这项工作得到了美国国立卫生研究院 （K99/R00 HL1431149;R01HL157803） 和美国心脏协会 （18CDA34110049）。我们还感谢来自美国国立卫生研究院 （R01DK134590;R24OD034189）、美国国家科学基金会 （EEC 1941543）、哈佛医学院 Eleanor 和 Miles Shore 奖学金、Polsky 家庭基金会、代表 MGH 研究执行委员会的 Claflin 杰出学者奖以及波士顿圣地兄弟会儿童医院 （Grant #BOS-85115）。

这项研究是在马萨诸塞州总医院机构动物护理和使用委员会 （IACUC） 之后进行的。
1. 系统设计
<ol><li>将系统与三个双夹套组件组装在一起，包括气泡收集器、储液器、氧合器、蠕动泵和水循环器。</li>
	<li>将所有带夹套的组件连接到一个夹具支架上。以两种不同的顺序用硅胶管依次连接组件（图 1A）。<ol><li>序列 1 - 水流经夹套的流型（ 图 1A 中的实线）：<ol><li>使用 36 G 管将水循环器的出口连接到气泡捕集器夹套的底部入口。这将保证灌注液在到达心脏之前保持在适当的温度 （37 °C），因为水在流经系统的其他组件时会失去热量。</li>
			<li>使用相同尺寸的管路将气泡捕集套的顶部入口连接到储液槽套的底部入口。</li>
			<li>随后，将储液槽夹套的顶部入口连接到氧合器夹套的底部入口。</li>
			<li>最后，将氧合器的顶部入口连接到水循环器的入口。</li>
		</ol></li>
		<li>序列 2 - 灌注液通过系统的流型（ 图 1A 中的虚线）<ol><li>将鲁尔接头接头连接到 16 G 管的两侧。将第一端连接到储液罐的底部，并通过蠕动泵头进料。将另一端连接到氧合器内硅胶线圈的入口之一。</li>
			<li>将第二根 16 G 管子（两端装有鲁尔锁连接器）连接到氧合器硅胶线圈的第二个入口处，连接到带有长突起的气泡捕集器的入口。</li>
			<li>将一根较短的 16 G 管子（配有鲁尔接头）连接到气泡捕集器未使用的出口，并连接到三通鲁尔锁阀。</li>
			<li>在三通阀的另一侧，将一根 16 G 管路与另一端的第二个鲁尔阀连接。第二个阀门位于储液罐的正上方。将阀的另一侧连接到更多的 16 G 管路，然后连接到压力传感器。</li>
			<li>将较小直径的管子 （̃3.7 mm） 连接到三通阀的垂直端口，并带有套管 （14 G 血管导管） 的连接器。灌注液通过气泡捕集器从储液器流向氧合器，然后通过主动脉插管连接再循环回储液器。</li>
		</ol></li>
	</ol></li>
</ol>2. 灌注液制备
<ol><li>制备基础灌注液、0.96% Krebs-Henseleit 缓冲液、9.915 mM 葡聚糖、25 mM 碳酸氢钠、1.054 mM 牛血清白蛋白、1% Pen 链球菌、0.13% 胰岛素、0.02% 氢化可的松、0.5% 肝素和 2.75 mM 氯化钙，并用蒸馏水定容。</li>
</ol>3. 灌注系统设置
<ol><li>将两个 10 mL 注射器连接到气泡捕集阱的顶部和侧面通气口。</li>
	<li>将基础灌注液 （75 mL） 添加到储液槽中。打开蠕动泵并将水循环器设置为 37 °C。</li>
	<li>将氧气（95% O2 和 5% CO2）管线连接到氧合器中的第三个入口，并将灌注液充氧至最小 pO2 为 400 mmHg。</li>
	<li>将进样口固定到三通阀的垂直端口上，紧接通过气泡阱。将带有 1 mL 注射器的带翼输液针连接到注射口（用于采样）。轻轻敲击进样口或用 1 mL 注射器抽取灌注液，以清除引入回路的任何气泡。</li>
	<li>一旦基础灌注液达到温度和氧气水平，对生化参数进行初始读数，以确保正确的离子浓度（表 1）和适当的氧合。 注：在溶液升温 （37 °C） 并用适当的气体混合物（95% O2、5% CO2）充氧后，必须读取离子和 pH 值。</li>
	<li>松开连接的管子，将压力传感器归零，让灌注液流通过打开的传感器和套管达到平衡。平衡后，按下传感器盒中的归零按钮并重新夹紧管路。在心脏连接到系统之前记录 1 mL/min 至 15 mL/min 的流量基础压力。</li>
	<li>低压灌注<ol><li>腺苷滴注：在基础灌注液中制备初始 20 mM 的腺苷储备液。将试管置于温水浴中并通过倒置混合来溶解腺苷。</li>
		<li>在基础灌注液中进一步稀释储备腺苷至 0.06 mg/mL 的浓度，并将其添加到 50 mL 注射器中。</li>
		<li>将带翼的输液针连接到 50 mL 注射器上，并将其连接到三通阀中的注射口。将注射器固定到注射泵上，并将其设置为 166.6 μL/min 的输注速度。 注意：轻轻敲击或轻弹输液口，气泡会从输液口中释放出来。</li>
	</ol></li>
	<li>高压灌注：<ol><li>浓缩红细胞 （pRBC） 分离：<ol><li>通过供体大鼠的心脏穿刺收集 10-12 mL 全大鼠血液。</li>
			<li>将血液以 2000 x g 离心 10 分钟。</li>
			<li>通过移液去除血浆和血沉棕黄层。</li>
			<li>通过倒置混合，以 1：1 的比例（例如，5 mL pRBC：5 mL 灌注液）将 pRBC 重悬于不含氯化钙的灌注液中。</li>
			<li>重复步骤 3.8.1.2-3.8.1.4 两次，总共洗涤 3 次。</li>
			<li>最后一次洗涤后，以 1：1 的比例将细胞重悬于灌注液中，并将混合物添加到灌注系统中，其中已包含 75 mL 基础灌注液。</li>
		</ol></li>
		<li>让细胞在系统中均匀分布，并使用血液学机器测量灌注液的血细胞比容。血细胞比容范围为 5%-7%。</li>
	</ol></li>
