本研究得到湖北省自然科学基金资助 （2022CFC057）。

该实验方案由江汉大学机构动物护理和使用委员会批准，并按照中国疾病预防控制中心发布的实验动物伦理指南进行。该方案使用成年雄性 C57BL/6J 小鼠，10 周龄，体重 24-26 g。将所有小鼠饲养在 12 小时的光照/黑暗循环受控环境中，随意进食和水 。
1. 术前准备
注意：该协议所需的关键仪器和设备如图 1 所示。
<ol><li>手术前，通过高压灭菌对手术器械进行消毒。</li>
	<li>手术前 60 分钟以 5 mg/kg 的剂量皮下注射美洛昔康用于镇痛。</li>
	<li>在诱导室中用 5% 异氟醚麻醉小鼠。</li>
	<li>用 75% 乙醇擦拭加热手术板，并激活加热开关，温度设置为 37 °C。 然后，将鼠标放置在板上的左侧位置，使头部远离外科医生，尾部面向外科医生。</li>
	<li>将面罩贴在小鼠脸上，提供 2% 异氟醚和 0.3 L/min 氧气的恒定供应，用于维持麻醉。将眼膏涂抹在角膜上，以防止它们在手术过程中干燥。</li>
	<li>用剃须刀剃掉从左眼眶到左耳道的毛发。使用脱毛霜去除剩余的皮毛进行消毒。</li>
	<li>通过涂抹聚维酮碘消毒剂和 75% 乙醇 3 次来准备手术区域。</li>
</ol>2. 远端 MCAO 模型
<ol><li>通过评估脚趾捏合的反射来检查鼠标是否被深度麻醉。</li>
	<li>使用无菌手术刀刀片在左眼眶和左耳道之间做一个垂直切口。</li>
	<li>用牵开器收缩皮肤的软组织，露出颞肌。</li>
	<li>使用直的显微镊子将颞肌的顶端和背段与颅骨分开。</li>
	<li>确定 MCA 的分叉，它在颞骨下方呈“Y”形，偏向颅底（图 2A）。 注意：如果 MCA 的分叉在视觉上无法辨别（由于解剖学上正常的变化），请确定最喙部的血管。</li>
	<li>使用电动颅钻将颅骨变薄，形成骨窗（直径 4 毫米），直到看到具有半透明质地的硬脑膜。 注意：间歇性地向颅骨中滴入生理盐水，以防止在此过程中过热。</li>
	<li>用弯曲的微镊子去除动脉上方的硬脑膜，露出 MCA 分支。</li>
	<li>将电烙术设置为 7 W。使用 电凝钳在 MCA 分叉的近端和远端部位凝固动脉（图 2A）。当由于解剖学变化而看不到分叉时，在相距约 1 毫米的两个不同位置对准确识别的 MCA 分支（如上所述）进行凝血13。</li>
	<li>使用激光散斑血流计监测左侧 MCA 皮质分支的血流。 注意：电凝后血流减少少于基线值 40% 的小鼠被排除在研究之外。</li>
	<li>使用 5-0 聚乙醇酸缝合线分别缝合肌肉和皮肤。使用无菌棉签将双氯芬酸钠凝胶和莫匹罗星软膏涂抹在皮肤切口上。</li>
	<li>将鼠标放入恢复室中。监测所有小鼠，直到它们完全清醒。每 24 小时提供美洛昔康 （5 mg/kg， SC） 用于镇痛，最长 72 小时。</li>
</ol>3. 假操作
<ol><li>重现与上述相同的程序，不包括动脉凝血过程。</li>
</ol>4. 行为测试
注意：在 dMCAO 之前，小鼠每天接受两次行为训练，持续 3 天。在 dMCAO 后第3 天， 将小鼠移至行为测试室进行 2 小时的环境适应，然后再进行测试。
