这项工作没有从公共、商业或非营利部门的任何资助机构那里获得任何具体的资助。

所有研究均得到华盛顿大学医学院机构动物护理和使用委员会的批准。根据美国国家科学院编写并由美国国立卫生研究院出版的《实验动物护理和使用指南》（第8 版 21 ）以及美国国家医学研究学会制定的《实验动物护理原则》，动物接受了人道关怀。
1.麻醉和插管
<ol><li>选择重量至少为 25 克的鼠标。由于声带之间的孔口较大，这将有助于更容易插管。</li>
	<li>腹膜内注射小鼠氯胺酮（剂量100mg / kg体重）和甲苯噻嗪（剂量10mg / kg体重）的混合物。该混合物应装入带有 1/2 英寸 29G 针头的 2/2 cc 注射器中。等待大约 5 分钟让氯胺酮生效 - 小鼠不应表现出自发运动并且对脚趾捏合没有反应，这证实了充分的麻醉。</li>
	<li>手术前皮下注射丁丙诺啡（剂量 0.05 mg/kg 体重），以进一步控制疼痛。</li>
	<li>将非药物眼药膏涂抹在双眼上，以避免手术期间角膜干燥。</li>
	<li>用剪子在左胸部和背部剃须鼠标（见 图1A），如果计划进行剖腹手术，则延伸到腹部（见步骤5.3）。</li>
	<li>一旦小鼠被麻醉，使用首选的插管设置进行插管，并在显微镜下在~37°C的加热垫上进行剩余的过程（以维持小鼠的正常体温）。</li>
	<li>在获得足够的声带视野后，将末端带有曲线的自制导引器（见 补充图 1）插入 1 英寸的 20G 血管导管中，该导管用作气管插管 （ETT）。引导导引器的尖端，然后引导 ETT 在声带之间并经过声带。 注意:重要的是要观察 ETT 穿过声带，以避免假插管进入食道。插管时应注意避免声带损伤（即，插管尝试次数应限制在 5 次以内，如果遇到阻力，则不应提前 ETT）。</li>
	<li>插入 ETT 后，取下导引器，并将 ETT 连接到小动物呼吸机。观察对称性胸部上升，以确认气管插管正确。呼吸机设置应为每分钟 100-105 次呼吸，吸入氧分数为 100%。潮气量为 0.35 mL，呼气末正压为 1 cm H2O。 注意:确保胸部而不是腹部上升，因为如果不及时纠正，意外食管插管将导致死亡。</li>
	<li>一旦确认就位，用一条 5 厘米的 1 英寸丝带将 ETT 固定在小鼠鼻子周围，确保与 ETT 和鼻子充分接触，这样 ETT 就不会从嘴里滑出（参见 图 1B-C）。</li>
	<li>为了在整个手术过程中保持足够的麻醉，应根据氧气流量给予 1%-1.5% 的异氟烷。</li>
</ol>2. 开胸手术
<ol><li>将鼠标置于右侧卧位，两只前爪都贴在左上角，右后爪贴在左下角，左后爪贴在右下角（见 图 2G 胶带）。</li>
	<li>用至少 3 轮交替的聚维酮碘对左胸皮肤进行消毒，然后使用 70% 酒精。</li>
	<li>在从前腋线到后腋线的第4肋间隙 上进行切口，使用剪刀打开覆盖的皮肤（见 图2A）。第 4 肋间隙位于腋窝中线腋窝下方约 1 厘米处。</li>
	<li>为尽量减少失血，请使用手持烧灼笔凝固皮下层和肌肉层内的可见血管。</li>
	<li>用剪刀沿着皮肤切口的长度将背阔肌和前锯肌急剧分开（见 图2B）。</li>
	<li>使用细弯曲的镊子，小心地抬高第 4肋骨（注意不要伤害下面的肺），并在第 5肋上方用细剪刀进行精确剪刀，以进入第4肋 间隙（见 图2C）。在第5 根肋 骨上方而不是第4 根肋 骨下方进行开胸手术，以避免损伤肋间神经血管束。</li>
	<li>一旦胸膜腔负压消失并且肺从胸壁回缩，就将开胸手术向前和向后延伸，位于第 5 肋骨上方的第 4 肋间隙（见图 2D-E）。切口的整个长度应足够长以暴露整个肺，通常为 ~1 厘米。 注意:如果开胸手术向前延伸太远，可能会发生乳内动脉出血 - 如果遇到，应进行烧灼以避免过多的血液流失。</li>
	<li>应用两个肋骨牵开器以展开肋骨空间，留出至少 1 cm2 的工作窗口以获得足够的可视化（见图 2F-G）。</li>
</ol>3、肺门钳的应用
<ol><li>使用双尖棉头涂抹器，通过抬高头侧，然后钝性地分开半透明的肺下韧带来动员左肺（见图 3A-B）。 注意:如果在不撕裂肺部的情况下执行此操作具有挑战性，也可以使用细剪刀将下肺韧带急剧分开。</li>
	<li>将肺向前反射，以便可以看到左肺门后部。将一块 6-0 丝带剪至 ~10 厘米，并将该带的中点放在肺门后方（见 图 3C）。</li>
	<li>然后，向后翻转左肺，放在领带上，并向前拉领带的两端（见 图3D）。</li>