</ol>4. 心脏移植物采购准备
<ol><li>在开始采购之前完全灌注灌注系统，以最大限度地减少冷缺血时间。</li>
	<li>准备手术工具。手术工具包括蓝垫、手术胶带、丝绸 5-0 缝合线、棉签、生理盐水注射器（50 mL 和 10 mL）、手术剪刀、镊子、微型剪刀、微型镊子、Halstead 夹、30 U 肝素、16 G 门静脉冲洗管、14 G 心脏管、16 G 血管导管、带袖带袖带的改良 14 G 血管导管、压力传感器、 带冰的冰桶，47 mm 培养皿，纱布。</li>
	<li>通过在 14 G 套管上插入一圈细管（内径 [ID] 0.167 毫米，外径 [OD] 2.42 毫米）来创建改良套管，从而产生袖带效果。<ol><li>取下套管的针头，在环下方加入一滴强力胶。小心地将环滑至套管底部上方 1/4 英寸处。使用前让胶水干燥。</li>
		<li>将套管尽可能靠近袖带切成一定角度，并去除锋利的边缘。</li>
	</ol></li>
	<li>用肝素化盐水 （0.03 U/mL） 填充 60 mL 注射器，用于门静脉冲洗。将注射器连接到压力传感器，然后连接到 16 G 冲洗管。</li>
	<li>将 10 mL 肝素化盐水 （0.03 U/mL） 注射器连接到 14 G 试管中。将管子的另一端连接到主动脉插管并冲洗以去除任何气泡。</li>
	<li>将主动脉插管放入带有纱布并充满盐水的 47 毫米培养皿中。将培养皿放在冰上，直到心脏连接到灌注系统。</li>
</ol>5. 心脏移植物采购
<ol><li>在含有 3% 异氟醚的麻醉室中麻醉大鼠。</li>
	<li>一旦没有注意到反射，将大鼠从腔室中取出，将其放入手术腔，并通过面罩连续输送异氟醚 （3%）。</li>
	<li>脚趾捏试验后，用 30 U 肝素通过静脉对动物进行肝素化。</li>
	<li>将大鼠的整个腹部和上胸部区域剃光。从手术区域去除毛皮屑。用胶带固定每个肢体，以确保手术过程中没有移动。</li>
	<li>在下腹部皮肤上做一个水平中线切口，露出腹部肌肉。</li>
	<li>在腹部肌肉上做第二个水平中线切口，露出内脏器官。</li>
	<li>露出胸骨，用止血钳固定，然后颅缩回以露出肝脏和门静脉。使用 16 号血管导管插管门静脉。</li>
	<li>将 60 mL 肝素化盐水注射器连接到血管腔上，并在下腔静脉和腹主动脉上切开一个切口进行通气。通过门静脉冲洗全量盐水。 注意：冲洗压力应保持在 10 mmHg 左右。</li>
	<li>在横膈膜上做一个水平切口，然后在胸骨两侧的肋骨上做一个近端切口，露出胸腔。</li>
	<li>从腔中取出心脏，并立即将其放在装有盐水的培养皿上。</li>
	<li>识别主动脉弓，用止血钳夹住，并通过清洁任何剩余的结缔组织来暴露降主动脉。</li>
	<li>在降主动脉的中间做一个水平切口，并用 14 G 血管导管插管。 注意：不要用套管破坏主动脉瓣。</li>
	<li>通过袖带上方的缝合线固定套管并松开止血钳。</li>
	<li>让心脏保持在冰上，直到放入灌注系统。</li>
</ol>6. 灌注启动
<ol><li>将蠕动泵的流量设置为 1.0 mL/min。 注意：主动脉插管始终与心脏成 90° 角处理，以避免将气泡引入冠状动脉（图 1B）。</li>
	<li>在将心脏连接到系统之前，用套管称量心脏。 注意：主动脉插管应完全没有任何气泡。</li>
	<li>将套管连接到系统中的连接器并启动计时器。</li>
	<li>一旦心脏完全收缩，以 0.2 mL/min 的增量增加流量，同时密切观察压力。</li>
	<li>当达到所需压力或直到达到最小 3.5 mL/min 时，停止增加流量。<ol><li>低压灌注<ol><li>当压力在 30-35 mmHg 之间时，使用 4.5 mL/min 的流速。</li>
			<li>启动腺苷注射泵。</li>
		</ol></li>
		<li>高压灌注<ol><li>当压力在 70-80 mmHg 之间时，使用最小流速为 5.0 mL/min。</li>
			<li>启动腺苷注射泵。</li>
		</ol></li>
	</ol></li>
</ol>7. 脑室内球囊：
<ol><li>将一个小 （50 μL） 乳胶球球连接到带有锥形尖端（直径 1.4 mm）的球囊导管（直径 2 mm，长 15 cm）上。</li>
	<li>通过鲁尔锁连接器将导管连接到压力传感器，并将整个装置固定到夹架上。</li>
	<li>通过连接到压力传感器顶端的注射器，向球囊/导管/压力传感器注入大约 200 μL 生理盐水，并去除传感器、导管和球囊内部的气泡。</li>
	<li>使用血压计校准压力传感器。</li>
	<li>在左心房上方做一个小的水平切口。通过在压力传感器顶部拉出注射器并将其插入左心室来给球囊放气。</li>
	<li>开始数据采集并给球囊充气，直到舒张压读数为 0 mmHg。</li>
</ol>8. 采样
<ol><li>在灌注的前 20 分钟后以及此后每小时收集一次心率、主动脉血流和冠状动脉压力。</li>
</ol>9. 结束/清理
<ol><li>在灌注结束时，从系统中取出心脏并称重以进行水肿估计。</li>
	<li>通过圆周切割切开心脏心尖，并在液氮中快速冷冻，用于灌注后分析。</li>
	<li>切下心脏的圆周部分用于组织学成像和染色。处理掉心脏和插管的其余部分。</li>
	<li>向水箱中加入大量去离子 （DI） 水并运行蠕动泵，冲洗系统的所有组件。将水收集在外部桶中。</li>
	<li>重复步骤 9.4 两到三次。</li>
	<li>彻底冲洗所有样品端口和压力传感器管路。</li>
	<li>在整个系统中，用 600 mL 去离子水和 3 mL 实验室级洗涤剂泵填充储液槽。</li>
	<li>关闭热水器、氧气罐和蠕动泵。</li>
</ol>