<ol><li>握力测试14 注意： 握力计包括一个推拉式应变计和一个金属水平杆。<ol><li>抓住鼠标尾巴的底部。逐渐将鼠标向金属握杆降低，直到它用双前爪抓住水平杆（图 2B）。</li>
		<li>以约 2 cm/s 的恒定速度向后拉鼠标，并保持其身体水平。</li>
		<li>记录鼠标从杆上松开前爪时的峰值力，以克 （g） 为单位。</li>
	</ol></li>
	<li>极测试15<ol><li>将鼠标放在垂直木杆（50 厘米高，直径 1 厘米）的顶部，头部朝上，让它们爬下杆子（图 2B）。</li>
		<li>记录鼠标转身 （T 形转弯） 所花费的时间和爬下杆子的总时间 （T-total），截止时间为 60 秒。</li>
	</ol></li>
	<li>胶带去除试验16<ol><li>握住鼠标并将一块胶带（2 × 2 毫米）贴在鼠标的右前爪上（图 2B）。</li>
		<li>将鼠标放回饲养笼中，让它自由移动。</li>
		<li>记录鼠标去除胶粘剂所花费的时间，限制为 60 秒。</li>
	</ol></li>
	<li>气缸测试17<ol><li>将鼠标放在一个敞开的顶部透明玻璃圆柱体（直径：14 厘米;高度：20 厘米）中，并使用相机记录其自发的站立探索行为 3 分钟（图 2B）。 注意：在实验过程中，两个镜子位于圆柱体旁边，以确保前肢运动的最佳记录。</li>
		<li>在测试后续鼠标之前，用 75% 乙醇擦拭圆筒以消除残留的嗅觉线索。</li>
		<li>将视频的播放速度降低到实际速度的 1/5，并计算用左 （L） 或右前爪 （R） 或同时使用双前爪 （B） 触墙的次数。将右前爪的使用率计算为 （L-R）/（L + R + B） × 100%。</li>
	</ol></li>
</ol>5. 灌注和样品制备
<ol><li>通过腹膜注射过量的戊巴比妥 （300 mg/kg） 对小鼠实施安乐死。将鼠标仰卧放置并固定四肢。</li>
	<li>在皮肤上做一个中线切口，从腹中腔开始，向胸部上部区域延伸。</li>
	<li>用手术剪刀打开胸部，然后用止血钳将剑突向上折叠。小心地取出肺和横膈膜，露出心脏。</li>
	<li>将灌注针尖插入左心室，用剪刀刺穿右心房。通过左心室用 20 mL 生理盐水灌注心脏。</li>
	<li>将鼠标斩首，沿着眼睛之间的中线切开头皮。沿两侧切骨，从颅底开始，一直到眼窝。</li>
	<li>提起颅骨，用镊子轻轻地将大脑从颅底分离。 注：一半小鼠的大脑进行 2,3,5-三苯基四唑氯化物 （TTC） 染色，而另一半小鼠的大脑进行组织学检查。</li>
	<li>将大脑浸泡在 4% 多聚甲醛溶液中过夜，以进行后续组织学分析。</li>
</ol>6. 梗死体积的识别
<ol><li>将大脑置于冰箱的 -20 °C 冷冻室中 20 分钟。</li>
	<li>将大脑放入 1 毫米的脑基质中，并使用切片机刀片将大脑切成厚度为 2 毫米的冠状动脉切片。</li>
	<li>将脑切片转移到 24 孔培养板中，并添加 2% TTC 以覆盖脑组织。将 24 孔培养板放入恒温烘箱中 30 分钟，温度设置为 37 °C。</li>
	<li>小心地取出脑切片，并在 4% 多聚甲醛溶液中固定 15 分钟。</li>
	<li>扫描仪以 1200 x 1200 dpi 的分辨率对这些脑切片进行光学扫描。</li>
