诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Sodium Chloride | Sigma Aldrich | S3014 | |
| Potassium Chloride | Sigma Aldrich | P9541 | |
| Magnesium Sulfate | Sigma Aldrich | 203726 | |
| Potassium Phosphate Dibasic | Sigma Aldrich | RES20765-A7 | |
| Calcium Chloride Dihydrate | Sigma Aldrich | C8106 | |
| Sodium Bicarbonate | Sigma Aldrich | S5761 | |
| D-Glucose | Sigma Aldrich | G8270 | |
| Octanoic Acid | Sigma Aldrich | C2875 | |
| 2,3,5-triphenyltetrazolium chloride | Sigma Aldrich | T8877 | |
| Medical Pressure Transducer | MEMSCAP | SP844 | |
| Masterflex Peristaltic Pump | Cole Parmer | EW-07521-40 | |
| Masterflex Easy Load Pump Head | Cole Parmer | EW-07518-10 | |
| Heated circulating water bath | Lauda | M3 | |
| Tubing Flow Module | Transonic | TS410 | |
| Modular Research Console | Transonic | T402 | |
| Inline flow sensor | Transonic | ME2PXN | |
| PowerLab Data Acquisition Device | AD Instruments | PL3508 | |
| LabChart data acquisition software | AD Instruments | MLU60/8 |
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