本刊物是由南卡罗来纳州的临床与转化研究（SCTR）研究所的支持下，与一个学术家在南卡罗来纳州医科大学，美国国立卫生研究院/ NCATS授权号UL1 TR000062。进一步的支持是DRM提供VA优异奖BX002327-01。 DJH是由美国国立卫生研究院/ NCATS授权号TL1 TR000061和美国国立卫生研究院资助号T32 GM008716的支持。大海是由美国国立卫生研究院资助号T32 HL07260支持。

这里列出的所有程序已经批准的机构动物护理和使用委员会在南卡罗来纳医科大学。这里所描述的实验是急性的，非生存实验。正因为如此，所以没有用眼膏，并且不需要无菌操作套件。安乐死放血过程中收获的心脏的实现。 1.实验准备<ol><li>设立无论是恒压或恒定流量的Langendorff灌注设备4。 </li><li>制备4L的改良Krebs Henseleit缓冲液（以mM计：112 NaCl的，5氯化钾，1.2 MgSO 4干燥，1 K 2 HPO 4，1.25氯化钙 ，25的NaHCO 3，11 D-葡萄糖，0.2辛酸，pH值= 7.4）。 <ol><li>使10倍的股票的缓冲区。 <ol><li>溶解氯化钠，氯化钾， 硫酸镁 ，和K 2 HPO 4在3升的超纯水。 </li><li>溶解氯化钙 500毫升超纯水无线个将40ml 1N HCl中。 </li><li>慢慢加入氯化钙溶液，以在1.2.1.1所取得的缓冲器。加入足够的超纯水至总量4 L.允许通过一个0.8微米的过滤器过滤之前搅拌1小时。保存在4℃。 </li><li>在4升超纯水溶解的NaHCO 3。允许通过0.8微米的过滤器过滤之前，搅拌1小时。商店在4℃ </li></ol></li><li>结合400毫升10倍的Krebs Henseleit缓冲液用400毫升10×的NaHCO 3缓冲液中。加入足够的超纯水至总量4 L.溶解葡萄糖和辛酸，同时搅拌。通过0.8微米的过滤器过滤。 </li></ol></li><li>准备系统心脏。 <ol><li>添加2升过滤缓冲液以一个4升瓶子置于38在附着到主动脉插管的活塞上方。这将提供70-80毫米汞柱的灌注压到心脏，这是推荐的灌注压4。 </li><li>插入气体分散鼓泡入在瓶子的缓冲器。 </li><li>通过该系统运行缓冲液，是确保消除任何气泡从管内。 </li><li>打开气体（95％O 2/5％CO 2）的至少30分钟前心脏要被放置在系统上。使用气流探测器和血液气体分析仪来验证氧饱和度。 </li></ol></li><li>准备手术用品和气球。 <ol><li>收获的心脏，准备1对6.75“手术剪，双4.5”剪刀，两对止血的，两套caramalt镊子，两个注射器用27G的针头，2枚0瓜哥丝线缝合（约15厘米长），一个1/16英寸的倒钩套管，和一个气管套管。 </li><li>要组装气管插管，粘上一段简短的1/16英寸直径的塑料管为Y型连接器。 </li><li>要组装气球装置：附加灌胃针一块1/16英寸管，然后连接油管压力传感器外壳上。附连到压力潮流的另一侧的小型注射器ansducer住房将允许该气囊充气和放气。 <ol><li>剪下的灌胃针在中心的保鲜膜和地点提示一个平方。环绕灌胃针的保鲜膜。 </li><li>气球填充用生理盐水并用两个缝线打结。膨胀的气球，以确保没有泄漏。盐水的膨胀的气球内的体积应为大约200微升，但可根据不同的大鼠的大小而变化。 </li><li>另外，使用乳胶指套代替保鲜膜。将灌胃针内手指套，填充以生理盐水和打结。 </li></ol></li></ol></li></ol> 2.收获的心脏<ol><li>克制使用decapicone或其他手动约束装置只SD大鼠。权衡老鼠。 </li><li>通过腹膜内注射施用氯胺酮/甲苯​​噻嗪混合物（0.85毫克/千克氯胺酮和0.15毫克/千克甲苯噻嗪）麻醉大鼠。这一剂量的麻醉剂将提供足够的麻醉20-40分钟，但程序应麻醉确认后，可迅速完成。 </li><li>确认通过测试脚趾捏反射适度麻醉。 </li><li>气管切开术<ol><li>拆下中间前爪皮到下巴的基础上，利用止血钳来提升毛皮和剪刀做切口。保持提升到它从底层肌肉分开毛皮，允许一个沿毛皮切割并除去它。 </li><li>使用止血，解除腺体组织，使横向切口筋膜逊色于腺体组织。使用止血蔓延开切口，注意不要割伤颈静脉。 </li><li>使用止血，捏的肌肉覆盖气管，并用剪刀作一横切口，在刚刚止血的尖端下方的肌肉。