	<li>通过使用 Image J 软件 （https://imagej.net/software/imagej/index）18 将每个切片的梗塞面积（右半球的面积减去左半球的未受伤面积）相加来测量脑梗死体积。将梗塞体积百分比计算为梗塞体积/右半球体积 × 100%。</li>
</ol>7. 组织病理学和免疫荧光染色分析
<ol><li>从多聚甲醛溶液中取出脑样品，用流水冲洗 1 小时。</li>
	<li>将大脑样本转移到自动化组织脱水机中。启动脱水、玻璃化和蜡浸泡的预设程序。将脱水的脑样品包埋在石蜡中，并通过切片机将其切成 5 μm 厚的切片。</li>
	<li>苏木精和伊红 （H&E） 染色<ol><li>用二甲苯脱蜡切片 3 次，每次 8 分钟。</li>
		<li>通过连续浸入梯度乙醇（100%、95%、90%、80%、70%）和蒸馏水中每次 5 分钟来再水化切片。</li>
		<li>用苏木精溶液对切片染色 5 分钟。</li>
		<li>用蒸馏水冲洗切片以洗涤残留的苏木精，然后将它们浸入 5% 盐酸醇中以进行 5 秒区分。用蒸馏水清洗切片 2 分钟。</li>
		<li>用伊红溶液对切片染色 30 秒，然后用蒸馏水冲洗掉多余的染色液。</li>
		<li>将切片连续浸入 90% 乙醇、95% 乙醇、100% 乙醇和二甲苯（2 次）中，每次 5 分钟。用中性香脂封固剂安装切片。</li>
	</ol></li>
	<li>免疫荧光染色<ol><li>用二甲苯脱蜡 3 次，每次 8 分钟。</li>
		<li>通过连续浸入梯度乙醇（100%、95%、90%、80%、70%）和蒸馏水中每次 5 分钟来再水化切片。</li>
		<li>将柠檬酸盐抗原修复溶液预热至在微波炉中煮沸。将切片浸入抗原修复溶液中。以低功率 （20 W） 重新加热抗原修复缓冲液 20 分钟。</li>
		<li>关闭微波炉，让抗原修复缓冲液冷却至室温 （RT）。</li>
		<li>用 0.1 mM 磷酸盐缓冲盐水 （PBS） 洗涤切片 3 次，每次 5 分钟。</li>
		<li>将切片与封闭溶液（5% 牛血清白蛋白）在 RT 下孵育 1 小时，以阻断免疫球蛋白的非特异性结合。</li>
		<li>将切片与兔抗 NeuN 抗体 （1：300）、兔抗 Iba-1 抗体 （1：500） 和小鼠抗 GFAP 抗体 （1：500） 在 4°C 下孵育过夜。</li>
		<li>用 0.1 mM PBS 洗涤切片 3 次，每次 5 分钟。</li>
		<li>在室温下，将切片与山羊抗兔 Alexa 594 偶联的 IgG （1：500） 或山羊抗小鼠 Alexa 488 偶联的 IgG （1：500） 孵育 1 小时。</li>
		<li>用 0.1 mM PBS 洗涤切片，并用含有 4'，6-二脒基-2-苯基吲哚 （DAPI） 的抗淬灭封固剂封固。</li>
		<li>使用 594 nm 和 488 nm 的荧光显微镜分别拍摄缺血半影内三个微观视野（300 μm × 300 μm）的图像。</li>
		<li>使用 ImageJ 软件 （https://imagej.net/software/imagej/index），通过平均缺血半影19 中的计数来估计阳性染色的细胞密度。</li>
	</ol></li>
</ol>