通过改良 Langendorff 灌注加强 CVD 研究

<ol>
	<li>Cardiovascular Diseases (cvds). World Health Organization Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds)">https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds)</a> (2021)</li><li>Amini, M., Zayeri, F., Salehi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trend+analysis+of+cardiovascular+disease+mortality,+incidence,+and+mortality-to-incidence+ratio:+Results+from+global+burden+of+disease+study+2017.">Trend analysis of cardiovascular disease mortality, incidence, and mortality-to-incidence ratio: Results from global burden of disease study 2017.</a> BMC Public Health. 21 (1), 401 (2021).</li><li>Aune, S. E., Yeh, S. T., Zelinski, D. P., Angelos, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hydrogen+peroxide+and+oxidant+stress+in+a+recirculating+whole+blood-perfused+rat+heart+model.">Measurement of hydrogen peroxide and oxidant stress in a recirculating whole blood-perfused rat heart model.</a> Resuscitation. 82 (2), 222-227 (2011).</li><li>Lateef, R., Al-Masri, A., Alyahya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langendorff's+isolated+perfused+rat+heart+technique:+A+review.">Langendorff's isolated perfused rat heart technique: A review.</a> Int J Basic Clin Pharmacol. 4 (6), 1314-1322 (2015).</li><li>Herr, D. J., Aune, S. E., Menick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+assessment+of+ischemia-reperfusion+injury+in+Langendorff-perfused+rat+hearts.">Induction and assessment of ischemia-reperfusion injury in Langendorff-perfused rat hearts.</a> J Vis Exp. (101), e52908 (2015).</li><li>Vervoorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+normothermic+ex+situ+heart+perfusion+without+functional+decline.">Extended normothermic ex situ heart perfusion without functional decline.</a> J Heart Lung Transplant. 43 (4), S156 (2024).</li><li>Moeslund, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex-situ+oxygenated+hypothermic+machine+perfusion+in+donation+after+circulatory+death+heart+transplantation+following+either+direct+procurement+or+in-situ+normothermic+regional+perfusion.">Ex-situ oxygenated hypothermic machine perfusion in donation after circulatory death heart transplantation following either direct procurement or in-situ normothermic regional perfusion.</a> J Heart Lung Transplant. 42 (6), 730-740 (2023).</li><li>Testai, L., Martelli, A., Cristofaro, M., Breschi, M. C., Calderone, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+effects+of+different+flavonoids+against+myocardial+ischaemia/reperfusion+injury+in+Langendorff-perfused+rat+hearts.">Cardioprotective effects of different flavonoids against myocardial ischaemia/reperfusion injury in Langendorff-perfused rat hearts.</a> J Pharm Pharmacol. 65 (5), 750-756 (2013).</li><li>Watanabe, M., Okada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langendorff+perfusion+method+as+an+ex+vivo+model+to+evaluate+heart+function+in+rats.">Langendorff perfusion method as an ex vivo model to evaluate heart function in rats.</a> Methods Mol Biol. 1816, 107-116 (2018).</li><li>Chang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+effects+of+salidroside+on+myocardial+ischemia-reperfusion+injury+in+coronary+artery+occlusion-induced+rats+and+Langendorff-perfused+rat+hearts.">Cardioprotective effects of salidroside on myocardial ischemia-reperfusion injury in coronary artery occlusion-induced rats and Langendorff-perfused rat hearts.</a> Int J Cardiol. 215, 532-544 (2016).</li><li>Neely, J. R., Liebermeister, H., Battersby, E. J., Morgan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pressure+development+on+oxygen+consumption+by+isolated+rat+heart.">Effect of pressure development on oxygen consumption by isolated rat heart.</a> Am J Physiol. 212 (4), 804-814 (1967).</li><li>Matsuura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+inotropic+effects+of+atp+released+via+the+maxi-anion+channel+in+Langendorff-perfused+mouse+hearts+subjected+to+ischemia-reperfusion.">Positive inotropic effects of atp released via the maxi-anion channel in Langendorff-perfused mouse hearts subjected to ischemia-reperfusion.