Name: isolated lung perfusion system in the rabbit model
Uploaded: 2021-07-15T14:30:00
Description: Watch this Scientific Journal Video about Isolated Lung Perfusion System in the Rabbit Model at JoVE.com

作者要感谢Bettina Sommer Cervantes博士对撰写本手稿的支持，并感谢Kitzia Elena Lara Safont对插图的支持。

兔模型中的分离灌注系统已在呼吸系统国家研究所的支气管反应亢进实验室中得到广泛应用。该协议包括新西兰兔子，体重约为2.5-3公斤。所有动物均按照墨西哥官方实验动物指南（NOM 062-ZOO-1999）和《实验动物护理和使用指南》（第8版，2011年）在标准动物饲养条件和随意喂养。该协议中提出的所有动物程序和动物护理方法之前都已获得呼吸系统国家研究所伦理委员会的批准。
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<ol>
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这项工作展示了孤立肺灌注系统的一般观点，这是肺生理学研究中的一项基本技术。隔离式肺灌注系统在用途上具有高度的多功能性，并允许评估与测试各种假设相关的几个参数15。孤立的肺系统是一种具有全球影响力的工具，在过去十年中，它进一步确立了其与器官特异性评估的相关性，并扩展了其作为涉及间充质干细胞16 和CRISPR / Cas9基因组工程等的最先进?...

