这项工作得到了教育、文化、体育、科学和技术部（MEXT）的科学研究援助赠款（18K09424，东芝库里原和20K18393对三和裕一郎）的支持。

这里描述的所有方法都得到了庆应大学医学院机构动物护理和使用委员会（IACUC）的批准。
1. 准备手术器械和动物
<ol><li>高压外科手术器械，并保持他们在70%的乙醇。在每一次新的外科手术之前，清洁手术器械小心使用70%的乙醇。</li>
	<li>在特定无病原体 （SPF） 室中准备雄性 BALB/cAJc1 小鼠（6 周大，26-28 公斤），以便在手术前、手术期间和手术后保持无菌状态。</li>
</ol>2. 瞬态双边常见胡萝卜动脉闭塞（tBCCAO）
<ol><li>通过腹内注射将鼠标置于麻醉下，结合中佐兰（40微克/100微升）、梅德托米丁（7.5微克/100微升）和丁醇酸酸盐（50微克/100微升），称为"MMB"，如前述的8，9。抓住老鼠的后皮，防止老鼠撞到眼睛，直到老鼠完全麻醉。<ol>
<li>判断麻醉的深度，捏鼠标脚趾，直到它没有反应，其中的方法通常用于检查完整的麻醉10。 注：一般来说，小鼠入睡需要不到5分钟。适当的全身麻醉配方可能因机构而异。</li>
	</ol>
</li>
	<li>将一滴0.1%纯化的透明质钠眼药水溶液涂抹在眼睛上，防止麻醉下眼睛干燥。</li>
	<li>将鼠标放在它的背上，用胶带固定鼠标的爪子。</li>
	<li>手术前使用70%的乙醇对老鼠的颈部区域进行消毒。 注：毛皮的额外剪裁没有进行，因为这可能导致随后的皮肤炎症11，12。</li>
	<li>用刀片（图1）对颈部进行下垂切口。 注：切口需要在颈部、胸骨和气管之间的中线进行。</li>
	<li>使用两个钳子小心地分离两个唾液腺，并动员它们来可视化潜在的CPA。</li>
	<li>在不损害其结构的情况下，小心地将右 CCA 与各自的血管神经和伴随的静脉隔离，并将两根 6-0 丝绸缝合线置于 CCA 下。将两条领带紧紧地系住，以阻止血流（图1）。 注：在手术过程中，小静脉可能会受损。如果看到出血，需要擦拭才能清楚地显示CCA。</li>
	<li>从各自的血管神经和伴随的静脉中仔细查找左CCA，而不会伤害其结构，并通过夹子将左CCA遮挡 2 秒（图 1）。 注：需要将 6-0 丝缝针放在左 CCA 下，以标记夹紧的部位。</li>
	<li>左CCA重新打开后，用6-0丝缝合颈部的缝合伤口，并在颈部涂抹抗生素（50 μL），以抑制细菌感染。 注：在左CCA重新打开时，请轻轻取下夹子以避免损坏动脉壁。</li>
	<li>将0.75毫克/千克盐酸阿提帕米佐尔注射到鼠体内，帮助小鼠迅速从深度麻醉中恢复过来。将鼠标返回带预热垫的鼠标笼中。 注意：不要让鼠标无人看管，直到鼠标恢复足够的意识，以保持体面休养。</li>
	<li>将 0.4 毫克/千克的布托帕诺酸酸注射到鼠标上，用于管理鼠标醒来时的疼痛。 注：协议可在此处暂停。作为成功tBCCAO的第一个提示，可以观察到鼠标的眼睑下垂（图2）。</li>
	<li>对于安乐死，向小鼠注射3倍的MB混合物，并牺牲它们进行实验。</li>
</ol>3. 一般观察（存活率和眼睑下垂）
<ol><li>手术后，检查所有死因的存活率在第0天（手术后），1，3和7。</li>
	<li>评估眼睑下垂 4 点评级尺度： 1 = 无下垂， 2 = 轻度下垂 （约 50%）， 3 = 严重下垂 （超过 50%）， 4 = 眼睛放电严重下垂。</li>
</ol>4. 视网膜输血
<ol><li>注射200μL的FITC-dextran（25毫克/mL）到小鼠的左心室，这是通常用于观察血液灌注小鼠视网膜血管13，14。</li>
