Name: modified technique for the use of neonatal murine hearts in the langendorff preparation
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NIH/NINDS R01NS112706 （R.L.）

哥伦比亚大学医学中心的机构动物护理和使用委员会对所有描述的方法都获得了批准。野生型C57Bl / 6雄性产后第10天小鼠用于研究。
1. 朗根多夫仪器的制备
<ol><li>为了最大限度地降低复杂性，请在朗根多夫装置内通过恒定流量或恒定压力使用非再循环含氧灌注液（参见材料表）。<ol>
<li>使用克雷布斯-亨赛莱特缓冲液（KHB），其中含有120毫摩?...

<ol>
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本工作描述了分离的新生儿小鼠心脏中成功的主动脉插管和逆行灌注。重要的是，它使研究人员能够克服年轻鼠龄和小心脏尺寸先前提出的障碍8。虽然在设计上并不复杂，但这种方法确实需要相当程度的技术技能。即使是技术最熟练的研究者，也不可避免地会面临挑战的关键步骤将是主动脉插管和将套管固定到位。新生儿插管困难不仅仅是由于主动脉腔体积小。升主动脉（主动...

一个多世纪以来，离体心脏制剂一直是生理学、病理生理学和药物学研究的主要内容。源于19世纪60年代Elias Cyon的工作，Oskar Langendorff将分离的青蛙模型用于逆行灌注，加压主动脉根部以提供带氧灌注的冠状动脉流动1。利用他的适应，朗根多夫能够证明冠状动脉循环与机械功能2之间的相关性。离体逆行灌注心脏，后来同名地被称为朗根多夫技术，仍然是生理学研究的基石，利用其简单性在没有潜在混杂物的情况下有力地研究了孤立的心脏。Langendorff制剂已被进一步修改，以允许心脏弹出（所谓的“工作心脏”）并允许灌注物再循环3。然而，感兴趣的主要生理终点保持不变。这些终点包括收缩功能、电传导、心脏代谢和冠状动脉阻力的测量 4。
为了评估他最初的青蛙心脏准备中的心脏功能，Langendorff使用连接在心脏顶点和力传感器之间的缝合线测量了纵轴心室收缩产生的张力。5 等距收缩以这种方式量化，在没有心室充盈的情况下将基础张力施加到心脏上。该方法的改进导致 通过 左心房将充满液体的球囊放入左心室，以评估等值宫收缩期间的心肌表现6。为了评估心律和心率，可以在心脏的两极上放置表面导线，以使研究人员能够记录心电图。然而，鉴于强制性去神经支配，相对心动过缓是可以预期的。外在起搏可能有助于克服这一点并消除实验1之间的心率变异性。另一个结果测量，心肌代谢，可以通过测量冠状动脉灌注物和流出物中的氧和代谢底物含量并计算它们之间的差异来评估7。冠状动脉流出物中的乳酸定量有助于表征厌氧代谢期，如缺氧、低灌注、缺血再灌注或代谢紊乱所示 7.
Langendorff的原创工作使研究体外哺乳动物心脏成为可能，以猫为主要受试者5。对孤立大鼠心脏的评估在1900年代中期随着霍华德·摩根（Howard Morgan）的普及而流行，他在1967年详细介绍了“工作心脏”大鼠模型5。小鼠的使用仅在25年前开始，由于技术复杂性，组织脆弱性和相对较小的鼠心脏尺寸。尽管存在与小鼠研究相关的挑战，但遗传操作的较低成本和易用性增加了这种小鼠离体制剂的吸引力和需求。不幸的是，该技术的应用仅限于成年动物，直到最近，4周龄的幼年小鼠才成为用于离体研究的最年轻的受试者8，9。虽然与成年小鼠相比，幼年小鼠“相对不成熟”，但它们作为发育生物学研究对象的效用有限，因为它们基本上已经从出生母亲那里断奶，很快就会开始青春期10。青春期发生在心肌底物利用从葡萄糖和乳酸盐到脂肪酸11的产后过渡之后。因此，关于新生儿心脏代谢变化的大多数信息历来来自较大物种（如兔子和豚鼠11）的离体工作。
事实上，存在朗根多夫准备的替代方法。这些包括缺乏整个器官功能数据和背景的 体外 实验，或 体内 研究。这在技术上可能具有挑战性，并且由于混淆变量而变得复杂，例如必需麻醉剂的心血管和呼吸系统效应，神经体液输入的影响，核心温度的后果，动物的营养状况以及底物可用性12，13。由于朗根多夫方法允许在没有这种混杂物的情况下以更可控的方式以离 体 方式研究分离灌注心脏，因此它一直并将继续被认为是一种强大的研究工具。因此，这里介绍的技术为研究人员提供了一种用于新生儿鼠心脏体 外 研究的实验方法，并限制了再灌注的时间。
鉴于心肌成熟期间发生的广泛的生化、生理和解剖转变，在发育期间检查心脏是一个重要的考虑因素。从厌氧代谢到氧化磷酸化的转变，底物利用的变化以及从细胞增殖到肥厚的进展是未成熟心脏中独特发生的动态过程11，14。心脏发育的另一个关键方面是，在必要时期遇到的压力源可能会在新生儿心脏中产生更高的反应，并改变未来在成年期对侮辱的易感性15。尽管先前的工作已经利用新生大鼠，羔羊和兔子来研究朗根多夫灌注的新生儿心脏，但鉴于该物种对发育生物学研究的重要性，允许小鼠使用的进步是必要的16。为了满足这一需求，最近建立了第一个使用10天大动物的小鼠Langendorff灌注新生儿心脏模型6。这里介绍的是一种能够成功进行主动脉插管并建立孤立的新生儿鼠心脏逆行灌注的方法。这种方法可用于药理学、缺血再灌注或专注于整个器官功能的代谢研究，或者可用于心肌细胞的分离。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rodent Langendorff Apparatus</td>
