Ravi Alluri and Hilliard L. Kutscher are supported by Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant 1T32GM099607.

所有的材料和设备需要收集和适当前程序组织的。该过程是要以无缝转换从一个步骤到下一个，以便提供一致的，可重现的数据进行的。该协议遵循的机构动物护理和使用委员会在水牛城退伍军人医疗中心设置机构政策。 注意:两个雄性和雌性C57BL / 6小鼠用于这个协议。有两性之间白蛋白漏出无显著差异。 1.盐酸准备<ol><li>使56.2毫盐酸无菌加入1N盐酸281微升至4.72毫升的无菌生理盐水（SNS）。滴定至pH值为1.25用1N盐酸（≈0 - 50μL）。 </li><li>使这个准备新鲜之前受伤。由于低pH SNS是不是缓冲它保证了盐酸伤害侮辱是短暂的。 </li></ol> 2.麻醉气管曝光技术注意:确保受伤过程与麻醉气体清除木炭过滤通风橱内进行。的无菌条件保养是必要的，因为这是一个生存手术。使用无菌手套和消毒的玻璃珠手术器械消毒所有仪器手术对每个动物之前。利用窗帘来隔离手术部位的建议保持无菌。使用异氟醚作为麻醉气体，因为它提供一个相对低的血液气体溶解度允许麻醉快速诱导，以及损伤后迅速恢复。 <ol><li>通过与在以2升/分钟的流速氧2.75％的异氟醚的室诱导麻醉开始。 </li><li>一旦老鼠被麻醉充分（不响应脚趾捏强大到足以不会破坏皮肤或引起深部组织损伤），暂停用1-0编织丝线缝合15厘米长的门牙优越鼠标在60仰卧位Ø倾斜的夹层板。&lt;/ LI&gt; <li>确保麻醉是通过鼻锥递送异氟烷在整个手术保持。对小鼠的眼睛适用兽医药膏，以防止干燥。 </li><li>用电动修剪剃颈部的腹侧部分和应用局部消毒剂溶液（ 即 ，聚维酮碘）。用纱布去除多余的防腐剂，让干燥（约15 - 30秒）。 </li><li>渗透颈部从胸骨切迹到颚的劣部分与100μL的0.5％布比卡因中线（以提供预，围，和手术后止痛）。 </li><li>做一个1 - 用手术刀或弯曲精剪颈部1.5厘米纵向正中切口。有2个弯曲的锯齿状钳穿透钝性分离唾液腺之间的筋膜膜，露出气管（由白色软骨环标识）。 </li><li>依然采用了2锯齿钳，把握气管旁肌肉的一侧，它拉边，而亩之间解剖第一百纤维气管纵向下一步。 </li><li>一旦切割平面的建立，继续缩回气管旁肌肉作为第二钳子是气管下工作。使用这些镊子1-0编织丝线缝合的15厘米的链放置到钳子的前端与拉通过放置气管背后的缝合。 </li></ol> 3.气管内滴入程序<ol><li>通过填写附着到22克针用0.2毫升的空气的0.5毫升的注射器接着3.6毫升/公斤的盐酸制备损伤滴注注射器。空气将充当在车辆的分散，以及一个肺泡复张，以帮助对麻醉给药期间可能经历肺不张的肺泡。 注:按照有关手术后镇痛当地IACUC的指导方针，并与当地兽医咨询。请小心使用任何止痛药与雇用前抗发炎的特性ING无菌肺损伤的这种模式。 </li><li>要管理受伤，第一停止异氟醚管理。让鼠标的麻醉深度上涨约10 - 15秒，直到它开始镊脚趾捏反应。 </li><li>立即开始伤害滴注步骤。如果麻醉深度太深动物不会损伤滴注，这是含有盐酸损伤尤其如此后自发呼吸。 </li><li>有一个助理挤压胸腔驱逐肺活量。 </li><li>使用1-0编织丝线缝合围绕气管以前放置的优用它拉来牵引，然后将注射器针头插入1 和第 2 次喉软骨下方环之间气管。针斜面应面对医生和推进，直到斜面是刚刚过去的针与气管尽可能平行气管。 </li><li>只是单次给药前释放胸部盐酸以保证分散和沉积到远端气道和肺泡。存款丸大致0.5 - 0.75秒。保持针在气管直到第一自发呼吸取为保证整个损伤丸药体积被吸入到肺部。 </li><li>关闭切口使用是1 - 2皮肤钉或缝线。 </li></ol> 4.恢复<ol><li>用5毫升注射器用＆G针，注射1毫升SNS皮下脖子颈背液体复苏。虽然过程中最小的液体流失，手术后的鼠标往往不喝酒，并有可能脱水。可替代地， 通过腹膜内注射提供的SNS。 </li><li>在加热腔鼠标放置在37℃灌注100％O 2。确保鼠标被放回外壳之前充分的恢复。监测动物在此期间，直到门诊和充分R请勿不闻不问ecovered。 </li></ol> 5.支气管肺泡灌洗和肺处理<ol><li>诱发和按步骤2.1中所述维持异氟醚麻醉和步骤2.2。确认使用步骤2.2中所述脚趾捏法麻醉足够平面。 </li><li>固定在解剖板仰卧位置并使用钩在优越门齿1-0编织丝线缝合的一个循环来延伸颈部。对眼睛应用兽医药膏，以防止干燥。 </li><li>去除颈部皮肤钉后，请通过从下腹壁皮肤正中纵形切口，通过前面的颈部切开用剪刀下颌骨。 </li><li>使用剪刀腹腔肌肉正中切口，并收回与海绵腹腔脏器暴露腹膜后间隙。如果需要的血液，收集此时腹主动脉或下腔静脉。 </li><li>由横断abdom通过安乐死放血的动物INAL主动脉和下腔静脉。通过利用在下面的步骤双边开胸安乐死的二次验证方法，通过IACUC准则，将建立以人道完成。 </li><li>穿孔隔膜造成肺萎陷。然后使用骨骼切割剪刀切掉膜片，并通过笼平行肋切割和胸骨的两侧。使用的锁定止血缩回胸骨襟翼以方便注射5毫升温暖（37℃）的Hank平衡盐与钙和镁溶液进入右心室冲洗肺脉管。 <ol><li>作为得到从主动脉血液之前本文中描述的方法评估周边细胞因子水平，使得在左心室的小切口，以提供必要的流出血液离开循环到足够冲洗肺脉管系统如果不能获得血。 </li></ol></li><li>道具夹层板60°倾斜，并使用日E工作步骤步骤描述2.2，以方便插入20国祥½"不锈钢在气管插管，安全的以1-0缝线。 </li><li> 水平放置的夹层板，并进行支气管肺泡灌洗（BAL）。 <ol><li>要执行BAL连接两个5毫升注射器到3路阀。填写一个针筒5毫升的SNS，然后拧入活塞的端口。安装一个空的5毫升注射器到第二端口，最后附上活塞的开放端口放置在步骤5.6气管内插管。灌输1毫升生理盐水到肺和灌洗第二注射器。 </li><li>重复5总共1毫升生理盐水灌注小心了气管插管保持安全且平行于气管，因此它不会撕裂气管。 </li></ol></li><li>用于组织学分析，导管插入气管如步骤5.7。通过在20 cm H2O的，用10％中性福尔马林缓冲液吹入24小时修复肺。 </li><li>删除每个肺叶carefully用剪刀和镊子由横断支气管肺门附近。在组织学盒各肺叶（共5个）将被石蜡包埋之前。切割5微米组织切片用切片机，并用苏木精和曙红使用标准组织学技术染色。 6形态特征也可以在组织学载玻片进行量化的严重性炎症。 4 </li><li>通过在4℃以1500 xg离心离心3分钟处理回收的BAL。分装在等效量的无细胞BAL上清液到2.0毫升离心管以供将来分析。存储在-80℃。 </li><li>分析通过ELISA白蛋白浓度，或感兴趣的任何其它生物产品的BAL。 11从BAL的沉淀的细胞进行计数，生成cytoslide标本，并与基于赖特-吉姆萨染色对白血球分类计数测定来评估肺&#39;染色炎症反应。 </li></ol>