</a> Front Cell Dev Biol. 9, 597997 (2021).</li><li>Louradour, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+assessment+of+mechanical+and+electrical+function+in+Langendorff-perfused+ex-vivo+mouse+hearts.">Simultaneous assessment of mechanical and electrical function in Langendorff-perfused ex-vivo mouse hearts.</a> Front Cardiovasc Med. 10, 1293032 (2023).</li><li>Ueoka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone+does+not+shorten+action+potential+duration+in+Langendorff-perfused+rabbit+ventricles.">Testosterone does not shorten action potential duration in Langendorff-perfused rabbit ventricles.</a> Heart Rhythm. 19 (11), 1864-1871 (2022).</li><li>Reichelt, M. E., Willems, L., Hack, B. A., Peart, J. N., Headrick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+coronary+function+in+the+Langendorff-perfused+mouse+heart+model.">Cardiac and coronary function in the Langendorff-perfused mouse heart model.</a> Exp Physiol. 94 (1), 54-70 (2009).</li><li>Abel, R. M., Reis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+coronary+blood+flow+and+perfusion+pressure+on+left+ventricular+contractility+in+dogs.">Effects of coronary blood flow and perfusion pressure on left ventricular contractility in dogs.</a> Circ Res. 27 (6), 961-971 (1970).</li><li>Arnold, G., Morgenstern, C., Lochner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autoregulation+of+the+heart+work+by+the+coronary+perfusion+pressure.">The autoregulation of the heart work by the coronary perfusion pressure.</a> Pflugers Arch. 321 (1), 34-55 (1970).</li><li>Iwamoto, T., Bai, X. J., Downey, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+perfusion+related+changes+in+myocardial+contractile+force+and+systolic+ventricular+stiffness.">Coronary perfusion related changes in myocardial contractile force and systolic ventricular stiffness.</a> Cardiovasc Res. 28 (9), 1331-1336 (1994).</li><li>Rossello, X., Hall, A. R., Bell, R. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Langendorff+perfused+isolated+mouse+heart+model+of+global+ischemia-reperfusion+injury:+Impact+of+ischemia+and+reperfusion+length+on+infarct+size+and+LDH+release.">Characterization of the Langendorff perfused isolated mouse heart model of global ischemia-reperfusion injury: Impact of ischemia and reperfusion length on infarct size and LDH release.</a> J Cardiovasc Pharmacol Ther. 21 (3), 286-295 (2016).</li><li>Headrick, J. P., Peart, J., Hack, B., Flood, A., Matherne, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+and+responses+to+ischaemia-reperfusion+in+Langendorff+perfused+mouse+heart.">Functional properties and responses to ischaemia-reperfusion in Langendorff perfused mouse heart.</a> Exp Physiol. 86 (6), 703-716 (2001).</li><li>Noly, P. E., Naik, S., Tang, P., Lei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+ex+vivo+murine+biventricular+function+in+a+Langendorff+model.">Assessment of ex vivo murine biventricular function in a Langendorff model.</a> J Vis Exp. (190), e64384 (2022).</li><li>Garcia-Manzano, A., Gonzalez-Llaven, J., Lemini, C., Rubio-Poo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+rat+blood+clotting+tests+with+reagents+used+for+humans.">Standardization of rat blood clotting tests with reagents used for humans.</a> Proc West Pharmacol Soc. 44, 153-155 (2001).</li><li>Lewis, J. H., Van Thiel, D. H., Hasiba, U., Spero, J. A., Gavaler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+hematology+and+coagulation:+Studies+on+rodentia+(rats).">Comparative hematology and coagulation: Studies on rodentia (rats).</a> Comp Biochem Physiol A Comp Physiol. 82 (1), 211-215 (1985).</li><li>Motayagheni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+Langendorff+technique+for+mouse+heart+cannulation:+Improved+heart+quality+and+decreased+risk+of+ischemia.">Modified Langendorff technique for mouse heart cannulation: Improved heart quality and decreased risk of ischemia.</a> MethodsX. 4, 508-512 (2017).</li></ol>