布罗迪和迪克森于1903年首次描述了 离体 肺灌注系统 1。从那时起，它已成为研究肺的生理学，药理学，毒理学和生物化学的黄金标准工具2，3。该技术提供了一种一致且可重复的方法来评估肺移植的可行性，并确定炎症介质（如组胺，花生四烯酸代谢物和P物质等）的影响，以及它们在肺现象（如支气管收缩，肺不张和肺水肿）中的相互作用。分离的肺系统是揭示肺在消除一般循环中的生物胺方面的重要作用的关键技术4，5。此外，该系统已用于评估肺表面活性剂的生化6。在过去的几十年中， 离体 肺灌注系统已成为肺移植研究的理想平台7。2001年，由Stig Steen领导的一个团队描述了 离体 肺灌注系统的首次临床应用，用它来修复一名19岁供体的肺部，该供体最初因其受伤而被移植中心拒绝。收获左肺并灌注65分钟;之后，它被成功移植到一名患有COPD8的70岁男性身上。对使用 离体 灌注进行肺修复的进一步研究导致开发了多伦多延长肺灌注技术，以评估和治疗受伤的供体肺9，10。临床上， 离体 肺灌注系统已被证明是通过治疗和修复不合格的供体肺来增加供体池的安全策略，与标准供体供体相比，风险或结局没有显着差异10。 
隔离肺灌注系统的主要优点是，可以在人工实验室设置下保持其生理功能的完整功能器官中评估实验参数。此外，它允许测量和操作肺机械通气，以分析肺生理学的成分，如气道阻力，总血管阻力，气体交换和水肿形成，迄今为止无法在 实验室 动物体内精确测量2。值得注意的是，可以完全控制灌注肺部溶液的成分，从而能够添加物质以实时评估其效果并从灌注中收集样本以进行进一步研究11。使用孤立肺系统的研究人员应该记住，机械通气会导致肺组织腐烂，缩短其有用时间。在实验期间，这种机械参数的渐进性下降可以通过偶尔使肺部过度充气而显着延迟4。尽管如此，准备工作通常不能持续超过八个小时。 离体 肺灌注系统的另一个考虑因素是缺乏中枢神经调节和淋巴引流。它们缺失的影响尚未完全理解，并可能成为某些实验中偏见的来源。 
分离肺灌注系统技术可以在兔模型中进行，具有高度的一致性和可重复性。这项工作描述了在墨西哥城国家呼吸测量研究所为兔子模型开发的 离体 孤立肺灌注技术的技术和外科手术程序，旨在分享见解，并为该实验模型应用的关键步骤提供明确的指导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Stop Tygon E-Lab Tubing, 3.17 mm ID, 12/pack, Black/White</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-1864</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter for Positive Pressure Ventilation on IPL-4</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4312</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter for Positive Pressure Ventilation on IPL-4</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4312</td>
			<td></td>
		</tr>
		<tr>
			<td>Alternative Pressure-Free Gas Supply for IPL-4: To supply the trachea with gas mixture different from room air during negative ventilation</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4309</td>
			<td></td>
		</tr>
		<tr>
			<td>Base Unit for the Rabbit to Fetal Pig Isolated Perfused Lung</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4138</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum A2:D41albumin lyophilized powder</td>
			<td>sigma</td>
			<td>3912</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Calcium chloride, CaCl2&#183;2H2O.</td>
			<td>JT Baker</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Corning</td>
			<td>430659</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>D-glucosa, C6H12O6.</td>
			<td>sigma</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential Low Pressure Transducer DLP2.5, Range +- 2.5 cmH2O, HSE Connector</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-3882</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential Pressure Transducer MPX, Range +- 100 cmH2O, HSE Connector</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-0064</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute HPLC grade</td>
			<td>Caledon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td></td>
			<td></td>
			<td>14 mL</td>
		</tr>
		<tr>
			<td>Harvard Peristaltic Pump P-230 (Complete with Control Box and P-230 Motor Drive)</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>70-7001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Linear Pneumotachometer 0 to 10 L/min flow range</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>59-9349</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater Controller for Single Pneumotachometer 230 VAC, 50 Hz</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>59-9703</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>PISA</td>
			<td></td>
			<td>5000 UI</td>
		</tr>
		<tr>
			<td>HPLC Column (C18 100A 5U)</td>
			<td>Alltech</td>
			<td>98121213</td>
			<td>150 mm x 4.6 mm</td>
		</tr>
		<tr>
			<td>Hydrophilic Syringe Filter</td>
			<td>Millex</td>
			<td>SLLGR04NL</td>
			<td>4 mm</td>
		</tr>
		<tr>
			<td>IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4296</td>
			<td></td>
		</tr>
		<tr>
			<td>IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230 V</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4296</td>
			<td></td>
		</tr>
		<tr>
			<td>Jacketed Glass Reservoir for Buffer Solution, with Frit and Tubing, 6.0 L</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-0322</td>
			<td></td>
		</tr>
		<tr>
			<td>Lauda Thermostatic Circulator, Type E-103, 230 V/50 Hz, 3 L Bath Volume, Temperature Range 20 to 150&#176;C</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-0125</td>
			<td></td>
		</tr>
		<tr>
			<td>Left Atrium Cannula for Rabbit with Basket, OD 5.9 mm</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4162</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Range Blood Pressure Transducer P75 for PLUGSYS Module</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate, MgSO4&#183;7H2O</td>
			<td>JT Baker</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube</td>
			<td>Corning</td>
			<td>430909</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative Pressure Ventilation Control Option with Pressure Regulator for IPL-4</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4298</td>
			<td></td>
		</tr>
		<tr>
			<td>New Zeland rabbits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PISABENTAL (Pentobarbital sodium)</td>
			<td>PISA</td>
			<td>Q-7833-215</td>
			<td></td>
		</tr>
		<tr>
			<td>PLUGSYS Case, Type 603* 7</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>PLUGSYS TCM Time Counter Module</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-1750</td>
			<td></td>
		</tr>
		<tr>
			<td>PLUGSYS Transducer Amplifier Module (TAM-A)</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-0065</td>
			<td></td>
		</tr>
		<tr>
			<td>PLUGSYS Transducer Amplifier Module (TAM-D)</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-1793</td>
			<td></td>
		</tr>
		<tr>
			<td>PLUGSYS VCM-4R Ventilation Control Module with Pressure Regulator</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-1755</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, KCl.</td>
			<td>JT Baker</td>
			<td>3040-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate, KH2PO4</td>
			<td>JT Baker</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>PROCIN (Xylacine clorhydrate)</td>
			<td>PISA</td>
			<td>Q-7833-099</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulmonary Artery Cannula for Rabbit with Basket, OD 4.6 mm</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4161</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel knife</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serotonin 5-HT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Servo Controller for Perfusion (SCP</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-2806</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap Microcentrifuge Tube</td>
			<td>Costar</td>
			<td>3620</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, NaHCO3</td>
			<td>sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, NaCl.</td>
			<td>sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves No. 7 1/2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves No. 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taygon tubes</td>
			<td>Masterflex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tracheal Cannula for Rabbit, OD 5.0 mm</td>
			<td>Hugo Sachs Elektronik (HSE)</td>
			<td>73-4163</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolated lung perfusion system in the rabbit model