	<li>用领带的两个自由端（A 和 B）系一个器械，用镊子和弯曲的蚊子夹系一个可逆的滑结。请参阅步骤 3.4.1 和 3.4.2 中的详细说明。温暖的缺血时间从打结开始。一旦打结，左肺就不应再随着呼吸机的呼吸而充气。它应该变成淡白色，意味着灌注停止。 注意:应注意确保结位于肺门的中间，避免无意中夹住左心房中央和肺实质外侧（见 图3F）。<ol><li>用夹子在惯用手，镊子在非惯用手，用镊子握住A的末端，并将A的中点绕在闭合的夹子上一次（见 图3E）。</li>
		<li>用夹子抓住 B 的中点，用双手拉动以收紧滑结。不要让 B 的末端穿过结，否则会使结不可逆。打结后，B 应该有一个从结发出的环，而 A 应该是直的（见 图 3F）。可以通过拉动 B 的末端来解开结。</li>
	</ol></li>
	<li>通过手动闭塞从 ETT 到呼吸机的流出管来确认支气管的充分闭塞，这应导致左肺不会充气（见 图 3G）。鉴于与软骨支气管相比，肺动脉和静脉的塌陷性增加，支气管闭塞被认为是血管闭塞。</li>
	<li>使用肺门钳夹后，使用 6-0 尼龙缝合线用一个简单的中断缝合缝合皮肤切口，以尽量减少热缺血期间的不显性液体流失。</li>
</ol>4. 松开肺门钳
<ol><li>在所需的热缺血期之后，轻轻拉动丝带的短自由端（ 图3F 和步骤3.4中的B）松开滑结。松开滑结后开始再灌注时间。 注意:松开系带后，可以通过再次手动闭塞 ETT 流出来确认肺通气，这现在应该会导致左肺扩张。同样，肺的充气暗示了灌注，但是，它也可以通过肺粉红色来确认（见 图4A）。</li>
	<li>在再灌注期间，分三层闭合胸部，以尽量减少不显性损失。首先，用 6-0 尼龙缝合线单针闭合第 4 肋骨空间，在第4 根肋骨上方咬一口，在第6 根肋骨上方咬一口（见图 4B-C）。将其与最大肺充气联系起来，以最大限度地降低医源性气胸的风险（见图4D-E）。</li>
	<li>接下来，使用带有 6-0 尼龙缝合线的简单缝合线闭合肌肉层。</li>
	<li>最后，用 6-0 尼龙缝合线进行简单的断续缝合，闭合皮肤切口（见 图 4F）。避免在皮肤上缝合，因为有清醒的动物会抠其切口并导致伤口完全裂开的风险。</li>
	<li>从这一点开始，可以在所需的再灌注时间结束时直接收获肺。如果需要进行动脉血气分析，请参阅步骤 5。如果需要在处死前进行额外的静脉注射，请参阅步骤 6。这是一项终末手术。</li>
	<li>如果再灌注时间超过几个小时，请关闭异氟醚，让小鼠从麻醉中醒来并拔管。拔管标准包括爪子抽搐、捏合以及自主呼吸和运动。从氯胺酮和异氟醚麻醉中唤醒小鼠通常需要 30-45 分钟。这是一项生存手术。<ol><li>对于生存手术，皮下注射 1 mL 温盐水，以解决手术造成的液体流失。手术前皮下注射丁丙诺啡（剂量 0.05 - 0.1 mg/kg 体重）以控制疼痛。手术后至少 3 天每 4-6 小时重复麻醉一次。考虑沿切口使用布比卡因阻滞剂，以进一步控制疼痛。</li>
	</ol></li>
</ol>5. 动脉血气分析
注意:如果需要 ABG 测量，最好通过左心室的动脉血抽吸来获得。为了确定动脉血气图仅反映左肺功能，应在右肺门夹夹约 4 分钟后获取动脉血22,23，在此期间只有左肺进行氧合和通气。
<ol><li>如果小鼠在肺门钳夹后从麻醉中醒来，则根据步骤 1 重新麻醉并插管小鼠。</li>
	<li>在所需的再灌注时间结束前约15-20分钟，将小鼠置于仰卧位，用胶带固定所有四个肢体。 注意:在再灌注结束之前，应为以下步骤分配足够的时间。打开腹部和胸部的具体时间将根据外科医生和他们的经验而有所不同。</li>
	<li>从耻骨到剑突进行中线剖腹手术，用剪刀切开皮肤，然后沿着白线切开腹壁（见图5A-B）。</li>
	<li>在剑突处，沿着最下肋骨的曲线，将剖腹手术向左和向右延伸到前腋线（见 图5C）。</li>
	<li>从腹部切开中线的前横膈膜进入胸部，注意不要太深，以免伤害心脏（见图5D-E）。然后，将前横膈膜切口左右延伸到前腋线，沿着最下方的肋骨（参见图5D中的白色虚线）。</li>
	<li>沿前腋线分割双侧肋骨，向上延伸至腋窝，进行翻盖式开胸术。然后，反射前胸壁（胸骨和双侧前肋骨）头侧，使心脏和双侧肺完全暴露（参见 图5F中的白色虚线）。</li>
	<li>将夹子夹在前胸壁上，该胸壁呈头侧翻转，以利于回缩。用弯曲的蚊子夹将横膈膜向下缩回amp 在中线以改善胸部的可见性（见 图 5F）。</li>
	<li>为了完全动员右肺（有 4 个肺叶），将延伸到左胸部中线的副肺叶返回到右胸部（见图 5G-H）。有一条细韧带将这个耳垂连接到左胸部 - 用棉头涂抹器钝切地分开，或者用剪刀尖锐地分开。 注意:该副叶穿过下腔静脉 （IVC） 后方的中线，因此在此操作过程中必须注意不要伤害 IVC。</li>
