受溶栓治疗的患者，通过 Koizumi 方法制备的永久性脑缺血模型可能提供更完全的闭塞和更简单的外科手术。
诱导大脑中动脉闭塞/再灌注 （MCAO/R） 的 Longa 方法可以通过保留颈总动脉 （CCA） 衍生的血管再通导致更严重的再灌注损伤23,24。然而，需要注意的是，在 Longa 方法期间需要切断颈外动脉 （ECA） 并永久结扎，这会破坏大脑的完整血流结构，与临床患者不一致。此外，舌动脉、上颌动脉和枕动脉是 ECA13 的分支。因此，切割或永久结扎 ECA 会导致饮食失调，从而导致体重减轻25。先前的研究表明，遵循 MCAO 建模的小鼠死亡主要是由于食物或水摄入不足，而不是脑缺血损伤14,26。此外，ECA 的结扎会减少视网膜血流，并以细胞死亡的形式诱导视网膜损伤27。
此外，中风研究最重要的指标是梗死量;然而，即使遵循严格的手术和数据收集方案，缝合方法诱导的梗死体积已被证明具有显着的标准差。在以前的报道中，梗死体积与再灌注程度相关28，小泉方法制备的卒中模型的再灌注取决于 Willis 循环 29,30,31。然而，已经证明 Willis 循环具有很高的解剖变异性，尤其是在 C57Bl/6 小鼠中，其中 90% 的动物可能缺乏一条或两条后交通动脉32，这是缺血性损伤高变异性的关键决定因素33。因此，保留 CCA 的灌注可以减少梗死体积的变异性34。据报道，中风试验中梗死面积的平均变异系数为 29.5%31，这导致中风试验的统计功效低。这也对潜在的药物发现构成了巨大的挑战。这无疑增加了开发和临床转化的成本，并可能导致研究人员错过可能有效的药物。它还增加了实验统计分析中要包括的动物数量，这进一步违反了动物伦理的 3R 原则。
因此，本研究对建模方法进行了修改，以确保脑缺血后脑组织血流结构的完整性和足够的再灌注。主要修改包括两个步骤：（1） 使用 5 mL 注射器针头将缝合线插入 CCA，而不是做一个小切口;此步骤减少了 CCA 的切口，更有利于结扎切口;（2） 拔出缝线，用较细的丝线结扎 CCA 的针口，不影响 CCA 的血流灌注。当然，这也需要仔细的外科手术，但与 通过 ECA 引入缝合不同;这种方法保留了脑血流结构的完整性，同时允许血流通过 CCA 完全再灌注，使手术程序更简单，减少了梗死体积的可变性。一些研究人员也使用过类似的研究方法，用组织胶修复切口，但这有血栓形成的可能，会带来不可控的影响。并且需要更长的时间来稳定切口25。
总之，本研究提供了一种改进的脑缺血再灌注模型的制备方法，该方法允许在细丝撤出后进行足够的血液灌注，并可能导致显着可重复的梗死体积和神经损伤。以上结果表明，改进后的方法是可行且可重复的，可为从业者准备模型提供参考，以促进脑缺血药物的有效临床转化。

