Name: bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury
Uploaded: 2018-05-21T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

作者宣布没有利益冲突。

总的协议需要2天, 包括第一天的败血症诱导和分离从动物的球液, 第二天的外来体隔离从老鼠 BALF。所有程序都已由亚特兰大 VA 医疗中心的机构动物护理和使用委员会审查和批准。
1. 小鼠急性脓毒性肺损伤模型
<ol><li>使用 6-8 周-老雄性野生型 C57BL/6J 小鼠 (重量 20-22 克) 的动物模型, 并执行所有程序使用无菌技术和仪器。高压釜所有手术器械, 包括镊子和剪刀, 20 分钟121摄氏度。</li>
	<li>将小鼠随机分布到实验和对照组。在手术板上抑制老鼠。用腹腔途径治疗小鼠与大肠杆菌脂多糖 (LPS, 15 毫克/千克). 根据个人体重, 稀释适当的 lps 剂量为每只老鼠0.1 毫升 PBS, 然后注射 lps 溶液使用1毫升注射器与27克针和注射一个同等数量的 PBS 控制动物。</li>
	<li>保持老鼠在动物 BSL2 设施, 其中包括干净的笼子在适当的室温。</li>
	<li>24小时后, 弄死小鼠与 CO2吸入。</li>
	<li>选择鼠标颈部中心的手术部位。做一个小切口 (大约5毫米), 并轻轻地分开肌肉, 使用钝钳进入气管, 直到气管暴露。</li>
	<li>用27克针提取10毫升无菌注射器的小鼠球液。首先, 轻轻地, 插入针入气管和注射1毫升的不育 PBS 慢慢进入气管, 然后绘制 BALF 成一个3毫升一次性注射器与27克针。程序完成后, 在生化利器容器中处理注射器和针头。</li>
	<li>用二氧化氯治疗外科器械, 如前所述的 Chauret et 。27, 然后用热处理20分钟在121摄氏度消毒所有手术器械。</li>
</ol>2. 外来体隔离-串联超离心和表征
<ol><li>收集15毫升锥形管中的小鼠球液。为了收割肺泡细胞, 离心机样品10分钟在1000克, 在4摄氏度。</li>
	<li>将上清到新的15毫升圆锥管, 然后离心样品30分钟在 4000 x g 在4°c 去除细胞碎片。</li>
	<li>收集在 ultracentrifuge 管和离心样品60分钟在 1万 x g 4 摄氏度的上清, 以消除较大的细胞颗粒。</li>
	<li>将上清液转移到新的锥形管, 然后通过0.2 µm 注射器过滤器过滤样品, 以除去剩余的较大颗粒。</li>
	<li>收集 ultracentrifuge 管和平衡管的样品。离心样品2小时, 在 14万 x g 在 4°c, 以颗粒的外来体。 注: 外来体的大小变化为 20-200 nm。0.2 µm 过滤器用于去除大小超过200纳米的较大粒子。</li>
	<li>轻轻丢弃上清液。用100µL PBS 或溶解溶液溶解颗粒, 并将外来体样品转移到1.5 毫升管, 并贮存在-80 摄氏度直至使用。 注: 建议使用透射电镜验证隔离外来体。</li>
	<li>按照标准 TEM 示例处理协议28。用透射电镜分析外来体样品, 并拍摄图像, 缩放条 200 nm。</li>
</ol>3. 制备 miRNA 和执行 miRNA-q-rt-pcr
注: miRNA-q-rt-pcr 是分析 miRNAs 表达水平的建议。
<ol><li>遵循标准的 miRNAs 隔离协议, 并确保在单链反向转录中使用等量的纯化 rna24。</li>
	<li>按照标准的实时 PCR 协议检测 exosomal microRNA 水平。使用特定的实时 PCR 引物对 miR-155, miR-146a 和 U6 SnRNA。用定量 pcr 仪进行 pcr 反应, 将表达值规范化为内部控制 U6 snRNA。 注: 每个组由三个样本24组成。</li>
</ol>4. 外来体标记和纯化
<ol><li>悬浮外来体颗粒与100µL 悬浮液在1.5 毫升聚丙烯管, 然后添加0.4 µL 的外来体染料进入另一管100µL 的悬液。</li>
	<li>快速添加外来体溶液中的染料溶液, 并将其彻底混合吹打。在室温下保持样品5分钟, 免受光照。</li>
	<li>在1.5 毫升洗脱管中放入一个外切旋转柱。将解决方案混合物转移到外切旋转列的中心。</li>
	<li>在室温下, 在 800 x g 处旋转柱2分钟。</li>
	<li>丢弃该列并将洗脱样本存储在-20 摄氏度, 直到使用。</li>
</ol>5. 接受方细胞外来体的验证
<ol><li>种子 1 x 104在8井室幻灯片中的 12/单元格, 以及在37°c、5% CO2上的培养单元。</li>
	<li>在细胞中添加10µL 荧光标记的外来体。在37摄氏度孵育1小时。</li>
	<li>去除培养基, 用 PBS 冲洗细胞, 室温下固定1% 多聚甲醛的细胞10分钟。</li>
	<li>用 DAPI 装入装入介质中的幻灯片。</li>
	<li>通过荧光显微镜分析样品, 拍摄图像, 放大40X。</li>
</ol>6. 验证外来体与接收细胞之间的相互作用
<ol><li>种子 2 x 105 MLE12/well 在包含适当培养基的12井培养板中, 在37°c、5% CO2上培养细胞。</li>
	<li>将20µg 外来体从小鼠的体液中添加到 MLE12 细胞中, 并建立每个实验组的复制。</li>
	<li>24小时后, 用 PBS 冲洗细胞, 并将600µL 溶解缓冲剂放入井中。轻轻摇动盘子, 在1.5 毫升管内用吸管收集裂解样品。</li>
	<li>遵循标准的总 RNA 隔离协议和 cDNA 反向转录协议, 完成单链 cDNA 反向转录29。</li>
	<li>按照标准实时 PCR 协议检测收件人单元格中的 mRNA 表达式级别29。使用特定的实时 PCR 引物抗肿瘤坏死因子α, IL-6 和 ZO-1。用定量 pcr 仪进行 pcr 反应, 将表达值规范化为内部控制 GAPDH。 注: 每组由三份样品组成。</li>
</ol>

