该研究由 科学与创新部 （MCIN）/Agencia Estatal de Investigación （AEI）、 Gobierno de España/10.13039/501100011033 和“欧洲区域发展基金 （ERDF）”资助的赠款 PID2020-113202RB-I00 支持。一种创造欧洲的方式“。NCC 和 MAR 拥有由 MCIN/AEI/10.13039/501100011033 和“欧洲社会基金 （ESF） 投资您的未来”资助的博士前奖学金（分别为 PRE2021-099481 和 PRE2018-085737）。我们感谢弗朗西斯卡·鲁伊斯-哈恩和莱昂纳多·马尔克斯-基西努斯基的技术支持。我们感谢 Institut d'Investigacions Biomèdiques August Pi i Sunyer （IDIBAPS） 的 MRI 成像设施的支持。加泰罗尼亚政府加泰罗尼亚研究中心（CERCA）计划支持IDIBAPS。

动物工作是按照加泰罗尼亚和西班牙法律（Real Decreto 53/2013）和欧洲指令进行的，并得到了巴塞罗那大学伦理委员会（Comité Ètic d'Experimentació Animal，CEEA）和加泰罗尼亚政府当地监管机构的批准。研究报告符合ARRIVE指南。该程序旨在在成年小鼠中进行，从 8 周龄开始，没有年龄限制。这里提供了在10-12周龄的C57BL / 6小鼠中开发的外科手术的示例。应考虑取决于小鼠品系的解剖学差异。
1.动物准备
<ol><li>在开始外科手术之前，请收集并消毒所有必需的材料和工具。用所有必要的手术材料（在 材料表中列出）设置手术台。</li>
	<li>使用异氟醚吸入氧气和一氧化二氮（30%/70%）的混合物麻醉动物。</li>
	<li>皮下注射丁丙诺啡（见 材料表），剂量为0.05mg / kg BW，以提供镇痛并减轻任何疼痛和不适。 注意：镇痛是强制性的，但接受不同的方案。在MCAo后的第一天，疼痛和不适的体征也必须得到控制（见步骤4）。必要时应用纠正解决方案。</li>
	<li>将动物置于含有5%异氟烷的麻醉诱导箱（见 材料表）中，直至达到深度麻醉状态（爪子穿刺和眼反射反射丧失）。</li>
	<li>将鼠标放在手术台上，将异氟烷的含量降低到1.5%，通过面罩给药。涂抹兽医软膏以避免在手术过程中眼睛干涩。</li>
	<li>将体温保持在37±0.5°C，由连接到加热垫的直肠探头控制（参见 材料表）。</li>
	<li>用电动剃须刀剃掉颈部和头部的腹侧部分（颅骨）。小心地清除毛皮碎屑，并用碘基消毒剂和 70% 酒精以打圈的方式对皮肤区域进行三次消毒。 .</li>
</ol>2. 使用激光多普勒血流法 （LDF） 进行脑血流 （CBF） 评估
<ol><li>用剪刀在头部皮肤上切开一个切口，沿着矢状缝线的方向，从耳朵到眼睛之间的区域。</li>
	<li>缩回皮肤并去除颅骨右侧的骨膜。</li>
	<li>找到坐标（距 Bregma 侧 2.5 毫米）并使用氰基丙烯酸酯连接多普勒支架（参见 材料表）。胶水干燥后，连接多普勒探头并检查信号读数是否正确。</li>
</ol>3. 短暂性大脑中动脉闭塞 （tMCAo）
<ol><li>将鼠标翻转到仰卧位，并用医用胶带将其固定在手术台上。</li>
	<li>在脖子上做一个中线切口。使用牵开器侧向拉回皮肤和唾液腺（见 材料表），以暴露颈动脉区域。</li>
	<li>识别颈总动脉 （CCA）、ICA 和 ECA 的血管解剖结构，以及由它们衍生的不同动脉（上颌动脉和舌动脉、甲状腺上动脉、枕动脉和翼腭动脉）（图 1A）。</li>
	<li>将主动脉与相邻的结缔组织分离，以便处理它们。 注意：特别注意不要损伤神经，尤其是与 CCA 平行的迷走神经。</li>
	<li>在上颌/舌分叉处将 6-0 真丝缝合线（见 材料表）缠绕在 ECA 周围。紧紧打结以永久中断循环。</li>