	<li>一旦所有肺叶都回到右胸，将整个右肺向前反射，并在右肺门后方放置另一根 6-0 丝带（切至 ~10 厘米）。然后，将右肺置换回胸腔，穿过领带。</li>
	<li>使用与步骤 3.4 中描述的相同技术在右肺门上系另一个滑结，注意环绕右肺的所有四个肺叶。这个右肺门钳夹步骤的时间应在再灌注结束前约 4 分钟。</li>
	<li>在 1 cc 结核菌素注射器上涂有约 200 μL 1000 单位/mL 肝素的 1/2 英寸 31G 针头。为此，吸取肝素的体积并反复来回拉动柱塞 3-4 次，以使肝素覆盖注射器的整个内部。这样做是为了最大限度地降低从手术站运输到 ABG 机器时吸入的血液凝固的风险。</li>
	<li>右肺门钳夹4分钟后，将左心室的动脉血吸入肝素包被的注射器中（见图5I）。注意避免刺穿室间隔和无意中抽吸右心室静脉血。较深的右心室和较亮的左心室之间存在明显的颜色差异（见图5I插图）。将针头朝向左侧颈部。可能需要多次穿刺才能获得足够的血液来进行 ABG （~150 μL）。 注意:在右肺门钳夹的 4 分钟内，小鼠可能会开始表现出痛苦的呼吸，这预示着即将死亡。如果发生这种情况，应在心脏骤停之前迅速抽吸动脉血。不可能从不跳动的心脏中吸出血液。</li>
	<li>在动脉血气分析机上运行动脉血，以获得氧饱和度、氧分压、二氧化碳分压等测量值。在ABG收集和/或抗体治疗后对小鼠实施安乐死（参见步骤6）。</li>
</ol>6.静脉注射抗体，用于测量细胞外渗
注:该技术可用于通过在处死前将荧光染料标记的抗体静脉注射到 IVC 中来确定细胞外渗，然后进行流式细胞术分析，如先前发表的18。简而言之，可以使用中性粒细胞特异性抗 Ly6G 抗体来区分血管内中性粒细胞和间质性中性粒细胞。异硫氰酸荧光素标记（FITC 标记）抗 Ly6G（克隆 1A8）在处死前 5 分钟静脉注射，标记循环血管内中性粒细胞。使用的 FITC-Ly6G 抗体的浓度为 100 ng，在 200 μL 磷酸盐缓冲盐水中稀释。然后，在制备来自左肺的单细胞悬液用于流式细胞术后，用别藻蓝蛋白标记（APC标记）的抗Ly6G（克隆1A8）标记所有中性粒细胞。因此，APC-Ly6G+FITC-Ly6G+ 中性粒细胞是血管内粒细胞，而 APC-Ly6G+FITC-Ly6G- 是血管外或间质细胞。例如，该技术可以适用于具有抗Ly6C抗体的单核细胞，具有抗CD19抗体的B细胞。
<ol><li>将所需的抗体装入带有 5/16 英寸 31G 针头的 3/10 cc 注射器中，注意尽量减少注射器内的气泡。在步骤 6.5 中将其用于静脉注射到 IVC 中。</li>
	<li>根据步骤 5.3-5.4 进行剖腹手术。</li>
	<li>剖腹手术后，使用两个尖头棉头涂抹器钝性地进行右内内侧内脏旋转。在小鼠中，这是通过切除腹部左侧的所有肠道来完成的，这将允许清楚地看到IVC（见 图6A）。</li>
	<li>用棉签涂抹器钝性地清除覆盖在 IVC 上的脂肪。</li>
	<li>通过静脉穿刺将抗体溶液注射到IVC中（见 图6B）。拔出针头后，立即用棉签轻轻按压静脉穿刺部位，直至止血（通常约2-3分钟）。</li>
	<li>通过棉签将内脏肠放回腹部，继续对IVC施加压力（见 图6C）。</li>
	<li>按照步骤5.5-5.7进行翻盖式开胸手术，收获左肺。在处死和收获之前，让抗体至少有 5 分钟的全身循环。在 ABG 收集和/或抗体治疗后对小鼠实施安乐死。</li>
</ol>7. 组织学 （H&E） 染色
<ol><li>在福尔马林固定和左肺石蜡包埋并切片至5μm厚度后，在二甲苯中脱蜡载玻片（两次10分钟洗涤）。</li>
	<li>顺序乙醇洗涤中的脱水:用 100% 洗涤 2x 5 分钟，用 95% 洗涤 1x 2 分钟，用 70% 乙醇洗涤 1x 2 分钟。然后，用去离子水冲洗。</li>
	<li>用苏木精染色载玻片 2-4 分钟，然后用去离子水洗涤 5 分钟或直到它变得清澈。苏木精染色的精确持续时间需要根据组织和所需的核染色强度进行优化。</li>
	<li>在定义溶液中洗涤 30 秒以区分污渍，然后在水中洗涤 2 分钟。在蓝色形成溶液中洗涤 30 秒，然后在水中洗涤 2 分钟。</li>
	<li>通过浸入 95% 乙醇 15 次进一步脱水。在曙红中染色 1 分钟。</li>
	<li>用2分钟乙醇洗涤（2x）脱水，然后用2分钟二甲苯洗涤（2x）脱水。涂抹安装介质并盖上盖玻片。</li>
</ol>

