（; EJD EY022383和EY022683）和Core补助（P30EY001765），成像和显微镜核心模块这项工作是由美国国立卫生研究院的研究资助。

伦理学声明:所有程序均按照由约翰霍普金斯大学机构动物护理和使用委员会规定的准则进行。 注意:在拍摄过程中使用的小鼠是从杰克逊C57BL / 6小鼠，虽然也可以使用其他啮齿动物品系或物种。当使用其他菌株或物种，应注意麻醉剂量和伤害时间表可能会有所不同。它适应I / R的条件，以适应应变，物种和实验的变化是重要的。 1.准备麻醉鸡尾酒<ol><li>结合1.25毫升氯胺酮，0.625毫升赛拉嗪，0.375毫升乙酰丙嗪，和22.75毫升磷酸盐缓冲盐水在50毫升离心管中。 注意:对于手稿的其余部分，该解决方案将被称为鸡尾酒。 </li><li>过滤到鸡尾酒用60毫升注射器和0.20微米的过滤器的新的无菌50ml离心管中。标签和日期这个新涂是。 </li><li>完全包住鸡尾酒管在铝箔以防止麻醉剂的光诱导降解。 注:鸡尾酒可在室温下保存和再使用直至其最早的过期的成分的到期日。 </li></ol> 2.准备麻醉助推器<ol><li>在50ml离心管中，结合4毫升氯胺酮和16毫升磷酸盐缓冲盐水。 注:该解决方案将作为今后助推器被称为。 </li><li>过滤成助推器用60毫升注射器和0.20微米的过滤器的新的无菌50ml离心管中。标签和日期这一新管。 注:助力器可以在室温下保存和再使用直至其最早的过期的成分的到期日。 </li></ol> 3.准备手术套房<ol><li>设定18℃，21℃之间的室温。 </li><li>打开手术表，并调整其表面热到解释第1条ST温度。 </li><li>覆盖手术底衬所有的工作表面。 </li><li>排列在手术台的空笼或其他容器温暖。 </li><li>安排一个规模上台面的耳朵恶搞。 </li></ol> 4.准备肝素钠的平衡盐溶液<ol><li>将0.5ml肝素钠到500ml四瓶平衡盐溶液（HBSS）中的。 </li><li>将prepierced Y形管插入瓶中0.1％肝素钠的主要集的尖底。 </li></ol> 5.设置输液架<ol><li>挂在输液架上延伸0​​.1％肝素钠的瓶子，并扣打开prepierced Y形管的主要集空气过滤器盖。 </li><li>提升0.1％瓶肝素钠到163厘米（120毫米汞柱）。测量从桌面到肝素钠滴的峰的高度。 </li><li>通过手动弹prepierced Y形管的主要集中移除在静脉输液管的气泡。 </l我&gt; </ol> 6.设置肝素钠滴灌<ol><li> prepierced Y形管的主要组连接到五阀歧管。 </li><li>连接30号½使用露儿男性对男性露儿管接头英寸针到五端口歧管。 </li><li>将每个30号½英寸针到30号管自己的10寸段。 </li><li>使用止血，打破新的30口径，½英寸针针尖和他们的钝端插入30号管。针尖无菌消毒或将是必要的步骤8.3。 </li><li>使用磁带，安排与针，使得连接到内口管被定位在连接到外端口的管的顶部的30号管。这种安排将防止前房插管过程中管缠绕。 </li><li>打开0.1％肝素钠流至五个阀歧管和每个单独的端口。 <ol><li>确保0.1％肝素钠是为每个端口强烈流动。如果一个端口弱流动，更换端口或无菌注射器与空气清除。 </li><li>允许0.1％肝素钠流动为2 - 3分钟以从30号管和5-阀歧管除去气泡。 </li></ol></li><li>关闭于5阀组的所有端口。 </li></ol> 7.准备手术的小鼠<ol><li>把老鼠的外科手术室。供水瓶每个网箱以防止手术过程中动物脱水。 </li><li>记录每只小鼠的体重。 </li><li>注射每只小鼠腹腔注射每克体重0.02毫升鸡尾酒。 </li><li>标记和记录各小鼠的数目。 </li><li>将所有的老鼠进入上手术台的空容器。允许所有的老鼠5-10分钟，以达到深度麻醉由无踏板戒断反应到脚趾捏的证实。 </li><li>对于每个小鼠，施用一滴托到每个眼睛的瞳孔扩张和短期润滑。 </li><li>对于每个小鼠，施用丙美卡因一滴到每个眼的局部麻醉和短期润滑。 </li><li>安排在麻醉的顺序小鼠，使得第一鼠标被麻醉将是第一个鼠标被插管。 </li><li>允许约2分钟的眼药水才能生效。 </li><li>由磁带上紧紧拉制备胶带的直4英寸件。预留每鼠一块胶带。 注意:如果不紧密地拉在磁带上会导致磁带的卷曲。 </li></ol> 8.导管插入前房<ol><li>安排手术显微镜下的第一个鼠标和聚焦显微镜的首选角膜。 注:可以插管在任的眼睛进行。右手主导的外科医生可能会发现我吨最容易导管插入左眼，而左侧优势的外科医生可能更喜欢的权利。 </li><li>打开第一个0.1％肝素钠端口上的五阀组。 </li><li>在手术显微镜，用一双镊子轻轻proptose眼球。大约中途插入30号套管针进入前房的小带纤维和角膜的顶点之间。 <ol><li>小心避免划伤或刺破虹膜，晶状体，或内角膜表面。 </li><li>避免穿透角膜第二次。 </li><li>使用温和的扭转运动，克服了套管和角膜之间的摩擦，在所述前房深深插入插管。 </li></ol></li><li>使用胶带的条带的30号管件固定到该表。为了尽量减少套管插入运动，按30号管对桌面同时达到磁带。 </li><li>记录手术的开始时间。 </li><li>使用显微镜，verify表示无泄漏是明显的。如果泄漏存在，流体的运动将是眼睛附近可见。 </li><li>通过观察，该I / R眼睛比对侧眼大目视确认眼部腹胀。一起，由于没有泄漏和眼腹胀的存在表明眼压的一个成功的高程。 </li><li>应用羟丙甲纤维素到双眼。羟丙甲纤维素起到润滑角膜和密封中微小。根据需要（大约每30分钟）持续润滑再涂羟丙甲纤维素。 </li><li>重复步骤8，直到所有的动物已经被插管。 </li></ol> 9.监控麻醉<ol><li>使用脚趾捏，晶须，或尾部的视觉观察的视觉观察，以确认每个小鼠保持麻醉。如果鼠标演示踏板戒断反应，晶须抽搐，或尾部的运动，立即进行步骤9.2。 </li><li>如果一只老鼠开始在手术过程中，电梯唤醒尾到腹膜内注入0.05毫升增压到下腹部从鼠标后面。允许1 - 2分钟，助推器生效。 </li><li>如果鼠标需要额外的镇静，必要时重复步骤9。 </li></ol> 10.前房取出套管<ol><li>对于每个动物，90分钟后已经过去了，轻轻拉动从前房插管。 </li><li>从手术表Untape鼠标，注意不要扰乱相邻动物的套管管道。 </li><li>润滑双眼具有润滑果冻。 </li><li>随后的套管被删除，安排在手术台上的空容器中的小鼠从手术中恢复。不要关闭手术台的热量。 </li><li>允许，至少，2 - 3小时于加热手术台复苏。经常观察，直到他们充分从麻醉中恢复的老鼠。 </li></ol> 11.清洁式uipment <ol><li>用消毒酒精擦拭插管。 注意:为消毒或灭菌的其它方法，如高压灭菌，可以被取代。 </li><li>用60毫升注射器填充有空气排出从5阀组0.1％肝素钠，30号针头，30号管和套管。 </li><li>用60毫升注射器充满蒸馏水冲洗5阀组，30号针头，30号管和套管。 </li><li>用60毫升注射器充满空气从5端口歧管，30号针头，30号管和插管排出的蒸馏水。 </li><li>消毒插管和冲洗管道设备后，存放该设备的再利用。 </li><li>商店，放弃或关闭所有其它设备。留在手术台的热量。 </li></ol> 12.返回所有小鼠饲养笼<ol><li>后小鼠从手术唤醒（后2 - 3小时），每个动物返回其家笼。每个笼子提供凝胶的食品。返回所有笼子到其指定的房间。 </li><li>关掉手术台的热量。丢弃所有垃圾，并擦拭手术套件。 </li></ol> 13.执行视网膜评估<ol><li>适当时，收集组织学分析或暗适应小鼠视网膜电图记录视网膜。 </li></ol>