根据世界卫生组织的数据，在过去十年中，中风一直是全球第二大死因，发病率高、死亡率高、残疾率高 1,2。随着全球人口老龄化，预计发展中国家的中风发病率将增加，可能成为成人过早死亡和残疾的主要原因。此外，中风有发生在年轻年龄的趋势3。中风后劳动力的流失也给家庭和社会带来了沉重的负担4。因此，开发安全有效的治疗方法对中风研究构成了重大挑战。
动物模型是研究人类疾病预防和治疗的重要工具。卒中治疗策略的成功转化取决于卒中动物模型的可重复性和可靠性 5,6。大脑中动脉 （MCA） 是临床卒中的常见部位，使 MCAO 模型成为最接近人类缺血性卒中的模型。使用缝合法制备的 MCAO 模型因其无需开颅手术和易于控制缺血时间等优点而受到研究人员的青睐。它已被用于超过 40% 的神经保护实验7。然而，尽管它有许多优点，但该模型的操作细节对许多研究人员来说仍然是一个有争议的话题。
对于缝合诱导的大脑中动脉闭塞 （MCAO） 模型，通过撤回缝合线进行再灌注。目前，缝合插入主要采用两种方法：小泉的方法8 和 Longa 的方法9。在 Koizumi 的方法中，缝合线主要通过颈总动脉 （CCA） 切口进入颈内动脉 （ICA），而在 Longa 的方法中，它通过切断的颈外动脉 （ECA） 进入 ICA。在再灌注过程中，小泉法需要永久结扎 CCA 切口，并依靠 Willis 环进行再灌注10。然而，一些研究表明，在失去 CCA 供应后，仅通过对 Willis 环的补偿供应，不能仅通过补偿性供应来实现有效的再灌注。此外，Willis 环表现出很高的解剖变异性，尤其是在 C57Bl/6 小鼠中，增加了梗死变异性并降低了实验数据的可靠性。因此，这种方法越来越多地受到研究人员的质疑11。
Longa 的方法包括通过切断的 ECA 插入缝合线，然后在缝合线拔出后永久结扎颈内动脉 （ICA）。这保持了 CCA 通畅性，允许血液灌注高达基线值的 100%。然而，这种方法需要将颈外动脉和小动脉分支分开，切断它们或电凝它们，使手术具有挑战性。它还会破坏大脑的全血流结构，这与临床患者的状态不同12。重要的是，研究表明，切割或结扎 ECA 会导致控制咀嚼和吞咽的肌肉出现缺血性病变，影响动物饮食并导致大鼠术后动物死亡和严重的感觉和运动损伤13,14。
因此，迫切需要一种改进的模型制备方法来解决这些问题。本研究介绍了一种改进的 MCAO 建模方法，该方法可修复 CCA 插入切口并实现有效的再灌注。该程序简单、实用、可行，可诱导严重的神经损伤和可复制的梗死病变，为卒中研究人员提供有价值的指导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal anesthesia system</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R500IE</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal temperature maintainer</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 secondary antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB21303</td>
			<td></td>
		</tr>
		<tr>
			<td>DAP1 antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>G1012</td>
			<td></td>
		</tr>
		<tr>
			<td>DCX antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB13434</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Beyotime Biotechnology Co., LTD</td>
			<td>C0265</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>GraphPad Prism 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>ImageJ software</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser speckle blood flow imaging system</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>PeriCam PSI NR</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP-2 antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB11128</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature hand-held skull drill</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>87001</td>
			<td></td>
		</tr>
		<tr>
			<td>monofilament suture</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>250-280g</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN antibody</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>GB11138</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT embedding agent</td>
			<td>BIOSHARP</td>
			<td>BL557A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin sodium</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>17121709-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Antigen Retrieval Solution for Frozen Sections</td>
			<td>Beyotime Biotechnology Co., LTD</td>
			<td>P0090</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats</td>
			<td>SPF ( Beijing ) Biotechnology Co.,Ltd.</td>
			<td>250-280g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Beyotime Biotechnology Co., LTD</td>
			<td>ST795</td>
			<td></td>
		</tr>
		<tr>
			<td>TTC</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>2019030101</td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified model preparation for middle cerebral artery occlusion reperfusion

大脑中动脉闭塞再灌注 （MCAO/R） 模型对于理解卒中的病理机制和药物开发至关重要。然而，在常用的建模方法中，小泉法由于结扎颈总动脉 （CCA） 且无法实现足够的再灌注而经常面临审查。同样，Longa 方法因断开和结扎颈外动脉 （ECA） 而受到批评。本研究旨在介绍一种改进的模型制备方法，该方法保留了 ECA 的完整性，包括通过 CCA 插入单丝尼龙缝合线，修复结扎的 CCA 切口，并维持 CCA 的再灌注。使用激光斑点流成像确认血流再灌注。Longa 量表、改良神经严重程度评分、氯化三苯基四唑 （TTC） 染色和神经元免疫荧光标记等评估方法表明，这种方法可以诱导稳定的缺血性神经损伤。这种改进的 MCAO/R 模型方案简单稳定，为脑缺血领域的从业者提供了有价值的指导。