孤立的肺灌注系统已被广泛用于肺部研究，有助于从微观和宏观上阐明肺部的内部运作。该技术通过测量代谢活动和呼吸功能（包括循环物质与吸入或灌注物质的影响之间的相互作用）来表征肺生理学和病理学，如药物测试。虽然 体外 方法涉及组织的切片和培养，但孤立的 离体 肺灌注系统允许与完整的功能器官一起工作，从而可以在重建通气和灌注的同时研究连续的生理功能。然而，应该注意的是，缺乏中枢神经支配和淋巴引流的影响仍然需要充分评估。该协议旨在描述分离肺装置的组装，然后从实验实验动物中手术提取和插管肺和心脏，以及展示数据的灌注技术和信号处理。孤立肺的平均生存能力在5-8小时之间;在此期间，肺毛细血管通透性增加，导致水肿和肺损伤。保留的肺组织的功能通过毛细血管过滤系数（Kfc）来测量，用于确定肺水肿随时间的变化程度。

隔离的肺灌注系统允许器官操作活检，从灌注中收集样本以及实时收集生理参数的数据。分离的系统可用于测试涉及不同功能和肺部现象的许多假设，从代谢和酶活性到水肿形成和肺移植的保存期。
图1显示了完全组装的隔离式肺灌注系统以及通气系统和计算数据采集的图表。系统的灌注组件确保灌注液不断流过孤立的肺部。肺动脉管以提供流入灌注，?...

Watch this Scientific Journal Video about Isolated Lung Perfusion System in the Rabbit Model at JoVE.com