	<li>循环后2分钟，将眼睛核化，固定在4%的准甲醛中1小时。视网膜被仔细获得和平装，如先前描述的15，并通过荧光显微镜检查。</li>
	<li>以 4 倍的放大倍率拍摄视网膜整个坐骑的照片，并使用合并分析仪合并成一个单个，以前描述的16。</li>
	<li>通过 NIH 斐济/ImageJ 软件中的容器分析工具测量注入区域。</li>
</ol>5. 西方污点
<ol><li>3和6小时后，tBCCAO，获得小鼠的眼睛，并立即转移到含有冷PBS的培养皿，以隔离视网膜。</li>
	<li>视网膜隔离后，进行西印，如先前描述的9。</li>
	<li>与抗体一起孵化，用于缺氧诱发因子-1+（HIF-1+;一般缺氧标记）和β-Actin（内部负荷控制），然后孵化HRP结合的次要抗体。通过化学发光可视化信号。</li>
</ol>6. 定量 PCR （qPCR）
<ol><li>6，12和24小时后tBCCAO，处理获得的视网膜qPCR，如先前描述的17。</li>
	<li>通过实时PCR系统执行qPCR。所使用的底漆列在 表 1中。通过ΔΔCT 方法计算不同成绩单水平之间的折叠变化。</li>
</ol>7. 免疫化学（IHC）
<ol><li>3天后tBCCAO，获得小鼠的眼睛，并嵌入石蜡。</li>
	<li>用微切除术切割石蜡嵌入的眼睛，以获得眼部。</li>
	<li>去石蜡和染色眼睛部分的5μm厚度，如先前描述的13。</li>
	<li>与胶质纤维酸性蛋白（GFAP;星形细胞和网膜中的穆勒细胞的可靠标记）的抗体一起孵化过夜，然后孵化亚历克萨氟555结合二次抗体。</li>
	<li>使用DAPI（4+，6-迪亚米迪诺-2-苯丙酮）染色视网膜中的细胞核。通过荧光显微镜可视化信号。</li>
	<li>按 4 分评级等级评估形态评分，如之前描述的 13、18：0 = 无信号、1 = 在胶质细胞层 （GCL） 中很少有正胶质端脚、2 = 从 GCL 到外核层 （ONL） 的标记过程很少，以及从 GCL 到 ONL 的大多数标记过程。</li>
</ol>8. 电视网膜成像（ERG）
<ol><li>3和7天后tBCCAO，执行ERG使用甘兹费尔德圆顶，采集系统和LED刺激器，如前所述9。</li>
	<li>经过一夜的黑暗适应后，在昏暗的红灯下用MB组合麻醉小鼠。</li>
	<li>使用0.5%热带酰胺和0.5%苯肾上腺素的混合溶液来延长瞳孔。</li>
	<li>将活动电极放在隐形眼镜上，并将参考电极放置在口腔中。</li>
	<li>从每只动物的两只眼睛获得ERG响应。</li>
	<li>用各种刺激记录黑暗适应下的滑板反应。</li>
	<li>测量波从基线到波的最低点的振幅。</li>
	<li>测量 b 波的振幅，从波的最低点到 b 波的峰值。</li>
	<li>使用热垫在手术过程中保持所有小鼠的温暖。</li>
</ol>9. 光学连贯断层扫描 （OCT）
<ol><li>tBCCAO后2周，使用SD-OCT系统执行OCT，如先前报道的8，9。</li>
	<li>在测量中，通过0.5%的热带酰胺和0.5%的苯肾上腺素的混合溶液，让小鼠患上甲虫病，并通过MB的混合物进行全身麻醉。</li>
	<li>从赤道片的面对面扫描中获取 B 扫描图像。</li>
	<li>检查视网膜在0.2，0.4和0.6毫米从视神经头。</li>
	<li>测量视网膜厚度从视网膜神经纤维层 （NFL） 到外部限制膜 （ELM），并将测量值的平均值视为单个鼠标的视网膜厚度。</li>
	<li>将结果绘制为蜘蛛图。</li>
</ol>