			<td>Radnoti</td>
			<td>130102EZ</td>
			<td></td>
		</tr>
		<tr>
			<td>24 G catheter</td>
			<td>BD</td>
			<td>381511</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G needle on 1 mL syringe combo</td>
			<td>BD</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G steel needle</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Silk Suture</td>
			<td>Ethicon</td>
			<td>S1173</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Amp</td>
			<td>ADInstruments</td>
			<td>FE135</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Cable</td>
			<td>ADInstruments</td>
			<td>MLA1515</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating heating water Bath</td>
			<td>Haake</td>
			<td>DC10</td>
			<td></td>
		</tr>
		<tr>
			<td>curved iris scissor</td>
			<td>Medline</td>
			<td>MDS10033Z</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>find spring scissors</td>
			<td>Kent</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr>
			<td>Force Transducer</td>
			<td>ADInstruments</td>
			<td>MLT1030/D</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure tubing</td>
			<td>Edwards Lifesciences</td>
			<td>50P184</td>
			<td></td>
		</tr>
		<tr>
			<td>iris dressing forceps</td>
			<td>Kent</td>
			<td>INS650915-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Jeweler-style curved fine forceps</td>
			<td>Miltex</td>
			<td>17-307-MLTX</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Gilson</td>
			<td>Minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>straight dissecting scissors</td>
			<td>Kent</td>
			<td>INS600393-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Temporary cardiac pacing wire</td>
			<td>Ethicon</td>
			<td>TPW30</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Range Force Transducer</td>
			<td>ADInstruments</td>
			<td>MLT1030/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

modified technique for the use of neonatal murine hearts in the langendorff preparation