	<li>在同一条动脉周围，在第一个结和 CCA 分叉之间通过第二条缝合线，并保持这个结松动。</li>
	<li>在 CCA 周围放置第三根线并打一个可以轻松解开的滑结。 注意： 这也可以使用血管夹进行，但螺纹允许更多的运动和灵活性。在这个阶段，可以观察到LDF信号中CBF的首次降低。</li>
	<li>放置一个血管夹（见 材料表），中断ICA的血液循环。</li>
	<li>在 ECA 上做一个小切口，靠近紧结所在的区域。</li>
	<li>插入单丝，直到厚涂层完全进入动脉腔。</li>
	<li>拧紧第二个结，将单丝固定在动脉内，防止血液施加的压力将其推出（图1B）。</li>
	<li>从ICA上取下血管夹。</li>
	<li>将ECA切到第一个结下方，并旋转树桩以使其朝向ICA的方向（图1C）。</li>
	<li>通过 ICA 推进单丝，直到 MCA 分支点。 注意：闭塞反映在 LDF 读数中的血流量突然下降。当 CBF 下降大于基础值的 70% 时，我们认为闭塞成功。如果没有CBF测量系统，则可以通过前进阻力来记录闭塞点，在成年小鼠中，该阻力通常距离CCA的分叉约11毫米。<ol><li>如果在闭塞期间继续麻醉，请监视小鼠并持续观察45分钟。</li>
		<li>如果老鼠在闭塞期间被唤醒，请缝合几针的颈部皮肤。在不断开LDF探头的情况下，将鼠标放入温控盒中，以便从麻醉中恢复。 注意：在此期间，鼠标表现出自发的盘旋行为是很常见的，表明成功闭塞。</li>
		<li>40分钟后，按照与1.4,1.5和1.7点相同的麻醉和消毒程序再次麻醉小鼠。将其放回手术台上，然后从颈部拆下缝线。</li>
	</ol></li>
	<li>闭塞 45 分钟后，松开将单丝固定到位的结。缓慢而轻柔地拉动细丝，检查是否发生了组织再通。</li>
	<li>拉出灯丝并拧紧结以防止失血。</li>
	<li>解开 CCA 结。确保动脉壁没有损伤。</li>
	<li>取下牵开器并重新定位肌肉、腺体和皮肤。缝合皮肤（6-0）并涂抹消毒剂。</li>
	<li>断开多普勒探头，然后拆下支架。缝合和消毒头部皮肤。</li>
	<li>在麻醉恢复期间，将小鼠留在装有加热器的笼子中以保持温度。持续观察，直到它从麻醉中完全恢复。恢复后，老鼠可以放回笼子里。 注意：强烈建议提供具有社会丰富的住房。但是，切勿将操作的小鼠与未操作的小鼠混合在同一个笼子中，没有任何物理隔离，以防止攻击性。</li>
</ol>4. 术后护理
<ol><li>按照当地法规制定的程序和规定定期监督动物。按适当的时间表提供镇痛治疗，以尽量减少手术后的疼痛。 注意：在本研究中，在手术后 6 小时和 24 小时施用与干预开始时相同的镇痛剂（丁丙诺啡 0.05mg/kg BW）。</li>
	<li>当监督参数表明安乐死时，按照机构批准的协议进行安乐死。</li>
	<li>每天监测动物的体重。在手术后的最初几天为动物提供软食。此外，在手术后立即通过皮下注射生理盐水（200μL）来水化它们，如果观察到小鼠不自行水化，则定期水化。以动物容易获得的方式安排食物和水。</li>
	<li>体内研究完成后，麻醉小鼠，对它们实施安乐死，并取出脑组织进行进一步的组织病理学分析（如果需要）。</li>
</ol>

大脑中动脉闭塞的短暂性脑缺血模型

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作者声明没有利益冲突。

腔内 tMCAo 手术是基础研究中最常用的局灶性脑缺血再灌注模型。目前，由于转基因菌株的可用性，小鼠是首选的动物模型。然而，必须承认转基因小鼠及其遗传背景会影响脑血管形成。不同动脉区域之间侧支循环和吻合的存在可以显着影响实验程序的结果 11。
在执行此程序时，必须考虑某些关键点。损伤可能发生在 MCA 区域之外，累及海马体、丘脑或下丘?...