SNT 拥有与本研究相关的专利申请，并在 Sylvatica Biotech Inc. 的科学顾问委员会任职，该公司是一家专注于开发器官保存技术的公司。所有竞争利益均由 MGH 和 Partners HealthCare 根据其利益冲突政策进行管理。

Langendorff 灌注是一种极其柔韧的技术，可实现令人印象深刻的定制和调整，以满足各种实验需求。大多数灌注参数（包括灌注压力）的显著可调性允许这种定制。由于 Langendorff 的逆行性，灌注压相当于冠状动脉灌注压，后者在心脏功能中起着至关重要的作用。已知冠状动脉灌注压 （CPP） 直接控制心脏工作，因为多种心脏指标（即左心室压、收缩力 （dP/dtmax）、壁张力、心室僵硬）与 CPP 16,17,18 成正比。从历史上看，该领域利用 60 mmHg 和 80 mmHg 之间的灌注压（实际上是 CPP）来模拟生理条件 5,8,15,19,20,21。然而，逆行离体机器灌注的非生理性质，加上对工作的高需求，导致心脏功能随着时间的推移而丧失（图 3）。或者，较低的灌注压 （30-35 mmHg），尽管不能准确复制大鼠心脏在体内的生理条件，但本质上会降低心脏工作需求并实现延长的灌注时间（4 小时），而不会随着时间的推移而丧失功能（图 3），并减少移植物水肿（图 2C）。使用较低的灌注压，虽然它意味着偏离生理 CPP，但似乎比使用生理灌注压具有重要优势，因为消除 Langendorff 灌注过程中存在的技术依赖性功能丧失将该技术改进为更准确和可预测的模型系统，具有推进心血管研究的巨大潜力。特别是，有益和/或需要延长灌注时间以达到科学相关性的研究领域（即药物治疗、免疫反应分析、基因编辑、常温移植物保存等）在对抗 CVD 的斗争中变得越来越重要。
Langendorff 灌注无疑是心血管研究领域的重要工具。因此，除了这种科学技术为研究界带来的巨大好处外，它还具有重要的科学复杂性。实际上，该方案中有几个关键步骤需要仔细标准化，主要是为了避免在开始灌注之前、期间和之后立即发生心脏移植物损伤。在门静脉冲洗期间，移植物损伤的第一次机会并不明显。这种用肝素化盐水冲洗的目的是从心脏移植物中去除尽可能多的全血，具有双重目的。首先，它作为一种通过放血来安乐死的方式。其次，它最大限度地减少了取回、插管和运输过程中心脏移植物内凝固的机会，因为已知大鼠全血的衣帽时间极短22,23。然而，在数百次成功的心脏灌注之后，很明显，在冲洗过程中施加在大鼠生物体上的压力非常重要，理想的冲洗压力约为 10 mmHg。较高的门静脉冲洗压力似乎会导致心脏移植物的脉管系统受损，从而导致血管阻力增加 （<img alt="Equation 1" src="/files/ftp_upload/66815/66815eq01.jpg" style="margin: auto;">）。较高的血管阻力实际上导致在较低的流速下达到目标灌注压力。压力和冠状动脉血流之间的这种不平衡在产生的左心室脉压 （LVPP） 中传递，导致显着的变异性。
下一个可能的心脏移植物损伤实例是通过将气泡引入冠状动脉将移植物连接到系统期间。由于对空心管操作不当（图 1B）或从气泡捕集阱24 上游的灌注系统中去除气泡不当，很容易引入气泡。由于这种设置的逆行性质，任何空气的引入都会导致心脏空气栓塞，导致缺血性损伤、纤颤，以及非常常见的移植物死亡。最后，确保方案成功的最后一个关键步骤发生在灌注开始时。与大多数报告使用 Langendorff 作为技术的手稿不同，该方案中的灌注开始以相对较低的流速 （1 mL/min） 进行，并逐渐增加 （+0.2 mL/min），这保证了对灌注压力的完全控制 5,8,15,19,20,21 .这种流量和压力的增量增加至关重要，因为压力的突然变化会不可逆地增加血管阻力并改变微妙的流量/压力平衡。
压力控制的 Langendorff 灌注中的高血管阻力非常重要，因为在较低流量下达到目标灌注压力，并且移植物导致灌注不足。对流量和压力之间这种完美平衡的严重依赖可能是该方案的最大限制，因为任何先前的移植物损伤，无论是有意的（即延长保冷、热缺血损伤、心肌梗死等）还是无意的，都会导致血管阻力增加。实际上，该方案对于实验在灌注开始后开始的研究（即药物治疗、免疫反应分析、基因编辑、常温移植物保存等）但不是之前的研究特别有用。这种限制是一个 Langendorff 并不适合所有用途的完美例子，应特别注意定制灌注参数以更好地满足实验需求。