自一个多世纪前Oskar Langendorff开发 以来，使用离体 逆行灌注心脏长期以来一直是缺血再灌注研究的基石。 尽管该技术在过去25年中已应用于小鼠，但其在该物种中的使用仅限于成年动物。开发一种成功的方法以始终如一地对新生儿鼠主动脉进行插管，将允许在遗传可改变和低成本物种的心脏发育的关键时期对孤立的逆行灌注心脏进行系统研究。对朗根多夫制剂的修饰能够在新生儿鼠心脏中进行插管和再灌注，同时最大限度地减少缺血时间。 优化需要两人技术，以允许使用解剖显微镜和改良的市售针头成功插管新生小鼠主动脉。 使用这种方法将在3分钟内可靠地建立逆行灌注。由于新生儿小鼠心脏和心室腔大小的脆弱性阻止了使用球囊产生的脑室内压力的直接测量，因此有必要使用通过缝合线连接到左心室顶点的力传感器来量化纵向收缩张力。这种方法使研究人员能够成功地建立一种孤立的恒流逆行灌注新生儿鼠心脏制剂，从而能够以 离体 方式研究发育性心脏生物学。重要的是，该模型将成为研究新生儿心脏缺血再灌注的生理和药理反应的有力工具。

P10小鼠用于模拟人类婴儿期的时间点26，27。15个分离的C57Bl / 6新生小鼠心脏被收获并成功插管。心脏灌注2.5 mL min-1 的加热含氧KHB的连续流动。测量代谢参数，包括葡萄糖提取，氧气消耗，乳酸产生以及心率，灌注压力和冠状动脉阻力等生理参数。使用表面电极记录连续心电图，从而可以确定内在心率和节律（图2）。纵轴?...

Watch this Scientific Journal Video about Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation at JoVE.com

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

本方案描述了新生儿鼠心脏的体 外 旋插管和逆行灌注。 使用解剖显微镜和钝化的小针头的两人策略允许可靠的插管。纵向收缩张力的量化是使用连接到左心室顶点的力传感器实现的。