激光斑点血流成像显示，在单丝尼龙缝合线闭塞前，大脑中动脉 （MCA） 区血流充沛，记录大鼠基线血流值。MCA 闭塞后，大脑缺血侧的血流值迅速下降。在撤回缝线之前，重新检查缺血侧的血流值，以确认缝线是否阻塞了 MCA。结果表明血流仅发生轻微变化。撤回缝合线后，血流灌注迅速恢复（图2）。 视频 1 还显示了颈总动脉的强劲充盈脉搏，表明该方法可以在脑缺血后实现足够的血流灌注。
神经功能评分结果（图 3）显示，与假手术组相比，模型组的 Longa 评分为 1.80 ± 0.27 （p < 0.05），mNSS 评分为 7.4 ± 0.89 （p < 0.05）。这些分数表明，使用该方法制备的大脑中动脉闭塞/再灌注 （MCAO/R） 模型导致了严重的神经损伤。 视频 2 展示了模型组大鼠在爬行时旋转的特征行为，这是中风诱导损伤的标志，进一步证实了该方法的有效性。
TTC 染色显示，模型组大鼠梗死率分别为 5.58 ± 1.79 （p < 0.05），梗死区域主要影响纹状体和皮层，梗死面积保持相对稳定。免疫荧光染色显示缺血再灌注后皮质神经元丢失（图 4）。这些发现共同表明，该方法可以建立稳定的 MCAO/R 模型，适用于评价临床前药物疗效。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67060/67060fig01v5.jpg"> 图 1：CCA 连接器连接的示意图。 黄色圆圈表示导丝插头的插入点和结扎部位，而结扎位置（黄色箭头）表示颈总动脉 （CCA） 与导丝结扎的位置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67060/67060fig02v5.jpg"> 图 2：大鼠在缺血侧闭塞前、闭塞后以及再灌注前后的激光散斑血流图像。 （A） 大鼠的脑血流图像，其中蓝色区域表示低血流值，红色区域表示高血流值。比例尺：200 毫米。（B） 大鼠脑缺血再灌注的变化，表示为平均值±标准差，样本量为 n = 5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67060/67060fig03v5.jpg"> 图 3：假手术组和模型组的梗死面积和神经功能评分结果。 （A） TTC 染色描绘了假手术组和模型组的脑组织梗死区域，白色区域表示梗死组织，红色区域表示正常组织。（B） 介绍了 Longa 和 mNSS 评分的定量结果，以及梗死率。与假手术组相比，统计显着性用 *p < 0.05 表示，样本量为 n = 5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67060/67060fig04v5.jpg"> 图 4：假手术组和模型组皮层神经元损伤的结果。 NeuN （A） 、 DCX （B） 和 MAP-2 （C） 在皮层神经元中的表达。比例尺：100 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig04large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67060/67060fig05v5.jpg"> 图 5：Koizumi、Longa 的 MCA 闭塞和再灌注图以及修改后的方法。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/67060fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>电机测试/strong></td>
			<td>点</td>
		</tr><tr><td>掀起老鼠的尾巴</td>
			<td>3</td>
		</tr><tr><td colspan="2">1 前肢屈曲</td>
		</tr><tr><td colspan="2">1 后肢屈曲</td>
		</tr><tr><td colspan="2">1 个头部在 30 秒内向垂直轴移动 10° ></td>
		</tr><tr><td>将老鼠放在地板上</td>
			<td>3</td>
		</tr><tr><td colspan="2">0 正常行走</td>
		</tr><tr><td colspan="2">1 无法直走</td>
		</tr><tr><td colspan="2">2 向麻痹的一侧盘旋</td>
		</tr><tr><td colspan="2">3 跌倒到麻痹的一侧</td>
		</tr><tr><td>感官测试</td>
			<td>2</td>
		</tr><tr><td colspan="2">1 分班测试（视觉和触觉测试）</td>
		</tr><tr><td colspan="2">2 本体感觉测试（深部感觉，将爪子推向台边</td>
		</tr><tr><td colspan="2">刺激肢体肌肉）</td>
		</tr><tr><td>光束平衡测试</td>
			<td>6</td>
		</tr><tr><td colspan="2">0 以稳定的姿势保持平衡</td>
		</tr><tr><td colspan="2">1 梁的抓握侧</td>
		</tr><tr><td colspan="2">2 拥抱横梁，一条肢体从横梁上落下</td>
		</tr><tr><td colspan="2">3 拥抱横梁，两条肢体从横梁上掉下来，或在横梁上旋转 （>60 s）</td>
		</tr><tr><td colspan="2">4 尝试在横梁上保持平衡但脱落（>40 秒）</td>
		</tr><tr><td colspan="2">5 尝试在横梁上保持平衡但脱落（>20 秒）</td>
		</tr><tr><td colspan="2">6 脱落：没有尝试平衡或挂在横梁上 （<20 s）</td>
		</tr><tr><td>反射消失和异常运动</td>
			<td>4</td>
		</tr><tr><td colspan="2">1 次耳廓反射（触摸耳道时摇头）</td>
		</tr><tr><td colspan="2">1 角膜反射（用棉花轻轻接触角膜时眨眼）</td>
		</tr><tr><td colspan="2">1 惊吓反射（对啪啪作响剪贴板纸发出的短暂噪音的运动反应</td>
		</tr><tr><td colspan="2">1 癫痫发作、肌阵挛、肌张力障碍</td>
		</tr><tr><td>最高分</td>
			<td>18</td>
		</tr></tbody></table>表 1：改良的神经严重程度评分。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2">Zea-Longa 评分</td>