研究肺缺血再灌注损伤的小鼠模型

<ol>
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作者没有报告任何相关的披露。

我们描述了一种肺门钳夹技术，该技术涉及在左肺门上应用滑结，该滑结阻塞肺动脉、静脉和支气管以诱导热缺血，然后进行再灌注。肺门钳夹后，可以采集左肺用于各种实验技术，如组织学、流式细胞术、批量或单细胞测序以及定量聚合酶链反应。此外，血液和脾脏可用于研究全身效应，而非缺血性右肺可用作内部对照。在该协议中，我们还描述了获得ABG测量值和/或确定细胞外渗的详细方法。
小鼠肺门钳夹技术对于肺 IRI 的研究和更广泛的肺移植领域至关重要。例如，使用这种肺门钳夹模型，我们小组发现脾脏衍生的 CCR2+ 经典单核细胞介导中性粒细胞在 IRI18 后外渗到肺部。另一组显示，使用肺门钳夹模型19，II 型肺泡上皮细胞以 NADPH 氧化酶-IL-17-TNFα 依赖性方式产生 CXCL1（一种中性粒细胞趋化剂）。在鼠肺门钳夹模型中发现 TLR4 在水肿的发展中起作用20.
虽然原位鼠肺移植在临床上更适用，可以模拟温缺血和冷缺血，但它也会引入血管和支气管吻合口的手术创伤以及供体-受体错配的同种异体刺激。因此，肺门钳夹技术的主要优点是它能够将肺 IRI 的影响与这些其他因素隔离开来。此外，对于没有经验的小鼠显微外科医生来说，肺门钳夹手术在技术上要求不高，与肺移植相比，可以更快地掌握。肺门钳夹模型的一个局限性是难以实现冷缺血。已经通过左肺动脉顺位冲洗灌注对猪进行了原位冷缺血25，然而，由于小鼠的体型较小且在手术期间需要保持体温正常，因此这在小鼠中更具挑战性。然而，孤立性温缺血的研究很重要，因为它通常比冷缺血对器官功能更有害 4,5,6。此外，移植中心越来越多地采用 DCD 肺移植，与传统的 DBD 肺移植相比，DCD 肺移植的热缺血时间要长得多7.因此，对热缺血期间和随后的再灌注期事件的详细研究至关重要，并且可以使用这种肺门钳夹模型来完成。
在过去的几十年里，已经创造了许多肺门钳夹技术的变体。这些包括使用非创伤性微血管钳夹 8,9,10,11,12,13、Rumel 止血带 14,15 和在肺门16 周围打结的领带。我们描述了我们在小鼠中对滑结技术的适应。与其他肺门钳夹技术相比，该技术的主要优点是它允许在缺血期间闭合胸部，从而最大限度地减少不显性液体的损失，特别是在缺血时间较长的方案中。当将夹子或止血带放在肺门上时，这是无法实现的。
关于动物选择，我们选择在小鼠中开发该模型，而不是大鼠或大型动物模型，例如猪。原因有两个方面 - 我们的实验室在小鼠显微外科手术和原位小鼠肺移植方面拥有丰富的经验17.其次，虽然小鼠比大鼠更小，技术上更具有挑战性，但目前市售的转基因小鼠品系比大鼠品系更多26,27。小鼠的繁殖速度也更快，允许更快地开发新菌株，这在研究IRI的免疫机制时特别有用。由于这些原因，我们选择在小鼠中开发这种技术。
据我们所知，这是第一篇使用左肺门钳夹技术在小鼠中使用滑结技术的基于视频的文章。Saito 等人发表了一篇基于视频的文章，展示了使用微血管钳夹在大鼠中钳夹22。在小鼠中，Liao 等人发表了一个肺 IRI 模型，其中肺动脉 （PA） 用滑结夹住，然而在该模型中，由于支气管被排除在夹子之外，因此肺保持通气28。PA 钳夹技术已被用作研究肺栓塞29、肺动脉高压和/或右心室衰竭30,31 的模型。由于研究领域是移植中的肺 IRI，因此我们选择夹紧包括支气管在内的整个肺门，以尽量减少缺血性损伤顶部的气压伤。