Isolated Lung Perfusion System in the Rabbit Model

分离的兔肺制剂是肺部研究的黄金标准工具。本出版物旨在描述该技术，用于研究涉及气道反应性，肺保留以及肺移植和肺水肿的临床前研究的生理和病理机制。

兔模型中的孤立肺灌注系统

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

该技术可用于通过测量代谢活动和呼吸功能来表征肺生理学和病理学。孤立的肺灌注系统允许与完整的功能器官一起工作，从而可以研究连续的生理功能，同时重建通气和灌注。该方法可以深入了解肺组织的非呼吸能力的表征，例如代谢活动以及肺泡巨噬细胞和内皮组织等各种成分的活动。首先，组装工作装置，确保它包含安装在底板上的主钢柱，该底板带有气压计和重量传感器，位于柱上方和带有气泡陷阱的预热线圈后面。将所有传感器连接到中央电子单元，并准备除设备外的通风系统。剃除麻醉兔的手术部位后，从胸骨手部到颈部做一个三到五厘米的腹侧正中线切口。使用手术剪刀在软骨环之间切开气管的前2/3。通过气管纤维膜插入一个5毫米的气管套管，并用4-0丝线小心地固定套管。将镊子或镊子放在气管下方，以确保套管不会向气管弯曲。将呼吸泵连接到气管套管，将潮气量设置为每公斤10毫升，并在气管切开术后和胸部打开之前快速启动通气，以保持肺部的正压，以防止手术期间的肺塌陷。要进入胸腔，请使用手术刀或剪刀打开胸壁，并进行内侧胸骨切开术，直至胸部的上三分之一。用两个牵开器将胸部的一半打开。定位上层和下层腔静脉，并用线标记它们。在动物灭绝之前，确定右心室并注射每公斤肝素1， 000国际单位。注射后立即用预环线将上腔和下腔静脉固定。切开胸骨的手房，将内侧胸骨切开术延伸至气管套管，将两侧的气管从连接组织中释放出来。现在切除气管套管上方的气管，并在颅/尾轴上轻轻拉起套管。采血后收获心肺传导阻滞，使用直接数字解剖或弹簧剪刀分离结缔组织并从胸部取出肺部。接下来，解剖脉管系统和食道。将孤立的肺从胸部抬起，小心地将肺放在培养皿上的无菌纱布上。为预防肺不张，使用正压通气对肺部进行通气，呼气末正压设定在两厘米水柱处。在房室沟的水平处切断心脏的心室。打开两个心室后，将肺动脉套管通过右肺动脉引入，左心房套管通过二尖瓣进入左心房。用4-0丝线固定套管，包括肺动脉和左心房结扎中的周围组织，以避免结构性扩张。通过动脉套管注入250毫升盐水等渗溶液，以冲洗血管床上剩余的血液。对于灌注设置，将孤立的肺小心地放入肺腔。将气管连接到腔室盖上的转导器上，并将插管血管连接到灌注系统，然后用旋转锁关闭并固定腔室。接下来，安装腔室盖并切换旋塞阀，从正压通风切换到负压通风。用200毫升人造无血灌注剂灌注肺部，从每公斤每分钟3毫升的流量开始，然后在五分钟内缓慢增加流量，达到每公斤每分钟5毫升。在接下来的五分钟里，让流量达到每公斤每分钟8毫升，然后每公斤的最大通量为每公斤10毫升，持续五分钟。用加湿空气以每分钟30次的频率通气，潮气量为每公斤10毫升，呼气末压力为2厘米水柱。要达到区域三条件，等待10至15分钟以获得以ISO重量状态为特征的平衡，确保静脉压力在整个注册表中稳定。确保肺的重量保持恒定，动脉和左心房压力稳定，以达到三区条件，在实验过程中打开最大数量的肺血管并保持微血管床含量。对于电子控制和信号处理，确保呼吸流量，重量变化，微血管压力，潮气量，血管阻力等都记录在多个中央电子单元上，该单元集成了来自传感器的信号并将其显示在评估系统上。评估了参与肺代谢的5-HT和单胺氧化酶的浓度和血管通透性。5-HT和单胺氧化酶的浓度在保存15分钟后达到峰值，然后在接下来的6小时内降低。之后，灌注水平在24小时内显示出无统计学意义的增加。5-HT和单胺氧化酶的释放速率表明，在保存的第一个小时内，5-HT水平高于单胺氧化酶，并且在被内皮细胞和血小板重新捕获后6小时内下降，以及单胺氧化酶介导的分解代谢。中性内肽酶活性在9小时时增加，然后在12至24小时内降低并保持稳定。血管紧张素转换酶活性在四小时后降低，然后增加并保持稳定，直到24小时。肺保存对毛细血管通透性的影响评估了24小时，表明灌注的肺毛细血管过滤系数逐渐增加。检查不同条件下不同添加剂对分离肺灌注系统毛细血管通透性的影响，并用阿托品观察到通透性的最大增加。最重要的操作是将肺正确放入肺腔，并将插管血管连接到灌注系统。该技术的含义延伸到循环物质与吸入或灌注物质之间的相互作用，如药物测试和各种肺部病变。

Research

JoVE Journal

Medicine

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

显微静脉袋分岔动脉瘤在兔模型：技术问题

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

科研

医学

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 ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

蛛网膜下腔出血急性和后遗症的研究兔血分流型号：技术问题

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型：教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

隔离肝脏灌注作为治疗葡萄膜黑色素瘤肝转移

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Transthoracic Echocardiographic Examination in the Rabbit Model

兔子模型中的跨胸超声心动图检查

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...