心血管研究领域在心血管疾病 （CVD） 的诊断和治疗方面取得了重要进展。然而，尽管发病率和死亡率普遍下降，但 CVD 仍然是全球死亡的主要原因 1,2。这一令人震惊的事实凸显了增加研究和科学进步的必要性，这无疑取决于可用研究工具的准确性和可预测性。因此，研究工具集不断需要创新、改进和/或重新调整用途。例如，逆行或 Langendorff 心脏灌注是一种在该领域应用了一个多世纪的技术，可以很容易地进行修改，以涵盖更广泛的科学需求并实现更广泛的应用。
在 Langendorff 灌注过程中，心脏移植物与生物体的其余部分分离，为各种实验参数提供了重要程度的控制，包括温度、循环溶液、冠状动脉灌注压力等3,4,5,6,7。这些参数的操作有助于模拟大量心脏场景，这些场景可用于进一步推动科学进步 5,8,9,10。在这些参数中，灌注压可能是最容易被忽视的实验设置11。
在 Langendorff 期间，灌注压与心率、收缩压/舒张压峰值和耗氧量直接相关11。这种相关性提供了对心脏移植物产生的工作量的直接和精确控制，可以进行调整以满足个人实验需求。尽管具有这种宝贵的控制能力，但该领域历来倾向于使用更高的灌注压 （60-80 mmHg），无论实验需要如何，所有心脏移植物都受到高工作要求8,12,13,14,15。这种不必要的高工作要求的后果源于一个总体原则，即过度工作往往会导致过早失败。对于通过 Langendorff 灌注的心脏移植物来说，这似乎尤其如此，因为这种方法的非生理性质和体内缺乏恢复支持似乎加剧了移植物失败。这种移植物功能的过早丧失显着限制了灌注和实验时间。实际上，在较长的灌注时间与实验需求（即药物治疗、免疫反应分析、基因编辑、移植物保存等）更相关的情况下，可以负担得起较低的心脏功以换取更高的移植物耐久性。
该方案证明了在 Langendorff 期间使用较低灌注压力 （30-35 mmHg） 的可行性，以及与较高的灌注压 （60-80 mmHg） 相比，这些压力对心脏移植物功能随时间推移的显着影响。此外，本手稿中的研究结果强调了优先考虑定制各种灌注参数以更好地满足实验需求的重要性。

<table><tbody><tr><td>5-0 Suture</td><td>Fine Scientific Tools</td><td>18020-50</td><td></td></tr><tr><td>14 G Angiocath</td><td>Becton Dickinson</td><td>381867</td><td></td></tr><tr><td>16 G Angiocath</td><td>Becton Dickinson</td><td>381957</td><td></td></tr><tr><td>24 mm Heart Chamber adaptors</td><td>Radnoti</td><td>140132</td><td></td></tr><tr><td>Balloon Catheter&amp;nbsp;</td><td>Radnoti</td><td>170423</td><td></td></tr><tr><td>BD&amp;nbsp;Slip Tip Sterile Syringes- 10 mL</td><td>Fisher Scientific</td><td>14-823-16E</td><td></td></tr><tr><td>BD&amp;nbsp;Slip Tip Sterile Syringes- 1 mL</td><td>Fisher Scientific</td><td>14-823-434</td><td></td></tr><tr><td>BD&amp;nbsp;Slip Tip Sterile Syringes- 50 mL</td><td>Fisher Scientific</td><td>14-820-11</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Bubble Trap Compliance Chamber</td><td>Radnoti</td><td>130149</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>Clamp Holder</td><td>United Scientic</td><td>RTCLMP1</td><td></td></tr><tr><td>Dextran</td><td>Sigma</td><td>31389</td><td></td></tr><tr><td>DIN8 Extension Cable</td><td>Iworx</td><td>SKU&amp;nbsp;C-DIN-EXT</td><td></td></tr><tr><td>Falcon High Clarity 50 mL conical tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>GSC Go Science Crazy&amp;nbsp;Cast Iron Support Ring Stand</td><td>Fisher Scientific</td><td>S13748</td><td></td></tr><tr><td>Heart&amp;nbsp; Chamber</td><td>Radnoti</td><td>140160</td><td></td></tr><tr><td>Heated Water Circulator bath</td><td>Cole Parmer</td><td>N/A</td><td></td></tr><tr><td>Heparin sodium Injection</td><td>Medplus</td><td>G-0409-2720-0409-2721</td><td></td></tr><tr><td>Hydrocortisone</td><td>Solu-Cortef</td><td>MGH Pharmacy</td><td></td></tr><tr><td>Insulin</td><td>Humulin R</td><td>MGH Pharmacy</td><td></td></tr><tr><td>Insvasive Fluid Filled Blood Pressure Sensor</td><td>Iworx</td><td>SKU&amp;nbsp;BP-10x</td><td></td></tr><tr><td>Iworx Data Acquisition System</td><td>Iworx</td><td>IX-RA-834</td><td></td></tr><tr><td>Krebs-Henseleit Buffer</td><td>Sigma</td><td>K3753</td><td></td></tr><tr><td>Left Ventricular Pressure Balloon</td><td>Radnoti</td><td>170404</td><td></td></tr><tr><td>Masterflex L/S Easy-Load II Pump Head for Precision Tubing, PPS Housing, SS Rotor</td><td>VWR</td><td>MFLX77200-60</td><td></td></tr><tr><td>Masterflex L/S Standard Digital Pump Systems</td><td>VWR</td><td>MFLX07551-30</td><td></td></tr><tr><td>Membrane Oxygenating Chamber</td><td>Radnoti</td><td>130144</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Polyethylene Tubing</td><td>Fisher Scientific</td><td>14-170-12H</td><td></td></tr><tr><td>Precision Pump Tubing-16</td><td>VWR</td><td>MFLX96410-16</td><td></td></tr><tr><td>Sodium Bicarobonate</td><td>Sigma</td><td>5761</td><td></td></tr><tr><td>Standard PHD ULTRA CP Syringe Pump</td><td>Harvard Aparatus</td><td>88-3015</td><td></td></tr><tr><td>Tygon Transfer Tubing</td><td>VWR</td><td>MFLX95702-03</td><td></td></tr></tbody></table>