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这项工作得到 VA 价值审查2I01BX001786-06A1 资助 RS 的支持。

小鼠疾病模型通常用于评估特定基因的生理功能, 并降低实验费用2。这里描述的急性脓毒性肺损伤模仿人与 ARDS 的炎症反应。该模型与研究分子发病机制、生物标志物的发展以及测试潜在的新疗法5有关。
共培养系统与体内研究有关, 它是在对体外系统23、30中的生物分子 (如 RNA、DNA、脂质和蛋白质) 转移的潜在机制进行调查的背景下进行的。其中一些平台在评估机械理解和治疗复杂疾病疗法方面有很大效用22,23。如下所示, 外来体从灌洗细胞 (噬细胞, 中性粒细胞) 可以通过转移外来体影响结构上皮细胞的完整性。
外来体的 pathobiological 效应从诊断和治疗的角度和理解疾病的发病机制 (31, 32) 中都成为人类疾病的重要方面.为了将外来体从生物流体中分离出来, 最常用的技术包括差速离心和离心, 如下所述 33, 34.这项技术有几个优势, 虽然是有限的事实, 它可能是耗时和污染的风险与非 exosomal 蛋白。已经提出了新的方法来克服这些限制, 如外切隔离的密度梯度或免疫亲和性或大小依赖的方法35。外来体在临床研究中被调查为诊断和预后的标志物, 以及靶向治疗的发展。