作者没有什么可透露的。

在这项研究中，我们已经表明，tBCCAO，使用简单的缝合和夹子，可以诱导视网膜缺血和伴随视网膜功能障碍。此外，我们已证明，我们目前的协议，开发视网膜缺血小鼠模型是更容易和更快相比，其他以前的协议，开发视网膜缺血损伤模型2，3，7。
从解剖学上讲，左右脑动脉可以通过后沟通动脉（PCAs）连...

视网膜是一种神经感官组织，用于视觉功能。由于视觉功能需要大量的氧气，视网膜被称为身体中对氧气要求最高的组织之一。由于氧气通过血管输送，网膜易患血管疾病。各种血管疾病，如糖尿病视网膜病变和视网膜血管（静脉或动脉）闭塞，可诱发视网膜缺血。为了研究视网膜缺血的病理机制，认为有必要对视网膜缺血进行可重复和临床相关的实验模型。通过插入内脏丝进行中脑动脉闭塞（MCAO）是开发实验性脑缺血2、3体内啮齿动物模型的最普遍方法。由于眼动脉（OpA）接近MCA，MCAO模型也同时用于了解视网膜缺血的病理生理学4，5，6。为了诱导脑缺血和视网膜缺血，长丝通常通过普通胡萝卜动脉 （CCA） 或外部胡萝卜动脉 （ECA） 的切口插入。这些方法很难执行，需要很长时间才能完成手术（一只小鼠超过60分钟），并导致手术后结果的高变异性7。制定更好的模式以改善这些关切仍然很重要。
在这项研究中，我们只是使用短的瞬态双边CCA闭塞（tBCCAO）与针头和夹子诱导视网膜缺血在小鼠，并分析了视网膜缺血损伤的典型结果。在此视频中，我们将演示 tBCCAO 程序。

<table>
	<tbody>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Zenoaq</td>
			<td>Antisedan</td>
			<td>For anti-anesthesia</td>
		</tr>
		<tr>
			<td>Applied Biosystems 7500 Fast</td>
			<td>Applied Biosystems</td>
			<td>-</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Meiji Seika Pharma</td>
			<td>Vetorphale</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>BZ-II Analyzer</td>
			<td>KEYENCE</td>
			<td>-</td>
			<td>For an image merge</td>
		</tr>
		<tr>
			<td>BALB/cAJc1</td>
			<td>CLEA</td>
			<td>-</td>
			<td>Mouse strain</td>
		</tr>
		<tr>
			<td>&#946;-Actin (8H10D10) Mouse mAb</td>
			<td>CST</td>
			<td>3700</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Clamp Forcep</td>
			<td>World Precision Instruments</td>
			<td>WPI 500451</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Dojindo</td>
			<td>340-07971</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Envisu SD-OCT system</td>
			<td>Leica</td>
			<td>R4310</td>
			<td>For OCT</td>
		</tr>
		<tr>
			<td>FITC-dextran</td>
			<td>Merk</td>
			<td>FD2000S</td>
			<td>For retinal blood perfusion</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-9000</td>
			<td>For fluorescence detection</td>
		</tr>
		<tr>
			<td>Gatifloxacin hydrate</td>
			<td>Senju Pharmaceutical</td>
			<td>Gachifuro</td>
			<td>For anti-bacterial infection</td>
		</tr>
		<tr>
			<td>GFAP Monoclonal Antibody (2.2B10)</td>
			<td>Thermo</td>
			<td>13-0300</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Marukan</td>
			<td>RH-200</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>HIF-1&#945; (D1S7W) XP Rabbit mAb</td>
			<td>CST</td>
			<td>36169</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>GE Healthcare</td>
			<td>-</td>
			<td>For chemiluminescence</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Sandoz K.K</td>
			<td>SANDOZ</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Microtome Tissue-Tek TEC 6</td>
			<td>Sakura</td>
			<td>-</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Orion Corporation</td>
			<td>Domitor</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Handaya</td>
			<td>HS-2307</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>PuREC</td>
			<td>MAYO Corporation</td>
			<td>-</td>
			<td>For ERG</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sodium hyaluronate</td>
			<td>Santen Pharmaceutical</td>
			<td>Hyalein</td>
			<td>For eye lubrication</td>
		</tr>
		<tr>
			<td>Tropicamide/Penylephrine hydrochloride</td>
			<td>Santen Pharmaceutical</td>
			<td>Mydrin-P</td>
			<td>For mydriasis</td>
		</tr>
		<tr>
			<td>6-0 silk suture</td>
			<td>Natsume</td>
			<td>E12-60N2</td>
			<td>For surgery</td>
		</tr>
	</tbody>
</table>

a murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion

多种血管疾病，如糖尿病视网膜病变、视网膜静脉或动脉闭塞以及眼缺血综合征可导致视网膜缺血。为了研究视网膜缺血的病理机制，需要开发相关的实验模型。从解剖学上讲，主要的视网膜供血血管是眼动脉 （OpA）， OpA 起源于普通胡萝卜动脉 （CCA） 的内胡萝卜动脉。因此，CCA的中断可以有效地导致视网膜缺血。在这里，我们建立了视网膜缺血的鼠标模型，通过瞬态的双边常见胡萝卜素动脉闭塞（tBCCAO），用6-0丝缝合右CCA，并通过夹子暂时将左CCA遮挡2秒，并表明tBCCAO可以诱发急性视网膜缺血，导致视网膜功能障碍。目前的方法通过仅使用手术针头和夹子来减少对手术器械的依赖，缩短了闭塞时间，以尽量减少意外的动物死亡，这在中脑动脉闭塞小鼠模型中经常看到，并保持常见视网膜缺血结果的可重复性。该模型可用于研究小鼠缺血视网膜病变的病理生理学，并进一步用于体内药物筛选。

在FITC-dextran系统循环2分钟后，检查了假手术小鼠和tBCCAO手术小鼠左视网膜和右视网膜的视网膜血管（补充图1）。FITC-dextran在假手术小鼠的视网膜和tBCCAO操作的小鼠的左视网膜中完全可见，而在tBCCAO操作的小鼠的右视网膜中部分可检测到。
在tBCCAO之后，对眼睑下垂进行了检查（图2）。右眼显示温和（得分2;75%）和严重的眼睑（分数3和4：25%?...

Watch this Scientific Journal Video about A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion at JoVE.com

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

在这里，我们描述了一个鼠标模型的视网膜缺血通过瞬态的双边常见的胡萝卜动脉闭塞使用简单的缝合线和夹子。该模型可用于了解心血管异常引起的视网膜缺血的病理机制。

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

a-murine-model-ischemic-retinal-injury-induced-transient-bilateral

Research

JoVE Journal

Medicine

7.3K 次观看.  Keio University School of Medicine. 在这里，我们描述了一个鼠标模型的视网膜缺血通过瞬态的双边常见的胡萝卜动脉闭塞使用简单的缝合线和夹子。该模型可用于了解心血管异常引起的视网膜缺血的病理机制。

视频: A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

科研

医学

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成年小鼠静脉高压型号：颈总动脉，以颈外静脉吻合。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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, ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

体内兔颈总动脉内皮的基因转移

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

颈动脉介入移植中颈静脉持久性转基因表达的家兔模型

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

氯化铁诱导的犬颈动脉血栓形成: 一种大的血管损伤动物模型

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

颅内动脉闭塞允许经普通颈动脉修复再灌注的研究

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...