远端中动脉闭塞开发缺血性卒中小鼠模型

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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=Quantitative+analysis+of+histological+staining+and+fluorescence+using+ImageJ.">Quantitative analysis of histological staining and fluorescence using ImageJ.</a> Anat Rec. 296 (3), 378-381 (2013).</li><li>Donahue, J., Wintermark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+CT+and+acute+stroke+imaging:+foundations,+applications,+and+literature+review.">Perfusion CT and acute stroke imaging: foundations, applications, and literature review.</a> J Neuroradiol. 42 (1), 21-29 (2015).</li><li>Sun, 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=Long-term+L-3-n-butylphthalide+pretreatment+attenuates+ischemic+brain+injury+in+mice+with+permanent+distal+middle+cerebral+artery+occlusion+through+the+Nrf2+pathway.">Long-term L-3-n-butylphthalide pretreatment attenuates ischemic brain injury in mice with permanent distal middle cerebral artery occlusion through the Nrf2 pathway.</a> Heliyon. 8 (7), e09909 (2022).</li><li>Balkaya, M. G., Trueman, R. C., Boltze, J., Corbett, D., Jolkkonen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+outcome+measures+to+improve+experimental+stroke+research.">Behavioral outcome measures to improve experimental stroke research.</a> Behav Brain Res. 352, 161-171 (2018).</li><li>Hao, T., 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=Inflammatory+mechanism+of+cerebral+ischemia-reperfusion+injury+with+treatment+of+stepharine+in+rats.">Inflammatory mechanism of cerebral ischemia-reperfusion injury with treatment of stepharine in rats.</a> Phytomedicine. 79, 153353 (2020).</li><li>Pietrogrande, 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=Low+oxygen+post+conditioning+prevents+thalamic+secondary+neuronal+loss+caused+by+excitotoxicity+after+cortical+stroke.">Low oxygen post conditioning prevents thalamic secondary neuronal loss caused by excitotoxicity after cortical stroke.</a> Sci Rep. 9 (1), 4841 (2019).</li><li>Shi, 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=Stroke+subtype-dependent+synapse+elimination+by+reactive+gliosis+in+mice.">Stroke subtype-dependent synapse elimination by reactive gliosis in mice.</a> Nat Commun. 12 (1), 6943 (2021).</li><li>Chen, W. 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=Aryl+hydrocarbon+receptor+modulates+stroke-induced+astrogliosis+and+neurogenesis+in+the+adult+mouse+brain.">Aryl hydrocarbon receptor modulates stroke-induced astrogliosis and neurogenesis in the adult mouse brain.</a> JNeuroinflammation. 16 (1), 187 (2019).</li></ol>

作者没有利益冲突需要披露。

在目前的开颅电凝 dMCAO 模型方案中，外科手术以最小的侵入性进行，其中仅分离一部分颞肌以减轻对咀嚼功能的不利影响。小鼠在手术后都恢复得很好，没有观察到喂养困难的情况。MCA 可以在小鼠的颞骨中轻松辨别，从而有助于精确识别合适的开颅手术位置。这种 dMCAO 模型诱导的缺血性病变位于皮层的外侧部分，与 Llovera G 等人描述的先前报告一致13。此外，与该模型相关的梗...