根据世界卫生组织的数据，中风是一种毁灭性的疾病，每年影响全球约 1500 万人。大约三分之一的患者死于这种疾病，而另外三分之一的患者则经历永久性残疾。卒中是一种复杂的病理学，涉及各种细胞类型，例如神经和外周免疫细胞、脉管系统和全身反应1。目前无法使用 体外 模型复制由系统水平的中风引发的复杂反应网络。因此，实验动物模型对于深入研究疾病的机制以及开发和测试新疗法至关重要。目前，早期组织再灌注是唯一获批的干预措施，通过组织型纤溶酶原激活剂 （tPA） 溶栓或血管内血栓切除术1。
脑中动脉 （MCA） 闭塞在卒中患者中很常见。因此，最初在大鼠中开发了瞬时 MCA 闭塞 （tMCAo） 的啮齿动物模型 2,3,4。如今，转基因小鼠是实验性中风模型中最常用的动物。在这项研究中，我们描述了小鼠腔内 tMCAo 的微创模型。该方法通过颈部水平的颈动脉进行，无需开颅手术。
闭塞期的持续时间是决定缺血性病变程度的关键因素。即使是 10 分钟的短暂闭塞也会导致选择性神经元死亡，而不会出现明显的梗死，而较长时间的闭塞（通常持续 30 至 60 分钟）会导致一定程度的脑梗死。与供应皮层并具有侧支的 MCA 的近端和远端分支不同，向纹状体提供血液的透镜状体动脉缺乏侧支5。因此，在 tMCAo 之后，纹状体中的血流量比皮层中的血流量减少更大。因此，30 分钟或更短时间的闭塞通常影响纹状体但不影响皮质，而从 45 分钟开始的较长时间闭塞通常会在整个 MCA 区域（包括纹状体和背外侧皮层）产生缺血性病变。
为了确保小鼠的健康，我们在手术前使用镇痛药，并在手术过程中使用麻醉剂。然而，麻醉可能会在小鼠的生理机能中引入人为的改变，并影响一些结果测量6。当由有经验的人员进行手术干预时，通常持续约 15 分钟以诱导 MCAo。随后，麻醉下的总时间取决于闭塞期。对于最小化麻醉至关重要的实验，手术中的替代步骤是在闭塞期间停止麻醉，并将其限制在插入和拔出闭塞 MCA 的细丝的手术步骤中。这种方法缩短了麻醉的持续时间，并最大限度地减少了其对实验模型的潜在人工影响7,8。因此，诱导短暂性局灶性缺血的方法通过MCA的腔内闭塞呈现，具有两种变体：在整个闭塞期间小鼠麻醉或在此期间小鼠清醒。在任何一种情况下，假手术都应与对缺血小鼠进行的干预同时进行。此外，在再灌注后的不同时间点通过行为测试和 MRI 测量，提供有关结果评估的数据。最后，讨论了实施实验程序时要考虑的主要因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6/0 suture&#160;</td>
			<td>Arago</td>
			<td></td>
			<td>Vascular ligatures</td>
		</tr>
		<tr>
			<td>6/0 suture with curved needle</td>
			<td>Arago</td>
			<td></td>
			<td>Skin sutures</td>
		</tr>
		<tr>
			<td>9 mg/mL Saline</td>
			<td>Fresenius Kabi</td>
			<td>CN616003 EC</td>
			<td>For hydration</td>
		</tr>
		<tr>
			<td>Anaesthesia system</td>
			<td>SurgiVet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt retractors, 1 mm wide</td>
			<td>Fine Science Tools</td>
			<td>18200-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorfine</td>
			<td>Buprex</td>
			<td></td>
			<td>For pain relief</td>
		</tr>
		<tr>
			<td>Clamp applying forceps</td>
			<td>Fine Science Tools</td>
			<td>S&#38;T CAF4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont mini forceps</td>
			<td>Fine Science Tools</td>
			<td>M3S 11200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Loctite</td>
			<td></td>
			<td>To stick LDF probe to the skull</td>
		</tr>
		<tr>
			<td>Grip Strength Meter</td>
			<td>IITC Life Science Inc.</td>
			<td>#2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>B-Braun</td>
			<td>CN571105.8</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF Perimed</td>
			<td>Perimed</td>
			<td>Periflux System 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF Probe Holders</td>
			<td>Perimed</td>
			<td>PH 07-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRI magnet</td>
			<td>Bruker BioSpin, Ettlingen, Germany</td>
			<td></td>
			<td>BioSpec 70/30 horizontal animal scanner&#160;</td>
		</tr>
		<tr>
			<td>Needle Holder with Suture Cutter</td>
			<td>Fine Science Tools</td>
			<td>12002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filament</td>
			<td>Doccol</td>
			<td>701912PK5Re</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery cage with heating pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sirgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91401-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Small vessel cauterizer kit</td>
			<td>Fine Science Tools</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope and cold light</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>18025-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostat, rectal probe and mouse pad</td>
			<td>Letica Science Instruments</td>
			<td>LE 13206</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors (4mm cutting edge)</td>
			<td>Fine Science Tools</td>
			<td>15019-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>00396-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