阿里和 ARDS 是一种危及生命的呼吸衰竭, 其严重的低氧血症由非心源性肺水肿引起, 每年全球约有100万人感染1。急性呼吸窘迫综合征的病因包括感染或吸入对肺部的直接损伤和各种间接侮辱。在过去的十年中, 人们对 ards 的分子发病机制有了越来越多的了解, 但是, 针对 ards 的特定靶向治疗还有待开发2,3。
建立了几种急性肺损伤动物模型, 为人类研究的实验治疗提供了一座桥梁4,5。通常使用的模型包括油酸、细菌、LPS 和博莱霉素的本地安装。其他方法包括缺血再灌注、盲肠结扎穿刺、机械通气引起的伸展损伤、高氧或系统性细菌的管理和 LPS5。这些模型提供了一个有用的生物学系统来测试临床假说和发展潜在的治疗方法。为了模拟人 ARDS, 动物模型应重现炎症和急性损伤的上皮和内皮细胞的缺陷, 在屏障功能的肺部。
外来体是 20-200 nm 直径的膜泡, 其分子含量包含蛋白质、DNA、RNA 和脂质, 并通过分子组成转移促进组织微环境中的细胞间通信。外来体由多种类型的细胞分泌, 如内皮细胞、上皮细胞、平滑肌细胞和肿瘤细胞, 并存在于人体体液中。研究表明, 外来体调节免疫细胞和基质细胞在传染性和无菌炎症性疾病中的交叉交谈, 其异常释放似乎受到各种自然和实验性刺激的调节, 在生理和病理过程6。这种通信网络可能在肺部疾病的发病机制中发挥重要作用, 并可能影响病理生理学进展7,8。作为 18-22 核苷酸非编码 rna, miRNAs 存在于组织和体液, 血浆, 血清和调制 mRNA 表达在后平移水平9,10。
包装 miRNAs 在外来体影响分化和多种类型细胞的功能, 过多的水平是与各种疾病, 包括癌症, 肺部疾病, 肥胖, 糖尿病和心血管疾病, 11, 12,13,14,15,16。进入接受细胞和穿梭 exosomal miRNAs 促进细胞间通讯, 改变微环境的止血17,18。急性肺损伤是一个复杂的过程, 涉及多细胞类型与广泛的细胞间通讯通过外来体 8.miR-155 和 miR-146a 共享共同的转录调节机制, 并有助于炎症反应和免疫耐受性19,20。最近的研究表明, 两种调节炎症反应通过 exosomal miRNAs 穿梭在免疫细胞之间的21。然而, exosomal miRNAs 对内毒素的肺泡反应的调节作用的分子机制尚不清楚, 毫无疑问, 潜在的临床相关性和转化暗示值得进一步研究。
共用培养模型用于定义复杂环境中特定细胞类型的相互作用, 如炎症和癌症22,23。这些平台提供了一个替代的策略, 以询问细胞类型之间的交叉谈话, 特别是免疫和结构细胞。
内毒素的气管内、雾化、腹腔或全身管理被广泛用于诱发实验性肺损伤24,25,26, 并显示诱导上皮和内皮渗透性缺陷。在这里我们用腹腔内毒素诱导小鼠急性肺损伤的脓毒性模型。在内毒素腹腔内24小时内, 肺部出现通透性缺损, 并招募炎症细胞。此外, 我们还表明, 外来体从 miRNA-155 和 miR-146a, 和外来体从球液诱导促炎细胞因子表达的受体上皮细胞, 包括 IL-6 和 TNF α。这些数据是首次表明, exosomal miRNAs 是分泌的急性脓毒症肺损伤模型。

<table><tbody><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>Escherichia coli 055:B5</td><td></td></tr><tr><td>PBS pH7.2(1X)</td><td>Life technologies</td><td>20012-027</td><td></td></tr><tr><td>mirVana miRNA isolation kit</td><td>Thermo Fisher</td><td>449774</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher</td><td>4369016</td><td></td></tr><tr><td>mmu-miR-155 (002571) primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>has-miR-146a (000468) primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>U6snRNA primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>TNF-&amp;alpha;(Mm00443258_m1 ) primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>IL-6 (Mm00446190_m1&amp;nbsp;&amp;nbsp; )primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>ZO-1 (Mm00493699_m1&amp;nbsp; )primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>minimal essential medium (MEM)</td><td>Thermo Fisher</td><td>11095-072</td><td></td></tr><tr><td>Trysin-EDTA solution(0.05%)</td><td>Thermo Fisher</td><td>25300054</td><td></td></tr><tr><td>Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&amp;nbsp;</td><td>Sigma-Aldrich</td><td>MINI67-1KT</td><td></td></tr><tr><td>Exosome Spin Columns (MW3000)</td><td>Thermo Fisher</td><td>4484449</td><td></td></tr><tr><td>Beckman Coulter Optima L-100XP ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>L-100XP</td><td></td></tr><tr><td>Ultra-clear centrifuge tubes</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>Applied Biosystems 7500 Fast Real-Time PCR System</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>transmission electron microscopes (TEM)</td><td>JEOL Ltd</td><td>120 kV TEM</td><td></td></tr><tr><td>Olympus BX41 microscope</td><td>Olympus</td><td>BX41</td><td></td></tr></tbody></table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