中风是一种常见的急性脑血管疾病，其特征是残疾和死亡率高1。在所有中风病例中，近 80% 属于缺血性中风2。到目前为止，静脉溶栓仍然是治疗急性缺血性卒中的少数有效方法之一。然而，溶栓治疗的有效性受到有效时间窗窄和出血转化发生的限制3。在缺血性中风后的长期康复阶段，相当多的患者可能会出现持久的神经功能障碍4。迫切需要进一步研究以揭示缺血性中风的潜在病理生理机制，并促进针对缺血性中风的新型治疗策略的开发。建立可靠且可复制的缺血性中风模型对于缺血性中风领域的基础研究以及随后的转化研究至关重要。
1981 年，Tamura 等人通过在大脑中动脉 （MCA） 近端部位采用经颅电凝术开发了局灶性脑缺血模型5。从那时起，许多研究人员利用各种方法，如结扎、压迫或夹闭来诱导远端 MCA 闭塞 （dMCAO），以建立短暂性或永久性缺血性卒中模型 6,7,8。与细丝模型相比，dMCAO 模型表现出明显的优势，例如更小的梗死面积和更高的生存率，使其更适合研究缺血性中风后的长期功能恢复9。此外，与细丝模型相比，dMCAO 模型在老年啮齿动物中的存活率更高，使其成为研究老年和合并症动物模型中缺血性中风的有利工具10。光血栓形成 （PT） 卒中模型已被证明具有手术侵入性小和死亡率显着低的特点。然而，与 dMCAO 模型相比，PT 模型表现出更大程度的细胞坏死和组织水肿，导致没有侧支循环11。此外，值得注意的是，在 PT 模型中观察到的缺血病变主要由微血管闭塞引起，这与 dMCAO 模型中大血管栓塞诱导的脑缺血有很大不同12。
在本文中，我们提出了通过小骨窗开颅手术凝固远端 MCA 来诱导小鼠 dMCAO 模型的方法。此外，我们进行了组织学检查和行为评估，以全面表征该实验模型中的缺血性损伤和中风结局。我们的目标是让研究人员熟悉该模型，并促进对缺血性中风病理机制的进一步研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,3,5-Triphenyltetrazolium 
			Chloride (TTC)</td>
			<td>Sigma-Aldrich</td>
			<td>108380</td>
			<td>Dye for TTC staining</td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>Corning (USA)</td>
			<td>CLS3527</td>
			<td>Vessel for TTC staining</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Wuhan Servicebio Technology 
			Co., Ltd.</td>
			<td>G1101</td>
			<td>Tissue fixation</td>
		</tr>
		<tr>
			<td>5% bovine serum albumin</td>
			<td>Wuhan BOSTER Bio Co., Ltd.</td>
			<td>AR004</td>
			<td>Non-specific antigen blocking</td>
		</tr>
		<tr>
			<td>5-0 Polyglycolic acid suture</td>
			<td>Jinhuan Medical Co., Ltd</td>
			<td>KCR531</td>
			<td>Material for surgery</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Midmark Corporation</td>
			<td>VMR</td>
			<td>Anesthetized animal</td>
		</tr>
		<tr>
			<td>Antifade mounting medium</td>
			<td>Beyotime Biotech</td>
			<td>P0131</td>
			<td>Seal for IF staining</td>
		</tr>
		<tr>
			<td>Automation-tissue-dehydrating&#160; 
			machine</td>
			<td>Leica Biosystems (Germany)</td>
			<td>TP1020</td>
			<td>Dehydrate tissue</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet (France)</td>
			<td>20220328</td>
			<td>Material for surgery</td>
		</tr>
		<tr>
			<td>Diclofenac&#160;sodium&#160;gel</td>
			<td>Wuhan Ma Yinglong Pharmaceutical 
			&#160;Co., Ltd.</td>
			<td>H10950214</td>
			<td>Analgesia for animal</td>
		</tr>
		<tr>
			<td>Drill tip (0.8 mm)</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td></td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Eosin staining solution</td>
			<td>Wuhan Servicebio Technology 
			Co., Ltd.</td>
			<td>G1001</td>
			<td>Dye for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Guangzhou Pharmaceutical Co., Ltd</td>
			<td>H44023098</td>
			<td>Material for surgery</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus (Japan)</td>
			<td>BX51</td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>GFAP Mouse monoclonal antibody</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>3670</td>
			<td>Primary antibody for IF staining</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa 
			488-conjugated IgG</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>4408</td>
			<td>Second antibody for IF staining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa 
			594-conjugated IgG</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>8889</td>
			<td>Second antibody for IF staining</td>
		</tr>
		<tr>
			<td>Grip strength meter</td>
			<td>Shanghai Xinruan Information Technology Co., Ltd.</td>
			<td>XR501</td>
			<td>Equipment for behavioral test</td>
		</tr>
		<tr>
			<td>Hematoxylin staining solution</td>
			<td>Wuhan Servicebio Technology 
			Co., Ltd.</td>
			<td>G1004</td>
			<td>Dye for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Iba1 Rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab178846</td>
			<td>Primary antibody for IF staining</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td>R510-22-10</td>
			<td>Anesthetized animal</td>
		</tr>
		<tr>
			<td>Laser doppler blood flow meter</td>
			<td>Moor Instruments (UK)</td>
			<td>moorVMS</td>
			<td>Blood flow monitoring</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Boehringer-Ingelheim</td>
			<td>J20160020</td>
			<td>Analgesia for animal</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td>78001</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Microsurgical instruments set</td>
			<td>Rwd Life Science Co., Ltd.</td>
			<td>SP0009-R</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>HM325</td>
			<td>Tissue section production</td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Leica Biosystems (Germany)</td>
			<td>819</td>
			<td>Tissue section production</td>
		</tr>
		<tr>
			<td>Monopolar electrocoagulation generator</td>
			<td>Spring Scenery Medical Instrument 
			Co., Ltd.</td>
			<td>CZ0001</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Mupirocin ointment</td>
			<td>Tianjin Smith Kline &#38; French 
			Laboratories Ltd.</td>
			<td>H10930064</td>
			<td>Anti-infection for animal</td>
		</tr>
		<tr>
			<td>NeuN Rabbit monoclonal antibody</td>
			<td>Cell Signaling Technology Inc. 
			(Danvers, MA, USA)</td>
			<td>24307</td>
			<td>Primary antibody for IF staining</td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Absin Bioscience</td>
			<td>abs9177</td>
			<td>Seal for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Paraffin embedding center</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>EC 350</td>
			<td>Produce paraffin blocks</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>Euthanized animal</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Shanghai Beyotime Biotech Co., Ltd</td>
			<td>C0221A</td>
			<td>Rinsing for tissue section</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Shenzhen Codos Electrical Appliances 
			Co.,Ltd.</td>
			<td>CP-9200</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Sodium citrate solution</td>
			<td>Shanghai Beyotime Biotech Co., Ltd.</td>
			<td>P0083</td>
			<td>Antigen retrieval for IF staining</td>
		</tr>
	</tbody>
</table>