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A., Schoen, T., Kuwabara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+ischemia+induces+cell+mitosis+in+the+adult+rat+retina.">Experimental ischemia induces cell mitosis in the adult rat retina.</a> Invest Ophthalmol Vis Sci. 29 (7), 1050-1055 (1988).</li><li>Honjo, 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=Statin+inhibits+leukocyte-endothelial+interaction+and+prevents+neuronal+death+induced+by+ischemia-reperfusion+injury+in+the+rat+retina.">Statin inhibits leukocyte-endothelial interaction and prevents neuronal death induced by ischemia-reperfusion injury in the rat retina.</a> Arch Ophthalmol. 120 (12), 1707-1713 (2002).</li><li>Otori, Y., 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=Expression+of+c-fos+and+c-jun+mRNA+following+transient+retinal+ischemia:+an+approach+using+ligation+of+the+retinal+central+artery+in+the+rat.">Expression of c-fos and c-jun mRNA following transient retinal ischemia: an approach using ligation of the retinal central artery in the rat.</a> Surv Ophthalmol. 42, 96-104 (1997).</li><li>Liu, J., 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=Epac2-deficiency+leads+to+more+severe+retinal+swelling,+glial+reactivity+and+oxidative+stress+in+transient+middle+cerebral+artery+occlusion+induced+ischemic+retinopathy.">Epac2-deficiency leads to more severe retinal swelling, glial reactivity and oxidative stress in transient middle cerebral artery occlusion induced ischemic retinopathy.</a> Sci China Life Sci. 58 (6), 521-530 (2015).</li><li>Zhang, Y., Zhang, Z., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simvastatin+inhibits+ischemia/reperfusion+injury-induced+apoptosis+of+retinal+cells+via+downregulation+of+the+tumor+necrosis+factor-alpha/nuclear+factor-kappaB+pathway.">Simvastatin inhibits ischemia/reperfusion injury-induced apoptosis of retinal cells via downregulation of the tumor necrosis factor-alpha/nuclear factor-kappaB pathway.</a> Int J Mol Med. , (2015).</li><li>Li, B., Pang, I. H., Barnes, G., McLaughlin, M., Holt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+and+device+to+induce+transient+retinal+ischemia+in+the+rat.">A new method and device to induce transient retinal ischemia in the rat.</a> Curr Eye Res. 24 (6), 458-464 (2002).</li></ol>

The authors have no disclosures.

视网膜我/ R损伤的高眼压已经证明了其在模拟细胞损伤和功能障碍，神经退行性疾病特别是在啮齿动物的视网膜神经血管单位效用。这个程序提供了一个强大的控制组织在技术成熟度而言很方便。它已经在这方面和其他I / R损伤模型，增加的压力和缺血的持续时间可能会增加损伤程度24被注意到。出于这个原因，一些从业者选择使用缺血性压力和持续时间与此处4,6-10,12提出不同。?...

