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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</ol>

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。主动脉原位及时插管是必不可少的预防心脏或动物的放血中缺氧的开发之前心脏切除。总体而言，从第一切口，切除心脏的时间应不超过6-8分钟。左心室巴lloon必须检查每个实验前泄漏，并在必要时更换。系统必须精心维护，包括冲洗用蒸馏水整个装置在每次运行后，更换为必要磨损的管道，以防止泄漏的管道的内部或污染。 分离的心脏可被修改为任何数目的新颖用途，包括荧光成像13，核磁共振波谱7，和光学测绘14在许多其他。该系统还可以被修改，以灌注来自不同的动物，包括小鼠心。本变形例是特别有用的，因为它允许使用转基因小鼠的实验。修改系统对小鼠心脏，小套管以及一个较小的灌注柱必须使用，除了其他修改。如何利用的Langendorff方法小鼠心脏的详细描述已在别处16,17出版。其他修改该协议包括各种药物在不同时间点的管理。当调查药物中，以引起药物预处理或后适应的能力，有必要给予药物在不同时间点相对于缺血再灌注损伤。可将药物施用于动物的心脏切除之前，或混入两种之前的心脏被放置在柱或在缺血阶段使得药物是再灌注时存在的缓冲器。可替代地，一个侧开口可用于施用药物的推注在协议过程中的任何时间点。此外，该协议可以被修改，以改变局部缺血，再灌注，或二者的持续时间。这使得功能和生化数据在多个时间点的分析，可用于跟踪药物的急性效应的时间过程。如果再灌注期间被改变，需要注意的是在至少60分钟的再灌注是nece是很重要ssary为TTC染色有效地划定梗死区15。 在缺血再灌注损伤方面的离体心脏模型的主要限制是它没有考虑许多存在于缺血再灌注的体内设置的全身性因素。这消除系统性影响必须考虑在使用的Langendorff模型生成的数据的分析，但并不排除该模型的回答关于肌细胞，成纤维细胞和内皮细胞对缺血和随后灌注的响应新颖的问题的能力。离体心脏模型允许许多影响缺血再灌注损伤，除了过短的时间间隔对心脏的作用效果实时分析的变量的完整操纵。通过使用离体心脏系统产生的数据是非常宝贵的理解药物预处理或后调理心脏响应各种药物干预。

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。

<table><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>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压力范 ​​围可以从70到130毫米汞柱（ 图1A）。缺血性损伤后，显影压力降低和端舒张压升高（ 图1B）。当老鼠施用的已知的预处理剂，如I类和Ⅱb族HDAC抑制剂SAHA（伏立诺他）18之前，切除心脏，减少在发达国家的压力和与缺血再灌注损伤相关的舒张末期压力的升高被衰减（ 图1C）。左心室功能的其他措施，如压力产生（上升/ dt 最大值 ）的速率，压力松弛（-dP / dt 最大值 ）的速率，以及速率压力乘积（RPP）可直接得到，或从计算软件输出（ 图2）。缺血阶段也可以在实时监测，压力产生的局部缺血发作的几分钟内的明显停止。缺血性收缩，也可以通过测量到出现收缩和峰值收缩时间的时间监视。 氯化三苯基四唑（TTC）通常用于之间代谢一个区分莫如和非活动的组织，并在这里被用于梗塞染色。一旦吸收到组织的TTC减少由代谢酶，转动活性组织红色。非活动的组织不减少TTC，正因为如此会弄脏白色8。的Langendorff灌注尚未经受缺血损伤不显示任何白色区域（未示出）的大鼠心脏，而心中经受染色白缺血再灌注损伤显示大量区域，表示梗塞组织（ 图3）。 <img alt="图1" src="/files/ftp_upload/52908/52908fig1.jpg" /> 图1:从左心室气球压力记录通过在心室收缩的低压球囊代表压力痕迹 。没有受到缺血（A）的心经历一段时间的收缩能力的未成年人的损失。心中经受缺血（B）示IMMED莱特损失压力产生的，接着强直收缩。再灌注后，这些心体验EDP升高和降低的开发压力。心预处理用SAHA和经受缺血（C）表示衰减缺血再灌注损伤。 N = 1元组。 <img alt="图2" src="/files/ftp_upload/52908/52908fig2.jpg" /> 图2:心功能参数的计算压力产生（A），压力松弛（B），开发的压力（C），和速率血压乘积（D）的比率是心功能的一些参数可以从计算速度。压力监控软件。 <img alt="图3" src="/files/ftp_upload/52908/52908fig3.jpg" /> 图3:TTC染色梗死面积。 </stron缺血再灌注损伤后g&gt;的实施例心室横截面。虚线描绘的右心室（RV）左心室（LV）。焦点梗死是由白色区域表示，而可行的组织是由红染色显示。

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

Nanyang Technological University

Research

JoVE Journal

Medicine

22.9K 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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

生物学

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 ...

通过改良 Langendorff 灌注加强 CVD 研究

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.

作者聚焦：推进心血管研究 - 量身定制的 Langendorff 灌注技术以改善实验结果

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

改良 Langendorff 灌注延长啮齿动物心脏移植物的灌注时间

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and ...

生物工程

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, ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

<meta charset="utf8"></head><body>用于评估缺血/再灌注大鼠模型中心脏微血管内皮屏障通透性的荧光测定

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

用于评估缺血/再灌注大鼠模型中心脏微血管内皮屏障通透性的荧光测定

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

小鼠心肌梗死和心肌缺血再灌注损伤的诱导

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

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