induction of acute ischemic stroke in mice using the distal middle artery occlusion technique

缺血性中风仍然是全球成年人群死亡和功能障碍的主要原因。只有少数缺血性卒中患者有资格在最佳时间窗口内接受血管内溶栓或机械血栓切除术治疗。在这些中风幸存者中，约有三分之二的人在很长一段时间内患有神经功能障碍。建立稳定且可重复的实验性缺血性脑卒中模型，对于进一步研究缺血性脑卒中的病理生理机制和开发有效的治疗策略具有极其重要的意义。大脑中动脉 （MCA） 是人类缺血性中风的主要部位，MCA 闭塞是局灶性脑缺血的常用模型。在该协议中，我们描述了通过在 C57BL/6 小鼠中经颅电凝建立远端 MCA 闭塞 （dMCAO） 模型的方法。由于闭塞部位位于 MCA 的皮质分支，因此该模型会产生局限于皮层的中度梗死病变。在该模型中，神经行为学和组织病理学特征表明可见的运动功能障碍、神经元变性以及小胶质细胞和星形胶质细胞的明显激活。因此，这种 dMCAO 小鼠模型为研究缺血中风和普及价值提供了有价值的工具。

Stroke is a common acute cerebrovascular disease characterized by high incidences of disability and fatality1. Of all stroke cases, nearly 80% belong to ischemic stroke2. Up to now, intravenous thrombolysis remains one of a limited number of productive approaches for the treatment of acute ischemic stroke. However, the effectiveness of thrombolytic treatment is restricted by the narrow effective time window and the occurrence of hemorrhagic transformation3. In the long-term rehabilitation phase following an ischemic stroke, a considerable number of patients are likely to experience durable neurological dysfunctions4. Further investigation is urgently required to unravel the underlying pathophysiological mechanisms of ischemic stroke, as well as to facilitate the development of novel therapeutic strategies targeting ischemic stroke. The establishment of a dependable and replicable model of ischemic stroke is crucial for basic research as well as subsequent translational research in the field of ischemic stroke.
In 1981, Tamura et al. developed a focal cerebral ischemia model by employing transcranial electrocoagulation at the proximal site of the middle cerebral artery (MCA)5. Since then, numerous researchers have utilized various methodologies such as ligation, compression, or clipping to induce distal MCA occlusion (dMCAO) for establishing transient or permanent ischemic stroke models6,7,8. Compared to the filament model, the dMCAO model exhibits notable advantages such as smaller infarct size and higher survival rate, rendering it more suitable for investigating long-term functional recovery subsequent to ischemic stroke9. In addition, the dMCAO model demonstrates a higher survival rate in aged rodents compared to the filament model, making it an advantageous tool for investigating ischemic stroke in elderly and comorbid animal models10. The photothrombotic (PT) stroke model has been demonstrated to possess the characteristics of less surgical invasiveness and a significantly low mortality rate. However, the PT model exhibits a greater degree of cellular necrosis and tissue edema compared to the dMCAO model, leading to the absence of collateral circulation11. Furthermore, it is noteworthy that the ischemic lesions observed in the PT model predominantly arise from microvascular occlusion, which differs substantially from the cerebral ischemia induced by large vessel embolism in the dMCAO model12.
In this paper, we present the methodology for inducing the murine dMCAO model by coagulating the distal MCA via small bone window craniotomy. Additionally, we conducted histological examinations and behavioral evaluations to comprehensively characterize the ischemic insults and stroke outcomes in this experimental model. We aim to acquaint researchers with this model and facilitate further investigations into the pathologic mechanisms of ischemic stroke.