modified langendorff perfusion for extended perfusion times of rodent cardiac grafts

尽管心血管疾病 （CVD） 的诊断和治疗取得了重大进展，但该领域迫切需要增加研究和科学进步。因此，现有研究工具集的创新、改进和/或再利用可以为研究进步提供改进的测试平台。Langendorff 灌注是 CVD 研究领域一种非常有价值的研究技术，可以对其进行修改以适应广泛的实验需求。这种定制可以通过个性化大量灌注参数来实现，包括灌注压力、流量、灌注液、温度等。该方案证明了 Langendorff 灌注的多功能性以及通过使用较低的灌注压力 （30-35 mmHg） 实现更长的灌注时间 （4 h） 而不损失移植物功能的可行性。实现延长的灌注时间，而不会由技术本身引起移植物损伤和/或功能丧失，有可能消除实验结果中的混杂因素。实际上，在较长的灌注时间与实验需求（即药物治疗、免疫反应分析、基因编辑、移植物保存等）相关的科学环境中，较低的灌注压力可能是科学成功的关键。

收获成年雄性 Lewis 大鼠 （250-300 g 体重） 的心脏，并在高 （70-80 mmHg） 或低 （30-35 mmHg） 灌注压 （每组 n = 3） 下灌注。通过收集心率、水肿和左心室功能来确定灌注压对整体心脏功能和健康的影响。
确定了心率和灌注压之间的明显相关性（图 2）。与低压心脏相比，除第一个时间点外，高压心脏的心率在统计学上较高（60 分钟，图 2A，B）。有趣的是，低压心脏似乎在灌注开始时经历了一段时间的调整，心率大约需要 30 分钟才能稳定并达到其余灌注所维持的水平（图 2A）。两组之间也观察到左心室脉压 （LVPP） 存在很大差异，在每个时间点，高压心脏的 LVPP 在统计学上都高于低压心脏（图 3B）。这种持续的高工作需求导致高压心脏功能进行性丧失，灌注 2 小时后 LVPP 出现统计学下降（图 3A、B）。或者，在低压灌注的心脏中不存在功能丧失，LVPP 在整个灌注时间内保持不变（图 3A、B）。与 LVPP 类似，与低压心脏相比，高压心脏在整个灌注时间内表现出更高的心肌收缩 （dP/dtmax） 和松弛 （dP/dtmin）（图 3C、D）。相应地，高压心脏的收缩力和舒张能力逐渐丧失，与灌注的最后一小时相比，这两个参数在灌注时间 1 h 时均在统计学上更高。不同的是，低压组的心肌收缩力和松弛能力相对较低，并且在 4 小时的灌注时间内保持不变（图 3C，D）。除了功能影响外，长时间的高灌注压也会加剧心脏移植物内的间质液潴留，导致水肿。这种水肿以重量变化百分比进行半量化，与在低压下灌注的心脏相比，高压心脏的体重增加在统计学上更高（图 2C）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66815/66815fig01.jpg"> 图 1：灌注系统设置。 （A） 整体灌注设置。虚线表示系统组件为优化灌注液循环而连接的顺序。彩色实线表示组件连接的顺序，以优化灌注液温度。（B） 插管后正确处理心脏，以避免排空导管并将空气引入冠状动脉。 <a href="https://www.jove.com/files/ftp_upload/66815/66815fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66815/66815fig02.jpg"> 图 2：压力对心率和水肿的影响。 （A） 从脑室内球囊测量中获得的心率。实线是实验组的中位数。阴影区域是四分位距。（B） 每小时灌注的心率数据曲线下面积 （AUC）。（C） 在低压和高压下灌注 4 小时后体重增加的百分比。所有数据均表示为中位数±四分位距 （IQR）。*p < 0.01，**p < 0.05，***p < 0.001。 <a href="https://www.jove.com/files/ftp_upload/66815/66815fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66815/66815fig03.jpg"> 图 3：压力对左心室功能的影响。 （A） 随时间绘制的最大收缩压，表示为左心室脉压 （LVPP）。实线是实验组的中位数。阴影区域是四分位距。（B） 每小时灌注的 LVPP 曲线下面积 （AUC）。（C） 从压力脉冲的最大导数量化的心肌收缩力。（D） 从压力脉冲的最小导数量化的心肌松弛。所有数据均表示为中位数±四分位数范围。*p < 0.01， **p < 0.05， ***p < 0.001， ****p < 0.0001。 <a href="https://www.jove.com/files/ftp_upload/66815/66815fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>离子</td>
			<td>浓度 （mmol/L）</td>
		</tr><tr><td>钠+ </td>
			<td>135–145</td>
		</tr><tr><td>K + </td>
			<td><6.00</td>
		</tr><tr><td>钙 +2 </td>
			<td>1.0–1.3</td>
		</tr><tr><td>Cl - </td>
			<td>96–106</td>
		</tr></tbody></table>表 1：灌注液中离子浓度的可接受范围。