值得注意的是，选择温缺血 30 分钟，然后再灌注 1 小时，因为我们对 IRI 后早期肺浸润细胞的动力学感兴趣。例如，有研究报告称，细胞保护基因（如血红素加氧酶 1）在缺血 30 分钟后上调，随后再灌注 2 小时13。中性粒细胞以前被认为是 IRI32 后浸润肺部的中心细胞类型，但我们发现单核细胞在到达肺部时先于中性粒细胞18,24。最近，我们发现 B 细胞甚至先于单核细胞，并在再灌注后 1 小时内进入肺部（未发表的数据）。当采用这种技术时，可以根据研究问题和假设调整热缺血和再灌注的持续时间。在不担心过多的液体和热量损失的情况下，可以进行更长时间的热缺血，因为在此期间胸部可以闭合。
围手术期可能会出现几种并发症，最终的常见结果是围手术期死亡。我们观察到，手术期间动物死亡的发生率随着外科医生重复该技术而降低。我们估计外科医生至少需要 2 个月的实践和/或至少 20 个病例才能达到技能平台。在这一点上，我们估计术中死亡率约为 1/50。麻醉小鼠的死亡通常预示着痛苦的呼吸，这种呼吸涉及横膈膜和胸壁肌肉组织的有力收缩。在这些痛苦的呼吸开始时，如果不采取干预措施，将在 3-5 分钟内死亡。有几种原因可以诱发术中死亡，其中许多是可以识别和预防的。我们将在下面重点介绍该手术的关键步骤和故障排除技术，以实现成功的结果。
插管 第一个关键步骤是小心插管并固定气道，以防止意外拔管。在生存手术中，确保声带在初次插管期间不会受损尤为重要，因为拔管后小鼠将无法呼吸。ETT通过脊髓的推进应通过显微镜直接观察进行。如果在插管过程中遇到阻力，不应盲目推进 ETT。在用蚊子夹保持口咽部张开的同时，应注意不要刺穿口腔粘膜，因为血液会遮挡气道的视线。插管尝试次数应限制在五次以下。
意外拔管 如果在手术过程中的任何时候，肺部似乎没有适当地充气，则可能是 ETT 已经脱落。意外拔管的另一种长时间被忽视的表现是小鼠突然出现呼吸困难。初次插管后，应注意用丝带将 ETT 沿圆周固定在鼻子上，并应限制小鼠颈部和躯干的调整。如果左肺和右肺都没有充气，则 ETT 可能已经从气管中出来。可以尝试将 ETT 推回，但是可能需要迅速重新插管小鼠，以避免动物过早死亡。如果只有一个肺在预计两个肺都充气时，则 ETT 可能太深，应小心地拉回并重新固定。
肺动员 左肺动员应使用棉尖涂抹器轻轻按压肺实质，因为压缩性损伤可能会损伤肺泡并降低肺的顺应性。不应用镊子或夹子夹住肺部，因为这会损害肺实质。
肺门钳的应用 左肺门钳夹应置于直接视线下，注意不要将结放得太中心（以免夹住左心房）或太靠近周边（以免夹住任何肺实质）。用肺门钳夹住左心房可能会诱发心房颤动并随后死亡。通过确保足够的肋骨回缩以扩大视野，可以改善肺门的可视化。系好滑结后，通过执行肺复张操作来确保夹子完全闭塞是有用的 - 通过手动闭塞连接 ETT 和呼吸机的流出管，并确保左肺不会因正压而扩张。鉴于与软骨支气管相比，肺动脉和静脉的塌陷性增加，因此在确认支气管闭塞的情况下可推定血管闭塞。此外，肺门钳夹不应用力过猛，因为它会挤压左主支气管的软骨部分，并防止松开肺门钳夹后左肺扩张。一旦松开肺门钳夹，重要的是要通过执行另一次肺复张操作来检查肺是否仍然能够通气。
胸部闭合 闭合胸部时，当肺处于最大末末吸气时，应将第 4 和第5 根肋骨并拢。这将最大限度地减少胸膜腔中残留的空气量，一旦肋骨闭合，胸膜腔内就会导致气胸。肌肉层和皮肤应以密闭方式闭合，以避免张力性气胸的发展。
获取动脉血气组 最后，在获得动脉血气分析时，重要的是首先夹住右肺门，以便动脉血气分析结果仅反映左肺功能22,23。如果没有右肺门钳夹，正常通气的右肺将足以补偿受损的缺血性左肺，动脉血气分析结果将看起来正常。
综上所述，我们上面描述的肺门钳夹技术是研究肺移植的宝贵工具，重要的是可以在不引入同种异体性、手术吻合相关创伤或冷缺血贮积的情况下分离温 IRI 的研究。值得注意的是，这种所描述的肺门钳夹技术易于教授并被广泛采用，并且可以产生可靠和可重复的结果。