使用止血，组织传播开来揭示气管。 </li><li>小心地将止血钳下的气管，清除筋膜同时轻轻进退止血进展。 </li><li>一旦止血成功放置气管的背后，掐0-0丝线缝合止血与和hemisect气管具有体积小，锋利的剪刀。 </li><li>插入气管套管插入半横断气管，拉缝合气管的后面，并绑缝线围绕插管气管，锚定的缝合线于Y腹股沟与外科医生打结插管。 </li><li>用27G的针，注入1000单位/公斤肝素入颈静脉并允许循环30秒。 </li></ol></li><li>开胸<ol><li>捏用止血钳和剪刀剪取出皮从中间腹部中间区域的前爪。 </li><li>做一个¾英寸横切口进入腹腔肌壁，略低于隔膜。 </li><li>使一个纵切口向上腹壁和胸壁，沿着胸骨切割。 </li><li>切开膈肌双边回来，然后蔓延到胸腔心脏透露，钳胸腔与止血，以确保它仍然蔓延。 </li&gt; <li>用止血钳，轻轻取出胸腺，暴露升主动脉。 </li><li>通过主动脉环逗引止血，进退止血，轻轻地蔓延筋膜通过允许止血的一角。 </li><li>使用止血身后拉拽升主动脉0-0丝线缝合，缝合打结成一个松散的半平结。 </li><li>打开水龙头开始流灌注缓冲，hemisect主动脉和立即插入导管主动脉进入内腔。迅速收紧半平结，完成结，彻底收紧。 </li><li>用剪刀解剖心脏离大血管，从胸部取出心脏，并连接到灌注柱。 </li><li>修剪掉多余的肺组织，并允许心脏上均衡列插入气球前约15分钟。通过10-20毫升/分钟的心脏使用缓冲器的正常流率。 </li></ol></li></ol> 3. Langendorff离灌注和缺血再灌注损伤<ol><li> LV预安置ssure气球<ol><li>气球放气，而擀成圆锥状，把活塞，以确保气囊仍然瘪。 </li><li>海关左心房用小剪刀暴露访问二尖瓣。 </li><li>通过插入左心房和二尖瓣进入左心室气球。 </li><li>启动压力监控软件（LabChart专业版）。 </li><li>打开活塞，轻轻地增加盐的用量，直到气球舒张压从微压力计读数超过零充气气球。 </li><li>要检查长度张力反应，慢慢充气气球的75毫米汞柱舒张压，慢慢缩小，重复一次。 </li><li>通过调节在气球盐水的量设定舒张压至10毫米汞柱。打开活塞密封气球在通胀这个水平，保持LV气球和压力传感器之间的密闭高压系统。 </li><li>较低的进入心脏加热室与室盖塑料。 </li></ol></li><li>广告缺血再灌注损伤的职事<ol><li>塞被加热腔室的底部，并允许腔来填充吐露缓冲区。 </li><li>当缓冲区淹没心脏，把活塞停止缓冲区的流入心脏，诱发缺血。缺血时间可根据实验的目的而变化。 </li><li>缺血30分钟后，打开水龙头恢复畅通缓冲到心脏，再灌注开始。 </li><li>终止实验之前让心脏1小时再灌注。使用变再灌注时间，这取决于实验的目标。 </li></ol></li><li>终止实验和组织的收获<ol><li>利用活塞的侧口，辖10毫升磷酸盐缓冲液在4℃到心脏，以10ml / min的速度逮捕的心脏。 </li><li>从灌流柱取出心脏，修剪掉剩余的心房组织。 </li><li>将心脏成不锈钢组织切片矩阵，覆盖霸rafilm，并置于在-80℃下8分钟，或直至心脏具有棉花糖状稠度。 </li><li>从冰箱中取出块，并插入到剃须刀片基体切片切片心脏分为2 mm层厚。组织块也可以用大于2mm其他片大小购买。 </li><li>除去切片一次一个，并落入液氮闪烁冻结生化研究。从保留的顶点为梗死染色第三脑室片。 </li><li>除去从液氮和存储组织在-80℃下，直到在生化实验中使用。 </li></ol></li><li>染色梗塞<ol><li>在37℃下孵育第三脑室切片在10毫升1％w / v的氯化三苯基四唑（TTC）15分钟。 注：可为15或30分钟，在效力的第15无变化进行孵化TTC。 </li><li>从TTC转移组织切片至10％缓冲的福尔马林中，并且允许在室温下孵育过夜。为了达到最佳效果，听到TS应取出福尔马林和24小时内4成像。 </li><li>照片染色切片和使用的软件，如ImageJ的根据制造商的协议估计梗塞大小。拍照尽快固定后，作为染料会褪色随着时间的推移。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