		</tr><tr><td>得分</td>
			<td>症状</td>
		</tr><tr><td>0 积分</td>
			<td>无神经功能缺损</td>
		</tr><tr><td>1 积分</td>
			<td>轻度：抬起尾巴时无法完全伸展右前爪</td>
		</tr><tr><td>2 积分</td>
			<td>中度：行走时向右/向左转圈</td>
		</tr><tr><td>3 积分</td>
			<td>严重：行走时向右/向左翻滚</td>
		</tr><tr><td>4 积分</td>
			<td>无法自发行走并失去意识</td>
		</tr></tbody></table>表 2：Zea-Longa 分数。
视频 1：大鼠再灌注期间 CCA 切口结扎修复的关键步骤。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/video%201.zip" target="_blank">请点击此处下载此视频。</a>
视频 2：大鼠接受 MCAO/R 的行为视频。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67060/video%202.zip" target="_blank">请点击此处下载此视频。</a>

作者聚焦：脑缺血建模的新方法 – 增强再灌注和简化手术

该协议描述了准备通过颈总动脉进行大脑中动脉闭塞再灌注的过程。

大脑中动脉闭塞再灌注的改良模型制备

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug ...

a-modified-model-preparation-for-middle-cerebral-artery-occlusion

Nanyang Technological University

Research

JoVE Journal

Medicine

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

801 次观看.  Foshan University. 我们的研究范围是脑缺血的建模程序。我们尝试从颈总动脉引入丝线并用细丝线结扎切口，维持颈总动脉和颈外动脉的再灌注。小泉法被认为再灌注不足，但 Longa 法被认为会影响主肌的血液供应。操作复杂，对初学者不友好。我们的协议解决了上述问题，并提供了一种替代方法。我们的方案使更多从业者更容易掌握脑缺血建模，并快速发现潜?...

视频: 作者聚焦：脑缺血建模的新方法 – 增强再灌注和简化手术

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

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

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

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

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

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

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

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

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

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

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

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

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

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

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

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

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

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

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

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

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

栓塞性脑梗塞（缺血）的大鼠缺血性中风与血同源凝块

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

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

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

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

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

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

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

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

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

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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.