新生儿鼠心在朗根多夫制剂中的应用改良技术

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

这种方法在三分钟内可靠地建立了新生儿离体小鼠心脏的逆行灌注。从而将最年轻的学习年龄从几周减少到产后10天。这种孤立的逆行灌注模型允许在遗传上可改变和低成本物种的心脏发育关键时期研究新生心脏。虽然该模型有助于研究新生儿心脏缺血再灌注的生理和药理学反应。它也可用于心肌细胞分离。为了制造新生小鼠主动脉瓣插管，使用锋利的剪刀剪掉尖端并钝化26号不锈钢针的末端，注意不要卷曲或限制针管腔的直径。然后，使用来回运动轻轻刮擦实验室工作台上的钝端，使切割边缘平滑以消除任何毛刺。接下来，将制造的插管连接到朗根多夫装置上并评估流量和阻力。通过收集和测量缓冲液量，每隔一分钟测量一分钟的流速。为了量化克雷布斯-海内瑟利特缓冲液流动时整个套管的压差，测量连接和未连接套管的系统中的压力。然后使用以下公式计算套管阻力。接下来，从朗根多夫装置中取出26号套管，并将高压管连接到装置上的插管部位。然后将主动脉套管连接到管子的远端，并用含氧缓冲液使管子和套管除气，确保除去所有气泡。为了防止冠状动脉微血栓的形成，使用26号针头在一毫升注射器上使用26号针头向10天大的小鼠幼崽腹膜内注射肝素。在继续注射任何麻醉剂之前，让肝素循环几分钟。接下来，将麻醉的小鼠置于仰卧位，并在失去意识后立即固定四肢。一旦动物对脚趾捏没有反应，就开始收获，因为动物在最初的解剖过程中会自发地呼吸。使用直解剖剪刀在暴露腹腔的动物宽度上做一个横向的亚尖瓣切口。请助手用镊子抓住剑突，然后沿头颅方向沿腋窝中线双侧切开胸腔，颅内反射胸骨和肋骨，露出胸廓。在优越地识别隔膜后，完全切入前部。识别肝脏上方的膈下腔静脉，并用弯曲的虹膜剪刀将其横断，同时用虹膜镊子在近端节段上保持轻微的头颅骨张力。然后，使用弯曲的虹膜剪刀沿着脊柱内表面向后切，同时将下腔静脉向上拉出胸腔。当心脏被动员时，将剪刀向前倾斜，并优越地切断大血管，以完全切除心脏和肺部。立即将标本浸入冰冷的克雷布斯-亨塞莱特缓冲液或盐水中。心脏应该在几秒钟内停止跳动。在浅培养皿的底部放置一张纸巾，以提供摩擦力，以在插管期间稳定心脏。用冰冷的克雷布斯-亨瑟利特缓冲液润湿纸巾，以防止心脏粘附在纸巾上。将准备好的培养皿置于解剖显微镜下并调整焦点。现在，将连接到高压延伸管的主动脉套管置于解剖显微镜下，并在其轮毂周围松散地绑上5-0丝线。将切除的胸廓器官放入培养皿中后，通过其白色光泽和两个裂片识别胸腺，并使标本定向，使胸腺位于前部和上部。接下来，使用镊子钝地分离胸腺的裂片，露出大血管。然后通过定位主动脉弓的分枝特征来识别主动脉，并使用细尖的剪刀将其横断到锁骨下动脉起飞的近端。现在使用珠宝商风格的精细弯曲镊子轻轻抓住横断的主动脉，并用26号钝针小心地夹住主动脉，注意不要损坏主动脉瓣。使用精细弯曲的镊子，抓住套管周围的主动脉并开始逆行灌注以限制缺血时间。要求助手抓住松散绑扎的缝合线的末端，并小心地将主动脉缠绕在套管周围。将缝合线夹在细弯曲的镊子上方或下方，然后拧紧缝合线并确认冠状动脉血流的充分性。断开高压管与朗根多夫装置的连接，抓住套管的轮毂，并将钝针与高压延长管断开连接。然后迅速将套管的集线器连接到设备上。一旦心脏以通常的位置悬挂在朗根多夫装置上，就确认了足够的灌注。小心地修剪掉肺，胸腺和多余的组织。然后切入右心房，使冠状动脉窦流出物自由滴落。在5-0丝缝合线的末端打一个小结，然后用针刺穿一小块石蜡膜，将石蜡滑到打结的末端。小心地将针头穿过心室的顶端，并将缝合线拉过心脏，直到石蜡膜紧贴心室的侧壁。将针头穿过朗根多夫装置的充满水的保暖夹克的开口，以便可以包裹和温暖心脏。然后将针头连接到力传感器上，以避免冠状动脉窦滴落。调整缝合线以施加一到两克基础张力，如舒张张力或最低点意图示踪所示。然后将表面电极放在心脏的上极和下极上以记录心电图。最后，使用24号静脉导管对冠状动脉窦流出物进行取样以进行分析。表面电极用于记录连续心电图，从而可以确定内在速率和节律。所有充分灌注的心脏都以窦性心律自发地跳动。这种灌注策略满足了新生小鼠心脏的代谢需求，因为乳酸的产生可以忽略不计，氧气提取和葡萄糖消耗百分比低。还记录和计算了生理变量平均值，例如平均去神经支配的内在心率和平均观察到的主动脉灌注压力。必须考虑一些排除标准，例如开始再灌注所需的时间、心律失常持续时间和主动脉灌注压力，以确保新生儿准备的一致性。建立灌注后，可能发生干预，例如药物递送，诱导缺血再灌注损伤，代谢物改变或用于心肌细胞分离的酶剂的递送。

Research

JoVE Journal

Medicine

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

NADH的荧光成像隔离双心室工作离体兔心

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

科研

医学

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

用改进的袖套法小鼠颈部心脏移植模型

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

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

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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

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 ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

通过在小鼠心脏中应用光遗传学多位点光刺激进行高级心律管理

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Creating Radio-cephalic Arteriovenous Fistula in the Forearm with a Modified No-Touch Technique

使用改进的非接触技术在前臂中创建放射性头动静脉瘘

Autologous arteriovenous fistula (AVF) is the primary and best option to obtain vascular access for hemodialysis treatment; other options are ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

一种改良的小鼠心肌梗死诱导简易方法

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

Modified Posterior Vertebral Column Resection for Patients with Thoracolumbar Kyphotic Deformity

胸腰椎后凸畸形患者改良后椎柱切除术

Old compression vertebrae fracture or congenital kyphoscoliosis with abnormal vertebral body development and other diseases that invade the spine may ...