<a href="https://app-jove-com.remotexs.ntu.edu.sg/t/6603">This corrects</a> the article <a href="https://dx-doi-org.remotexs.ntu.edu.sg/10.3791/66232">10.3791/66232</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/66232">Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury</a>. The Protocol and Representative Results&#160;sections were&#160;updated.

Erratum: Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

器官移植期间的 IRI 是原发性移植物功能障碍和后续移植物排斥反应发作的主要危险因素 1,2。在移植过程中，热缺血时间定义为从供体主动脉交叉钳夹到开始冷灌注以及从器官从冰中取出到器官植入的时间段。冷藏时间定义为从开始冷灌注到器官从冰中取出的时间段3.温性缺血比冷性缺血对后期器官功能更有害 4,5,6，其潜在机制值得在临床前模型中进一步研究。此外，与传统的脑死亡后捐献 （DBD） 相比，心源性死亡 （DCD） 捐献的器官移植与更长的热缺血时间相关7。虽然使用 DCD 供体可以扩大供体库并提高肺利用率，但需要进一步的临床前研究来评估温缺血对移植后肺功能的影响。下面，我们描述了通过左肺门钳夹在小鼠中温 IRI 的模型。
在过去几年中，已经开发和适应了几种肺门钳夹的动物模型，可能包括使用无创伤微血管钳夹 8,9,10,11,12,13、Rumel 止血带 14,15 或缝合结扎16作为肺门夹。肺门钳夹的关键是它必须是可逆的，并且对肺门结构造成最小的损伤或不会造成损伤，以便实现再灌注。在这里，我们描述了我们在小鼠中的肺门钳夹技术，该技术涉及左肺门的可逆缝合结扎术，并用滑结结。这种方法阻塞了肺动脉流入、静脉流出和进出主支气管的气流。与血管夹、夹子或止血带相比，滑结的主要好处是，在长时间缺血期间，胸部可以闭合，从而最大限度地减少小鼠体内的不显性液体和热量损失。我们提供了一种方案，用于获得可靠的动脉血气 （ABG） 测量值和测量肺门钳夹后细胞外渗。
这种肺门钳夹技术在更广泛的肺移植研究中占有重要地位。与原位肺移植的小动物模型相比，肺门钳夹技术可以在不增加手术吻合口创伤或同种异体性的情况下隔离 IRI 的影响17。此外，与小鼠肺移植相比，肺门钳夹技术可以更容易、更快速地掌握。事实上，使用肺门钳夹技术，在过去十年中已经确定了IRI发病机制的几个重要机制，如TLR4、NADPH氧化酶和腺苷A2A受体14,18,19,20。在以下方案中，我们提出了一种可靠的、可教的和可重复的肺门钳夹方法，作为研究肺 IRI 的工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medications</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% povidone-iodine solution</td>
			<td>Aplicare</td>
			<td>NDC 52380-0126-2</td>
			<td>For disinfectant</td>
		</tr>
		<tr>
			<td>Buprenorphine 1.3 mg/mL</td>
			<td>Fidelis Animal Health</td>
			<td>NDC 86084-100-30</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Cronus Pharma</td>
			<td>NDC 69043-027-18</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Heparin 1000 units/mL</td>
			<td>Sagent</td>
			<td>NDC 25021-404-01</td>
			<td>For obtaining arterial blood</td>
		</tr>
		<tr>
			<td>Isoflurane 1%-1.5%</td>
			<td>Sigma Aldrich</td>
			<td>26675-46-7</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride 100 mg/mL</td>
			<td>Vedco</td>
			<td>NDC 50989-996-06</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Puralube Vet eye ointment</td>
			<td>Medi-Vet.com</td>
			<td>11897</td>
			<td>To prevent eye dessiccation</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Akorn</td>
			<td>NDC 59399-110-20</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Tools and Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Argent High Temp Fine Tip Cautery Pen</td>
			<td>McKesson</td>
			<td>231</td>
			<td>To coagulate blood vessels</td>
		</tr>
		<tr>
			<td>Curved mosquito clamp</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Fine curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>15040-11</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Intubation clamp set-up</td>
			<td>Fine Science Tools</td>
			<td>18374-44, 18144-30</td>
			<td>For holding mouse vertically by the tongue during intubation. See Supplementary Figure 1A.&#160;</td>
		</tr>
		<tr>
			<td>Magnetic rib retractors</td>
			<td>Fine Science Tools</td>
			<td>18200-01, 18200-10</td>
			<td>For retraction of thoracotomy. Magnetic fixator and retractor should be connected by micro latex tubing below.</td>
		</tr>
		<tr>
			<td>Optical Grade Plastic Optical Fiber Unjacketed, 500&#956;m</td>
			<td>Edmund Optics</td>
			<td>02-532</td>
			<td>To make the introducer for the endotracheal tube. See Supplemental Figure 1B. A 1.5-inch length of this optical fiber should have a piece of silk tape secured to one end. It can then be used as an introducer for the endotracheal tube. The end of the introducer should be curves slightly.</td>
		</tr>
		<tr>
			<td>Power Pro Ultra clipper</td>
			<td>Oster</td>
			<td>078400-020-001</td>
			<td>To clip hair</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14370-22</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Small animal heating pad</td>
			<td>K&#38;H Pet Products</td>
			<td>Thermo-Peep Heated Pad</td>
			<td>To maintain normothermia</td>
		</tr>
		<tr>
			<td>Small animal ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0000</td>
			<td>For ventilation (TV 0.35 cc, PEEP 1 cm H2O, RR 100-105/min, FiO2 100%)</td>
		</tr>
		<tr>
			<td>Spearit Micro Latex Rubber Tubing (1/8 in outside diameter, 1/16 in inside diameter)</td>
			<td>Amazon.com</td>
			<td>https://www.amazon.com/Rubber-Tubing-CONTINUOUS-Select-Length/dp/B00H4MT7V0?th=1</td>
			<td>For retraction of thoracotomy</td>
		</tr>
		<tr>
			<td>Stat Profile Prime Critical Care Blood Gas Analyzer</td>
			<td>Nova Biomedical</td>
			<td>https://novabiomedical.com/prime-plus-critical-care-blood-gas-analyzer/index.php?gad=1&#38;gclid=Cj0KCQjwmICoBhDx 
			ARIsABXkXlInZX--R3ezBkc304nS_GVGI9Z2T3Esr33 
			2aM8WGPiUVhicPQZ 
			Wj2AaAqhDEALw_wcB&#160;&#160;</td>
			<td>For retraction of thoracotomy</td>
		</tr>
		<tr>
			<td>Straight clamp</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Straight forceps</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td>For surgical procedure</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Wild Heerbrugg</td>
			<td>no longer produced</td>
			<td>For intubation and surgical procedure; recommend replacement with Leica surgical microscopes</td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#189; cc syringe with &#189; inch 29G needle</td>
			<td>McKesson</td>
			<td>942665</td>
			<td>For injecting ketamine/xylazine intraperitoneally</td>
		</tr>
		<tr>
			<td>&#189; inch 31G needle on a 1 cc tuberculin syringe</td>
			<td>McKesson</td>
			<td>16-SNT1C2705</td>
			<td>For aspiration of arterial blood from left ventricle</td>
		</tr>
		<tr>
			<td>1-inch 20G IV catheter</td>
			<td>Terumo</td>
			<td>SROX2025CA</td>
			<td>For endotracheal tube (ETT)</td>
		</tr>
		<tr>
			<td>1-inch silk tape</td>
			<td>Durapore</td>
			<td>3M ID 7100057168</td>
			<td>To tape ETT to nose and to secure limbs</td>
		</tr>
		<tr>
			<td>3/10 cc syringe with 5/16 inch 31G needle</td>
			<td>McKesson</td>
			<td>102-SN310C31516P</td>
			<td>For antibody injection into the inferior vena cava</td>
		</tr>
		<tr>
			<td>6-0 monofilament suture on a P-10 needle</td>
			<td>McKesson</td>
			<td>S697GX</td>
			<td>For closure of thoracotomy, muscle layer, and skin</td>
		</tr>
		<tr>
			<td>6-0 silk tie</td>
			<td>Surgical Specialties Look</td>
			<td>SP102</td>
			<td>To make slipknot for hilar clamp</td>
		</tr>
		<tr>
			<td>Pointed cotton-tipped applicators</td>
			<td>Solon</td>
			<td>56225</td>
			<td>To manipulate lung and for blunt dissection</td>
		</tr>
	</tbody>
</table>