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The authors have no competing financial interests to disclose.

<html>其目的是通过胃酸的愿望是非常类似于酸肺炎和急性呼吸窘迫综合征后续开发过程中发生在人类的病理生理发展的ALI动物模型。在开发的模型，我们选择了提供高吞吐量数据采集动物物种，由于其低成本，短生殖周期，并用大量的调查工具（ 即 ，单克隆抗体，转基因的菌株）一个很好的理解免疫系统。 ALI继人类尖刻地损害肺泡上皮细胞和血管内皮屏障功能产生?...

急性呼吸窘迫综合征的特点是广泛的肺部炎症和临床上被视为呼吸短促急性低氧血症。这些症状通常是煽动事件发生后不到24小时，如外伤，败血症，输血相关反应或愿望。它被定位于肺泡上皮细胞和血管内皮细胞，导致蛋白质漏出，随后透明膜形成中性粒细胞肺泡炎（ 即广泛的炎症）组织病理学特点。吸入被归类为化学性肺炎或吸入性肺炎。 1胃吸出的酸性成分有助于肺炎和偏好都制定了继发性细菌性肺炎。吸入性肺炎是ALI和ARDS的后续发展的主要风险因素之一。 2 胃愿望是定义为从T物料吸入急性事件他带或不带口咽部菌群到气管超出声带胃。在抽吸内容物可以包含低pH的胃流体，细菌，血液或食品颗粒。胃吸出患者常发生在重症监护病房（ICU），其中通常是在空腹状态，因此放置在一个质子泵抑制剂限制酸化胃内容物误吸。比起一般患者群高5倍 - 急性肺损伤在美国ICU的人口的发病率是2.5。 3遗憾的是，这些易患因素往往会导致胃内细菌过度生长的状态，可能会导致下面的一个愿望事件肺部更严重的后遗症，如胃的愿望是继发细菌性肺炎发展的一个独立危险因素（SBP） ，ALI和急性呼吸窘迫综合征。 胃抽吸具有两个主要部分组成:盐酸和胃内容物，其可以或可以不含有细菌或食物颗粒。在啮齿动物模型单独胃抽吸的酸成分产生一个初始炎症应答作为对气道上皮低pH的直接苛性损伤的结果。这之后是中性粒细胞浸润，并在4炎性反应 - 6小时。 4这两个因素最终导致的肺微血管完整性从而导致流体和蛋白质外渗进入肺泡和呼吸道破坏。要理解本病理生理学和进一步研究可能的治疗干预，开发和表征即阐明涉及的基本机制的动物模型是重要的。单独的酸性抽吸必须庞大或具有足够低的pH值以绕过呼吸树的缓冲能力和到达肺泡。如果这不发生，只发生瞬态上，传导气道损伤，从而不太可能导致ARDS的严重后遗症。 5 &lt;/ SUP&gt; 为了准确地与受伤的肺泡上皮仿真抽吸事件，绕过动物的天然防御很重要。利用小鼠模型的愿望，产生ALI模仿人类看到的胃酸的伤害，必须占气管支气管树的差异。开气管切开术，该方法利用绕过损伤重现ALI既生理学和组织学的方式鼠和人的呼吸的树木和模型之间的差异。从历史上看，气管内插管用于生成ALI，然而它被认为是困难的小鼠未经喉损伤来执行。因此，这种方法提供了已取得在多个研究人员和以最小的过程归因死亡率一致的结果的潜在替代。