离体灌注大鼠心脏可以成功地用于研究的药理干预对心肌预处理在缺血再灌注损伤9的效果。但是，也有一些重要的步骤，即必须以确保可重复的结果进行标准化的步骤。维持37.4℃，系统内的温度是至关重要的，因为即使是轻度低温和高温可引起心脏预处理10,11。的总时间，从注射麻醉剂的流逝对心脏的切除必须保持到最小，因为长时间暴露于氯胺酮可与心脏预处理12干涉?...

既缺血再灌注的心脏反应底层的事件的澄清是在提高的需要主动脉阻断2心肌梗塞1和心脏外科手术的治疗至关重要。而在体内模型缺血再灌注损伤的允许非常有用的端点分析，它们不是有效用于研究缺血再灌注损伤的作用效果敏锐的实时性。此外， 体内缺血再灌注模型一般在再灌注时产生的梗塞面积显著的变化，并直接递送药物对心脏是具有挑战性的。一个的Langendorff离体心脏系统用于研究缺血再灌注损伤的利用允许对药物治疗，梗塞组织的均匀区，并直接向心肌瞬时递送药物的实时功能评估。 第一所记载的BŸ奥斯卡的Langendorff于1895年3，Langendorff离心脏是一个强大的模型研究缺血再灌注损伤，已用于缺血再灌注研究，为过去40年4,5。在这里，一些改动，以优化离体心脏的功能分析， 在主动脉插管原地 ，而心脏跳动保证心脏不经历缺血预处理，这将改变缺血再灌注试验6的结果。为了便于此，气管切开术被执行，从而允许通风，并确保在手术过程中的鼠的生理稳定性。的心脏然后附着到玻璃水夹套螺旋柱通过它的Krebs Henseleit缓冲液经由逆行灌注直接传送到主动脉。甲生理盐水填充的气囊被插入左心室并连接到压力换能器，其允许压力的实时测量从心室的内ð计算的多个功能参数。在实验结束时，将心脏冲洗用冷盐水逮捕收缩和快速冷冻在液氮中，以使DNA，RNA和蛋白水平的下游分析。这样修改，灌注的Langendorff心脏在缺血再灌注损伤，随时作为一个有效的系统直接监测的药物干预的生理效应敏锐。

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	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td align="right">203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>RES20765-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>C2875</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Pressure Transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07521-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Easy Load Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated circulating water bath</td>
			<td>Lauda</td>
			<td>M3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Flow Module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Research Console</td>
			<td>Transonic</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline flow sensor</td>
			<td>Transonic</td>
			<td>ME2PXN</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart data acquisition software</td>
			<td>AD Instruments</td>
			<td>MLU60/8</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and assessment of ischemia-reperfusion injury in langendorff-perfused rat hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

左心室气囊装置允许订约左心室（ 图1）产生的压力的实时监测。如前面7描述的，该压迹可以用来计算许多心功能的参数。这些计算可以在基线阶段以及再灌注阶段进行，平均多条迹线的每个组内，并以确定的药理学干预是否导致心肌缺血预处理，如我们先前9做了比较。一个这样的参数是发展压，计算为收缩压和舒张末期压力之间的差。在正常灌注的大鼠心脏的开发...

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

induction-assessment-ischemia-reperfusion-injury-langendorff-perfused

Research

JoVE Journal

Medicine

22.6K 次观看.  Medical University of South Carolina. The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

视频: Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

科研

医学

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

在原位横向腹直肌肌皮瓣：肌缺血再灌注损伤模型大鼠

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

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 ...

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, ...

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 ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

前维沃·兰根多夫注入心脏的心电生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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, ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...