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。
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远端中动脉闭塞开发缺血性卒中小鼠模型

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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。

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中

Abstract