<table><tbody><tr><td>syringe, 1 cc</td><td>Becton Dickinson</td><td>309628</td><td></td></tr><tr><td>syringe, 5 cc</td><td>Becton Dickinson</td><td>309646</td><td></td></tr><tr><td>needle, 22 G x 1 1/2&quot;</td><td>Becton Dickinson</td><td>305159</td><td></td></tr><tr><td>needle, 26 G x 1 1/2&quot;</td><td>Becton Dickinson</td><td>305111</td><td></td></tr><tr><td>1-O Braided Silk Suture</td><td>Harvard Apparatus</td><td>517730</td><td></td></tr><tr><td>3&quot; Curved tissue serrated forceps</td><td>Fine Science Tools</td><td>11065-07</td><td></td></tr><tr><td>3&quot; Curved tissue &quot;toothed&quot; forceps, 1 x 2 teeth</td><td>Fine Science Tools</td><td>11067-07</td><td></td></tr><tr><td>4&quot; curved micro dissecting scissors</td><td>Fine Science Tools</td><td>14061-10</td><td></td></tr><tr><td>bone cutting spring scissors</td><td>Fine Science Tools</td><td>16144-13</td><td></td></tr><tr><td>3 1/2&quot; curved locking hemostat</td><td>Fine Science Tools</td><td>13021-12</td><td></td></tr><tr><td>Disposable Skin Stapler</td><td>3M</td><td>DS-25</td><td></td></tr><tr><td>tracheal cannula (20 gauge x 1/2&quot; stainless steal tubing adapter)</td><td>Becton Dickinson</td><td>408210</td><td></td></tr><tr><td>60-degree Incline Dissection Board</td><td></td><td></td><td></td></tr><tr><td>0.5% Bupivacaine</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Betadine and &quot;Q-tip&quot; cotton applicator</td><td></td><td></td><td></td></tr></tbody></table>

open tracheostomy gastric acid aspiration murine model of acute lung injury results in maximal acute nonlethal lung injury

酸肺炎是无菌的急性肺损伤（ALI）的主要原因在人类。酸肺炎跨越临床谱从无症状到急性呼吸窘迫综合征（ARDS），其特征在于嗜中性肺泡炎，和伤害到两个肺泡上皮和血管内皮。在临床上，急性呼吸窘迫综合征是由低氧血症急性起病，双侧斑片状肺浸润和非心源性肺水肿定义。人类研究为我们提供了有关引起ARDS肺中的生理和炎性变化，这导致了对下属机制各种假说有价值的信息。不幸的是，在确定ARDS的病因，以及广泛的病理生理学的困难导致缺乏可能是在开发治疗策略有用的关键信息。 平移动物模型是有价值的，当他们发病机制和病理生理学准确reproducEA概念在体外和临床证实。虽然大动物模型（ 如羊）人的气管，支气管树的解剖结构的共同特征，小鼠模型提供的其他优势，包括主机:成本低;繁殖周期短借贷本身更大的数据采集;一个很好的理解免疫系统;和充分表征的基因组，导致的各种基因缺失和转基因的菌株的可用性。低pH诱导的急性呼吸窘迫综合征的鲁棒模型需要鼠ALI靶向主要肺泡上皮，其次的血管内皮，以及小气道导致肺泡。此外，不同的伤害性和非伤害性的侮辱之间的巨大差异可重复的伤害是很重要的。 用盐酸这里介绍的鼠胃酸误吸模型采用开放式气管切开术，并再现了致病场景，再现低pH pneumonITIS在人体的伤害。此外，该模型可用于研究低pH损伤与其它肺损害的实体（ 例如 ，食物颗粒，致病菌）的相互作用。

酸吸入性肺炎的小鼠模型的肺组织病理如上所述，并取出肺24小时后的低pH侮辱，切片，H＆E染色（ 图1）小鼠受伤。坏死细胞，肺实质结构，细胞和碎片空域内和显著PMN浸润的损失被清楚地观察到。类似于临床心愿，我们在产生斑片状浸润中性粒细胞和纤维蛋白蛋白沉积的区域远端肺空气空间的异质性损伤模型的结?...

Watch this Scientific Journal Video about Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury at JoVE.com

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

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

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 ...

open-tracheostomy-gastric-acid-aspiration-murine-model-acute-lung

Research

JoVE Journal

Medicine

9.7K 次观看.  University at Buffalo, State University of New York. This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

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

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

压力控制通气诱导小鼠急性肺损伤

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

科研

医学

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

单方支气管内酸灌输自我限制急性肺损伤模型

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

免疫与感染

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

油酸诱导的急性呼吸窘迫综合征小鼠模型

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

口腔插管和通气肺缺血再灌注手术小鼠模型

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

评估肺炎消退的实验模型

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

重症监护病房型装置在猪模型急性呼吸窘迫综合征中的卤化剂递送

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...

研究肺缺血再灌注损伤的小鼠模型

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows for&#160;detailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

作者聚焦:缺血再灌注对肺移植影响的见解

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse models.