急性肺损伤 (ALI) 和急性呼吸窘迫综合征 (ARDS) 是一种不同种类的肺部疾病, 持续高发病率和死亡率。阿里的分子发病机制得到了更好的界定;然而, 由于疾病的复杂性质, 分子疗法还有待开发。在这里, 我们使用脂多糖 (LPS) 诱导小鼠急性脓毒性肺损伤模型, 描绘了外来体在炎症反应中的作用。使用这个模型, 我们可以表明, 暴露于腹腔内 LPS 分泌外来体的小鼠从肺部的支气管肺泡灌洗 (球) 液中, 包裹着 miRNA 和细胞因子, 调节炎症反应。进一步使用共培养模型系统, 我们表明, 外来体释放的巨噬细胞扰乱紧张连接蛋白的表达在支气管上皮干细胞。这些结果表明, 1) 通过 exosomal 穿梭的先天免疫和结构细胞之间的交叉交谈, 有助于炎症反应和破坏的结构屏障和 2) 针对这些 miRNAs 可能提供一个新的平台, 以治疗阿里和 ARDS。

为诱发脓毒性肺损伤, 小鼠采用腹腔内 LPS (15 毫克/千克) 治疗。在 LPS 管理的24小时内, 中性的涌入出现在肺部, 如图 1A所示。绘制了小鼠球液, 然后分离纯化了外来体。用电子透射显微镜 (图 1B) 证实了 BALF 外来体的形态学。
从纯化外来体中检测 miRNA-155 和 miR-146a 的选择性促炎 miRNAs 的表达。用 LPS 治疗小鼠 miR-155 和 miR-146a 的表达水平明显增高, 与 PBS 治疗的对照小鼠相比 (miR-155, 1, 0.16 vs 11.6 @ 0.6; miR-146a, 1 @ 0.12 vs 13.5 @ 1.186) (图 2)。
为确定外来体、小鼠支气管上皮细胞 (MLE12 细胞) 的功能效应, 分别用 LPS 或 PBS 处理的小老鼠的自体液中提取纯化外来体。首先, 我们证明 BALF 外来体进入受体上皮细胞。BALF 外来体被标记为 PKH-67, 并与 MLE12 细胞共培养, 然后荧光显微镜图像显示外来体的摄取, 上皮细胞(图 3A)。与对照组 (TNF α, 1 0.13 vs 5.01) 相比, 上皮细胞中的炎症介导、TNF α和 IL-6 的表达显著增加;IL-6, 1 @ 0.02 vs 1.72 @ 0.17) (图 3B)。我们还确定了紧密连接蛋白 ZO-1 作为上皮完整性的替代品的表达。用 lps 治疗小鼠外来体治疗的细胞显示 ZO-1 的表达明显减弱, 与 LPS 治疗的控制小鼠 (ZO-1, 1 @ 0.12 vs 0.66 @ 0.13) ( 图3C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57737/57737fig1.jpg"> 图 1:对经 LPS 处理的小鼠外来体液分离的特征。C57BL/6J 小鼠用腹腔内 LPS (15 毫克/千克) 注射 (n = 5), PBS 作为控制 (n = 5)。24小时后, 老鼠被安乐死, 并进行了体液。用 LPS 和 PBS 治疗小鼠肺组织的代表性图像, 显示急性肺损伤, 鳞片棒, 50 µm, A)。外来体从球液中提取。透射电镜 (TEM) 分析显示外来体, 鳞片杆, 200 毫微米, B)。<a href="/files/ftp_upload/57737/57737fig1large.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/57737/57737fig2.jpg"> 图 2: Exosomal miRNAs 在经 LPS 处理的小鼠体内分离的液中表达.C57BL/6J 小鼠受到腹腔内 LPS 的挑战。选择 microRNAs 的表达通过 q rt-pcr 和规范化的 U6 表达式.A) miR-155;B) miR-146a。n = 每组5只老鼠。学生考试 < 0.05。<a href="/files/ftp_upload/57737/57737fig2large.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/57737/57737fig3.jpg"> 图 3:外来体治疗小鼠上皮细胞炎性细胞因子和紧密连接蛋白的表达用 PKH-67 和荧光显微镜显示 PKH67 标记外来体 (绿色)、DAPI (蓝色) 和在外来体细胞中显示吸收 MLE12 的合并图像, 鳞片条, 50 µm, 如图所示, 外来体液衍生的。小鼠上皮细胞 MLE12 与脂多糖或 PBS 治疗的老鼠外来体分离。24小时后, 细胞被收获, 总 RNA 被分离。用 q-rt-pcr-PCR 法测定肿瘤坏死因子α和 IL-6 的表达, 规范化为 GAPDH 内控制。B)TNF α和 (C) IL6;(D) 用 q rt-pcr 测定 ZO-1 mRNA 的表达。* P < 0.05, 学生考试。<a href="/files/ftp_upload/57737/57737fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