视网膜缺血再灌注（I / R）表征许多人视网膜病变，包括青光眼，糖尿病性视网膜病和视网膜血管闭塞1。在视网膜我/ R，血流量减少（缺血）在视网膜血管造成视网膜过敏的氧气和其他营养物质，沉淀严重的氧化和炎症损伤的状态时，循环后来又恢复（再灌注）2。视网膜神经似乎特别容易受到这些变化，患有视网膜神经变性的也许就是我/ R引起的损伤最鲜明的特点。这里介绍的是在小鼠模型视网膜I / R损伤的协议。这项技术使研究人员能够检查视网膜神经血管单元的人类疾病的潜在机制和治疗策略。 通过寻求理解手术贫血3的神经退行性后果外科医生于1952年首创，罗登ŧ视网膜的I /高眼压（IOP）R于1991年重新建立缺血损伤后4标准化神经退行性端点的目的。用盐水贮存提高眼压以上收缩压的滴，这些研究表明，加压眼部插管足以中止视网膜循环，并由此引发的神经元变性。使用视网膜我/ R高眼压最近的努力已开始拟订底层I / R引起的视网膜神经性变性5-12机制。多组报道额外的病理改变包括炎症13,14，血管通透性15,16，和毛细血管变性14,17。总之，这些研究已经更加普遍建立高眼压视网膜I / R损伤视网膜神经血管疾病的模型。 表征I / R损伤的机制是对VA的研究必不可少scular疾病。视网膜我/ R损伤的高眼压是众多缺氧性损伤模型，包括I / R损伤肺18，19心脏，脑20，21肝脏，肾脏22，和小肠23中的一个。这些模型已经在推进我们的血管疾病的认识及临床补救措施至关重要。通过扩展I / R进程眼部组织，视网膜我/ R损伤的高眼压的调查有助于绘制的这些相关条件，更全面的了解。 在临床视网膜神经退行性疾病，视网膜我/ R损伤的高眼压密切相应提出了兴趣探索缺血的发病机制研究的宝贵工具。本文描述的协议是有针对性的，听话的，并且可以访问。它是通过在神经元变性的端点，例如视网膜神经元的定量，视网膜厚度的测量，和电ř补充井视网膜神经元功能的ECORDING。这种模式已经证明了其在促进神经血管查询工具，它显示了在收入中的视觉医学研究的一个基础性协议状态的承诺。

<table border="0" cellpadding="0" cellspacing="0" width="544">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Heparin Sodium Injection, USP</td>
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</table>

a mouse model of retinal ischemia-reperfusion injury through elevation of intraocular pressure

视网膜缺血再灌注（I / R）是有助于在多个眼部病症，包括青光眼，糖尿病性视网膜病和视网膜血管阻塞的细胞损伤病理生理过程。 I / R损伤的啮齿动物模型中提供显著见解为人类I / R损伤的机制和治疗策略，特别是关于在视网膜神经血管单元神经变性损伤。这里介绍的是通过眼内压（IOP）升高诱导小鼠视网膜I / R损伤的协议。在这个协议中，眼部前房插管用针，通过该流动升高​​盐水储层的滴注。使用该滴提高眼压上述收缩动脉血压，从业者暂时停止内视网膜血流（缺血）。当循环被除去套管的恢复（再灌注），严重的细胞损伤随之而来，在视网膜的神经变性，最终导致。最近螺柱IES表明炎症，血管通透性和毛细管变性作为该模型的另外的元件。相比于替代视网膜I / R方法，如视网膜动脉结扎术，视网膜我/ R损伤的高眼压有优势在其解剖的特殊性，实验可追踪性和技术辅助，呈现自己作为一个有价值的工具，用于检查神经元病机及治疗视网膜神经血管单元。

由高眼压视网膜的I / R的神经变性的影响通常进行评价使用两个标准的方法。神经核的NeuN免疫标记已经揭示以下I / R损伤（ 图1）显著神经元细胞的损失。简单地说，眼睛剜7天I / R固定后，多聚甲醛，与神经细胞的NeuN标记标记和全装。图像用共焦显微镜拍摄的，并标有的NeuN细胞通过计数11定量。减少在神经节细胞层的神经元计数指示I / R诱导的细胞死?...

Watch this Scientific Journal Video about A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure at JoVE.com

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

本文介绍了在小鼠高眼压诱导视网膜缺血再灌注损伤的过程。视网膜缺血再灌注损伤的高眼压用于人类疾病视网膜特点是妥协的氧气和营养物质的交付模式，使研究人员能够检查视网膜神经血管单元的人类疾病的潜在细胞机制和治疗。

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

a-mouse-model-retinal-ischemia-reperfusion-injury-through-elevation

Research

JoVE Journal

Medicine

14.8K 次观看.  Johns Hopkins University. 本文介绍了在小鼠高眼压诱导视网膜缺血再灌注损伤的过程。视网膜缺血再灌注损伤的高眼压用于人类疾病视网膜特点是妥协的氧气和营养物质的交付模式，使研究人员能够检查视网膜神经血管单元的人类疾病的潜在细胞机制和治疗。 

视频: A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

常温心脏骤停和心肺复苏术:缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

脑缺血再灌注的双血管闭塞小鼠模型

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

大鼠心肌缺血再灌注后冠状动脉流动储备评价

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...