murine left pulmonary hilar clamp model of lung ischemia reperfusion injury

肺移植期间的缺血再灌注损伤 （IRI） 是移植后并发症的主要危险因素，包括原发性移植物功能障碍、急性和慢性排斥反应以及死亡率。研究 IRI 基础的努力导致了可靠且可重复的左肺门钳夹小鼠模型的开发。该模型涉及在麻醉和插管小鼠中进行的外科手术。进行左胸切开术，然后仔细进行肺活动并清扫左肺门。肺门钳夹术涉及用滑结对肺门进行可逆缝合结扎术，以阻止动脉流入、静脉流出和气流通过左主支气管。再灌注是通过小心地移除缝合线开始的。在目前的研究中，我们的实验室使用 30 分钟的缺血和 1 小时的再灌注作为实验模型。但是，这些时间段可以根据特定的实验问题进行修改。在处死前，在右肺门钳夹 4 分钟后，可以从左心室获得动脉血气，以确保获得的 PaO2 值仅归因于受伤的左肺。我们还描述了一种用流式细胞术测量细胞外渗的方法，该方法涉及在处死前静脉注射对要研究的细胞具有特异性的荧光染料标记的抗体。然后可以收集左肺进行流式细胞术、冷冻或固定、石蜡包埋的免疫组织化学和定量聚合酶链反应。这种肺门钳夹技术可以详细研究 IRI 背后的细胞和分子机制。代表性结果显示肺门钳夹术后左肺氧合减少和肺损伤的组织学证据。无论有没有显微外科经验的人员都可以很容易地学习和复制该技术，从而获得可靠和一致的结果，并作为研究肺部 IRI 的广泛采用模型。