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

本文证明了通过在 Langendorff 期间采用低于生理 （30-35 mmHg） 的灌注压 （30-35 mmHg） 来实现小鼠心脏移植物灌注时间 （4 h） 而不丧失功能的可能性。

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 ...

modified-langendorff-perfusion-for-extended-perfusion-times-rodent

Research

JoVE Journal

Bioengineering

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

1.1K 次观看.  Harvard Medical School and Massachusetts General Hospital. 本研究旨在强调 Langendorff 灌注作为研究工具的活力和广泛应用，以及其在心血管疾病研究中的巨大效用。它还强调了定制不同灌注参数（如流速、压力、灌注液和温度）以更好地满足实验需求的重要性。该方案为 Langendorff 灌注的现状提供了一种替代方法。它表明，使用低于正常的灌注压力，例如 30 至 35 毫米汞柱，而不是通常的 60 至 80 毫米汞...

视频: 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

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 ...

科研

生物学

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermic的技术<em&gt;前体内</em&gt;肝脏灌注的存储，评估和修复边际移植肝

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

医学

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

小鼠常母离 体 肝机灌注

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

工程学

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.

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 ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循环死亡后心脏捐献的临床前模型

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

无静脉-静脉旁路术的猪肝移植作为扩展标准供体模型

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Langendorff 模型中离 体 小鼠双心室功能的评估

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

异位灌注异位心脏移植大鼠模型

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

<meta charset="utf8"></head><body>左心室负荷离体灌注中的增强心脏评估

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (&#707;6 h).

<meta charset="utf8"></head><body>作者聚焦：通过定量指标和创新储液库系统增强嫁接物活力评估

The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

使用被动左心房加压和被动后负荷进行移植物评估的心脏负荷

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. ...

改良 Langendorff 灌注延长啮齿动物心脏移植物的灌注时间

副本

摘要

探索更多视频

此视频中的章节

光学测绘的Langendorff灌注大鼠心脏

Subnormothermic的技术

小鼠常母离体肝机灌注

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

循环死亡后心脏捐献的临床前模型

无静脉-静脉旁路术的猪肝移植作为扩展标准供体模型

Langendorff 模型中离体小鼠双心室功能的评估

异位灌注异位心脏移植大鼠模型

使用被动左心房加压和被动后负荷进行移植物评估的心脏负荷

使用被动左心房加压和被动后负荷进行移植物评估的心脏负荷

改良 Langendorff 灌注延长啮齿动物心脏移植物的灌注时间

副本

摘要

探索更多视频

此视频中的章节

光学测绘的Langendorff灌注大鼠心脏

Subnormothermic的技术

小鼠常母离 体 肝机灌注

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

循环死亡后心脏捐献的临床前模型

无静脉-静脉旁路术的猪肝移植作为扩展标准供体模型

Langendorff 模型中离 体 小鼠双心室功能的评估

异位灌注异位心脏移植大鼠模型

使用被动左心房加压和被动后负荷进行移植物评估的心脏负荷

使用被动左心房加压和被动后负荷进行移植物评估的心脏负荷

小鼠常母离体肝机灌注

Langendorff 模型中离体小鼠双心室功能的评估