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

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中

In the present research, we aim to provide a comprehensive and detailed protocol for establish middle cerebral artery occlusion model in C57BL/6J mice using transcranial electrocoagulation and we evaluated the subsequent neurological behavior and histopathological features. The middle cerebral artery or MCA is recognized as the primary site of ischemic stroke in humans. The MCA occlusion methods, including technique, photochemical introduction, and occlusion have been extensively employed to induce focal cerebral ischemia in rodents.Compared to other ischemia stroke models, this model has the advantage of less surgical invasiveness, smaller infarct size, and higher survival rate, rendering it more suitable for investigation the long-term functional recovery after ischemic stroke. Overall, the current approach effectively generates a reliable experimental ischemic stroke model that exhibits high survivability and excellent repeatability. Consequently, this methodology serves as an invaluable tool for both foundation and translational research in the field of stroke.In the future, we will employ behavior paradigms such as novel object recognition and water maze to investigate the dynamic alterations in long-term learning and memory capabilities among distal MCA-occluded mice. This will enable us to ascertain the viability of utilizing them as animal model for cognition-impairment studies post-stroke.

用于执行 dMCAO 的关键器械是显微外科器械组、异氟醚蒸发器和单极显微外科电凝发生器， 如图 1 所示。本研究的实验过程如图 2 所示。简而言之，采用小骨窗开颅手术暴露远端 MCA，随后凝固以诱导 C57BL/6 小鼠永久性局灶性脑缺血。此外，在 dMCAO 后 3 天通过 TTC 染色、组织学检查和行为评估评估缺血性损伤和卒中结局。在手术过程中，只有 1 只小鼠死?...

Watch this Scientific Journal Video about Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique at JoVE.com

在这里，我们提出了一种方案，通过在 C57BL/6J 小鼠中经颅电凝建立远端大脑中动脉闭塞 （dMCAO） 模型，并评估随后的神经行为和组织病理学特征。

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

在本研究中，我们旨在为使用经颅电凝术在 C57BL/6J 小鼠中建立大脑中动脉闭塞模型提供全面而详细的方案，并评估随后的神经行为和组织病理学特征。大脑中动脉或 MCA 被认为是人类缺血性中风的主要部位。MCA 闭塞方法，包括技术、光化学引入和闭塞，已被广泛用于诱导啮齿动物的局灶性脑缺血。与其他缺血性卒中模型相比，该模型具有手术侵入性小、梗死面积小、生存率高等优点，使其更适合研究缺血性卒中后的长期功能恢复。总体而言，目前的方法有效地生成了可靠的实验性缺血性卒中模型，该模型表现出高生存率和出色的可重复性。因此，该方法可作为中风领域基础和转化研究的宝贵工具。未来，我们将采用新物体识别和水迷宫等行为范式来研究远端 MCA 闭塞小鼠长期学习和记忆能力的动态变化。这将使我们能够确定将它们用作中风后认知障碍研究的动物模型的可行性。

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Neuroscience

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

1.2K 次观看.  Jianghan University. 在本研究中，我们旨在为使用经颅电凝术在 C57BL/6J 小鼠中建立大脑中动脉闭塞模型提供全面而详细的方案，并评估随后的神经行为和组织病理学特征。大脑中动脉或 MCA 被认为是人类缺血性中风的主要部位。MCA 闭塞方法，包括技术、光化学引入和闭塞，已被广泛用于诱导啮齿动物的局灶性脑缺血。与其他缺血性卒中模型相比，该模型具有手术侵入性小、梗死面积小、生...