左肺门钳夹后，归因于左肺的动脉血氧合分压 （PaO2） 为 ~100 mmHg，与假开胸手术后的 ~500 mmHg 相比显着降低（图 7A，n=6-7）。值得注意的是，在 B6 小鼠中进行了假开胸手术，在右肺门钳夹 4 分钟后进行 ABG 测量，代表仅归因于左肺的值。肺门钳夹左肺的H&E染色显示浸润的炎症细胞充满气道和血管充血红细胞，而这些发现在幼稚的左肺中不存在（图7B，n = 2）。这些结果表明，在一段时间的缺血和再灌注后，立即出现组织损伤和细胞浸润。
如步骤 6 中所述，血管内和间质中性粒细胞可以通过在处死前 5 分钟静脉注射 FITC 标记的抗 Ly6G 抗体来区分，然后用 APC-Ly6G 抗体标记所有中性粒细胞18,24。代表性图显示，~75%的中性粒细胞在血管内（APC-Ly6G + FITC-Ly6G +）和~25%的中性粒细胞在左肺门钳夹后为间质（APC-Ly6G + FITC-Ly6G-）（图7C，n = 4）。这些结果表明，中性粒细胞外渗是评估中性粒细胞外渗的可靠方法，并且可以根据所使用的抗体（即，单核细胞的抗Ly6C，B细胞的抗CD19）适用于其他细胞类型。该技术可用于研究 IRI 后的细胞动力学。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66232/66232fig1v2.jpg"> 图 1:小鼠插管。 （A） 左胸部和腹部剃光的小鼠。（B） 气管插管与呼吸机导管相连。（C） 确认插管后，将管子沿圆周方向粘在小鼠的鼻子上。（D） 插管小鼠，具有从小动物呼吸机进出和流出的管道。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig1v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66232/66232fig02.jpg"> 图 2:左侧开胸手术。 （A） 第 4肋 间隙 （ICS） 的皮肤切口。（B） 肌肉层分布在第 4 肋 间隙。（C） 在第 5根 肋骨上方做一个小切口。（D） 在第 4个 ICS 中向前和向后扩展切口。（E） 在第 4个 ICS 中可见的肺。（F-G）放置了肋骨牵开器。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66232/66232fig03.jpg"> 图 3:左肺门钳夹。 （A） 用棉签活动左肺。（B） 肺下韧带 （IPL） 可见。（C） 左肺前翻转，将丝带置于肺门后方。（D） 左向后翻转在丝绸领带上，可以看到前肺门。（E） 左肺门上的器械打结系带。（F） 肺门上的滑结。（G） 手动闭塞流出物时，左肺不充气，表明肺门钳夹成功。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66232/66232fig04.jpg"> 图 4:松开左肺门钳夹和左胸部闭合。 （A） 松开滑结后，左肺重新充气。（B） 肋骨闭合，在第 4根 肋骨上方咬一口，（C） 在第6 根肋 骨上方咬一口。（D-E）胸部闭合，在吸气结束时缝合一针。（F） 用断续缝合进行皮肤闭合（在用流缝合缝合肌肉层后）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66232/66232fig05.jpg"> 图 5:剖腹手术、双侧翻盖式开胸手术和动脉采血。 （A） 将小鼠调整到仰卧位。（B） 在腹部中线做皮肤切口。（C） 剖腹手术从剑突延伸到耻骨，沿最下肋骨向左和向右延伸。（D-E）横膈膜开口进入胸腔，横膈膜切口沿最下肋骨（虚线）左右延伸。（F） 双侧翻盖式开胸手术，在前腋线（虚线）朝向胸部顶点切开。（G） 右肺副肺叶延伸到下腔静脉后方的左胸。（H） 副肺叶活动并返回右胸。（I） 左心室动脉血抽吸（插图:右心室和左心室之间的色差）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66232/66232fig06.jpg"> 图 6:静脉注射到下腔静脉。 （A） 右内侧内脏旋转以暴露下腔静脉 （IVC）。（B） 将溶液注射到 IVC 中。（C） 对 IVC 施加压力以达到止血效果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66232/66232fig07.jpg"> 图 7:左肺门钳夹后肺氧合、组织学和中性粒细胞外渗的代表性结果。 （A） 接受假开胸手术的 C57BL/6 小鼠动脉血中的氧分压与肺门钳夹。结果以 SEM ±平均值表示，n=6-7。通过 Mann-Whitney U 检验计算的 p 值。**第 <0.01 页。（B） 接受假开胸手术与肺门钳夹的 C57BL/6 小鼠福尔马林固定、石蜡包埋肺切片的 H&E 染色。比例尺代表 250 μm。 （C） 肺门钳夹后 C57BL/6 小鼠血管内 （APC-Ly6G+FITC-Ly6G+） Ly6G+ 中性粒细胞分布的代表性图。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/66232fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图1: 气管插管导引器。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66232/Supplemental_Figure_1_updated.pdf" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation at JoVE.com

作者聚焦:缺血再灌注对肺移植影响的见解

该协议概述了一种可行、可靠和可重复的左肺门钳夹方法，可用于研究小鼠模型中的肺缺血再灌注损伤。

小鼠左肺肺门钳夹模型治疗肺缺血再灌注损伤

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft ...

我们有兴趣研究缺血再灌注损伤对肺移植后结果的影响，目的是了解潜在的细胞和分子机制。进入肺部的中性粒细胞会导致缺血再灌注损伤（IRI）。我们最近发现 B 细胞使中性粒细胞进入肺部，它们在 IRI 后中性粒细胞的募集中发挥作用。小鼠左肺移植在技术上具有挑战性，需要持续数月的全职、专门培训才能获得一致的结果。此外，它还会引入致敏性和手术吻合口创伤。因此，我们需要一种更易于获得的技术，可以隔离缺血再灌注损伤的研究。使用我们的肺门钳夹模型，我们发现 B 细胞在缺血再灌注损伤后早期进入肺部，并且它们将 B 细胞受体中的经典单核细胞募集到 TLR4 依赖性方式。这最终导致中性粒细胞外渗到肺部的增加。我们的方案涉及左肺门的可逆缝合结扎术，并用滑结结扎。缝合线的柔韧和不显眼的性质允许胸部闭合，这对于研究较长时间的热缺血很有用，因为胸部闭合可以最大限度地减少小鼠的热量和不显性液体损失。

murine-left-pulmonary-hilar-clamp-model-lung-ischemia-reperfusion

Nanyang Technological University

Research

JoVE Journal

Medicine

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

1.2K 次观看.  Washington University, St. Louis. 我们有兴趣研究缺血再灌注损伤对肺移植后结果的影响，目的是了解潜在的细胞和分子机制。进入肺部的中性粒细胞会导致缺血再灌注损伤（IRI）。我们最近发现 B 细胞使中性粒细胞进入肺部，它们在 IRI 后中性粒细胞的募集中发挥作用。小鼠左肺移植在技术上具有挑战性，需要持续数月的全职、专门培训才能获得一致的结果。此外，它还会引入致敏性和手术吻合口创?...

视频: 作者聚焦:缺血再灌注对肺移植影响的见解

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

常温心脏骤停和心肺复苏术:缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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 ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

温缺血/再灌注损伤的大鼠肺移植模型:改善结局的优化

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

单侧肾缺血再灌注损伤的有效小鼠模型

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

心肌缺血再灌注损伤的改进啮齿动物模型

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...