transient middle cerebral artery occlusion model of stroke

中风是全球死亡或慢性残疾的主要原因。然而，现有的最佳治疗方法仅限于缺血性卒中急性期的再灌注治疗。为了深入了解中风生理学并开发创新的治疗方法，中风 的体内 啮齿动物模型起着重要作用。转基因动物的可用性特别推动了小鼠作为实验性中风模型的使用。
在卒中患者中，大脑中动脉 （MCA） 闭塞很常见。因此，最普遍的实验模型涉及 MCA 的腔内闭塞，这是一种不需要开颅手术的微创技术。该过程包括通过颈外动脉 （ECA） 插入单丝并将其推进通过颈内动脉 （ICA），直到它到达 MCA 的分支点。动脉闭塞 45 分钟后，移除单丝以允许再灌注。在整个过程中，监测脑血流以确认闭塞期间的减少和随后的再灌注恢复。使用行为测试和磁共振成像 （MRI） 研究评估神经和组织结果。

有不同的方法可以评估 tMCAo 程序的结果。这里使用 体内 神经成像方法 （MRI） 和行为测试。
小鼠在大脑中出现缺血性病变，主要影响MCA同侧提供给闭塞的区域，如纹状体和背外侧皮层。有几种方法可以确定病变范围，包括 2,3,5-三苯基氯化四氮唑 （TTC） 组织染色、组织学染色（苏木精/伊红素、乙酸亚硫氨酸）和 MRI 等 体内 神经影像学检查。这里之所以选择 MRI...

Watch this Scientific Journal Video about Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model at JoVE.com

作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

该方案描述了通过大脑中动脉腔内闭塞在小鼠中短暂性局灶性脑缺血的模型。此外，还显示了使用磁共振成像和行为测试的结果评估示例。

脑卒中短暂性中动脉闭塞模型

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

大脑中动脉闭塞模型被广泛用于模拟小鼠缺血性脑卒中。该模型允许研究缺血性损伤的时间进展，评估神经保护剂的有效性，并探索特定基因和通路在中风病理学中的作用，最终目的是寻找新的潜在治疗方法。MRI等神经成像技术的实施可以更好地诊断，改善结果评估，并减少每项研究中所需的动物数量。该协议对诱导短暂性大脑中动脉闭塞的最常用技术进行了清晰而详细的解释。此外，它通过引入闭塞期间麻醉退出的精确修改来实现该技术，从而减少了长时间麻醉时间的不利影响。

transient-middle-cerebral-artery-occlusion-model-of-stroke

Research

JoVE Journal

Neuroscience

Transient Middle Cerebral Artery Occlusion Model of Stroke

1.6K 次观看.  Spanish National Research Council (CSIC). 大脑中动脉闭塞模型被广泛用于模拟小鼠缺血性脑卒中。该模型允许研究缺血性损伤的时间进展，评估神经保护剂的有效性，并探索特定基因和通路在中风病理学中的作用，最终目的是寻找新的潜在治疗方法。MRI等神经成像技术的实施可以更好地诊断，改善结果评估，并减少每项研究中所需的动物数量。该协议对诱导短暂性大脑中动脉闭塞的最常用技术进行了清晰而详?...

视频: 作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

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

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

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

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

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

科研

神经科学

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

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

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

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

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

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

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

利用3D打印技术来合并MRI与组织学:一个大脑切片协议

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from &plusmn; 25 min to &plusmn; 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (&plusmn; 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (&plusmn; 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke.

脑缺血中风发作时间估计的磁共振成像协议

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

模拟小鼠中风:通过颈外动脉的短暂性脑中动脉闭塞

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

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

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

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

小鼠脑中动脉闭塞诱导脑卒中后脑肌粘连血管病治疗模型

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