视频: 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

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.

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.

科研

神经科学

Distal Middle Artery Occlusion Procedure to Induce Ischemic Stroke in Mice

To begin, wipe the heating surgery board with 75%ethanol and set the temperature to 37 degrees Celsius. Place the anesthetized mouse in a right lateral position on the board with the head positioned away. To protect the cornea from drying, apply eye ointment.After shaving the fur from the left orbit to the left ear canal, use a depilatory cream to remove the remaining fur. Disinfect the surgical area three times with povidone iodine and 75%ethanol. Perform a toe-pinch assessment.After making a vertical incision between the left orbit and the left ear canal, retract the soft skin tissue to expose the temporal muscle. Using straight micro forceps, separate the apical and dorsal segments of the temporal muscle from the skull. Identify the Y-shaped bifurcation of the middle cerebral artery, or MCA, beneath the temporal bone.Then using an electric cranial drill, thin out the skull to make a bone window until the dura mater becomes visible. Remove the dura mater above the MCA with curved micro forceps. Using electric coagulation forceps, coagulate the artery at the site's proximal and distal to the bifurcation.Monitor the blood flow of the left MCA cortical branch in a laser speckle blood-flow meter. Then suture the muscle and the skin separately with polyglycolic acid sutures, and apply diclofenac sodium gel and mupirocin ointment to the skin incision. Place the mouse in a recovery chamber.

远端中动脉闭塞手术诱导小鼠缺血性中风

Neurological Behavioral Tests in Distal Middle Artery Occluded Mice to Study Ischemic Stroke Outcomes

To perform the grip strength test in the previously developed distal MCAO model, grasp the posterior section of a mouse tail and gradually lower the mouse until it holds the horizontal bar with both forepaws. Keeping the body horizontal, pull the mouse backward at a constant speed of two centimeters per second. When the mouse releases its forepaws from the bar, record the peak force in grams.For the pole test, place the mouse vertically on the top of a wooden pole. Record the time taken by the mouse to turn around and the total time to climb down the pole with a 60-second cutoff time. To conduct the adhesive test, attach a patch of adhesive tape to the right forepaw of the mouse.Put the mouse back into the rearing cage and record the time taken by the mouse to remove the adhesive with a 60-second cutoff time. To conduct a cylinder test, wipe an open top clear glass cylinder with 75%ethanol, and place the mouse in it. Using a camera, record its spontaneous standing exploratory behavior for three minutes.The distal MCAO mice showed a significant reduction in grip strength and contralateral forepaw usage rate as compared to the sham operated group. Further, the occluded mice showed a significant increase in the descent time in the pole test, and in the time taken to remove the adhesive.

远端中动脉闭塞小鼠的神经行为测试研究缺血性卒中结局

Mouse Brain Histology to Study the Pathological Mechanisms of Ischemic Stroke Induced by Distal Middle Artery Occlusion

To begin, obtain the dissected brain of the euthanized distal MCAO mouse model after behavioral testing, and place it in a minus 20 degrees Celsius freezer for 20 minutes. Then position the brain into a one-millimeter brain matrix. And using microtone blades, slice the brain into two-millimeter-thick coronary sections.Transfer the brain sections to a 24-well culture plate and add 2%TTC to cover the brain tissue. Incubate the plate at 37 degrees Celsius for 30 minutes. Then carefully fix the brain section in 4%Paraldehyde solution for 15 minutes.Using a scanister, perform optical scanning of the brain sections. The histological analysis of the brain revealed the disordered arrangement of neuron cells and a significant reduction in the density of NeuN positive cells in the peri-infarct area of the distal MCA occluded mice.