暴露于腹腔内的 LPS 分泌外来体的小鼠支气管肺泡灌洗 (球) 液, 包装与 miRNAs。使用共培养系统, 我们表明, 外来体释放的球液扰乱支气管上皮细胞紧密连接蛋白的表达, 并增加支持炎症细胞因子的表达, 加重肺损伤。

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Nanyang Technological University

Research

JoVE Journal

Genetics

10.8K 次观看.  Atlanta VAMC. 暴露于腹腔内的 LPS 分泌外来体的小鼠支气管肺泡灌洗 (球) 液, 包装与 miRNAs。使用共培养系统, 我们表明, 外来体释放的球液扰乱支气管上皮细胞紧密连接蛋白的表达, 并增加支持炎症细胞因子的表达, 加重肺损伤。

视频: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

科研

免疫与感染

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

医学

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

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 ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

在合并臭氧和LPS诱导穆林急性肺损伤期间可视化肺细胞适应

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

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 ...

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. ...

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome (ARDS)

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% in severe cases. Further research efforts are needed to better understand the complex pathophysiology of this disease. There are different well-established animal models to induce acute lung injury but none has been able to adequately mimic the complex pathomechanisms of ARDS. The most crucial factor for the development of this condition is the damage to the alveolar capillary unit. The combination of two well-established lung injury models allow us to mimic in more detail the underlying pathomechanism. Bronchoalveolar lavage (BAL) leads to surfactant depletion as well as alveolar collapse. The repeated instillation of fluid volumes causes subsequent hypoxemia. Surfactant depletion is a key factor of ARDS in humans. BAL is often combined with other lung injury approaches, but not with a second hit followed by oleic acid injection (OAI) yet. Oleic acid injection leads to severely impaired gas exchange, a deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The OAI mimics most of the expected effects of ARDS consisting of extended inflammation of lung tissue with an increase of alveolar leakage and gas exchange impairment. A disadvantage of the combination of different models is the difficulty to determine the influence to the lung injury caused by BAL alone, OAI alone or both together. The model presented in this report represents the combination of BAL and OAI as a new double-hit lung injury model. This new model is easy to implement and an alternative to study different therapeutic approaches in ARDS in the future.

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome ARDS

Different, complex animal models exist to study the pathophysiology of acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage and oleic acid injection induced lung injury is suitable as a new double-hit animal model for studying the acute respiratory distress syndrome.

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% ...