Name: inducing acute lung injury in mice by direct intratracheal lipopolysaccharide instillation
Uploaded: 2019-07-06T15:00:00
Description: Watch this Scientific Journal Video about Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation at JoVE.com

作者感谢扬·克莱纳和苏珊娜·舒尔茨提供技术支持。作者肯定了波恩大学医学院对流式细胞学核心设施的出色支持。提交人没有得到任何外部组织的任何资助。 结果部分给出如图3所示的部分数据已在上一份出版物8中显示。

此动物协议由当地动物护理委员会(德国莱克林豪森的LANUV;第84-02.04.2015号协议)批准,并根据国家卫生研究院使用活体动物的准则(NIH出版物)执行第85-23号,1996年修订)。
1. 阿里公司感应
<ol><li>在10-12周的年龄使用成年C57BL/6小鼠。将动物安置在单独通风的笼子里,免费获得水和标准啮齿动物。然而,有可能对年轻的动物和其他小鼠菌株执行这种方法。</li>
	<li>在-20°C下以5mg/mL的浓度将...

<ol>
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微创性、简单处理和良好的可重复性是所呈现的方法的关键特征,用于在小型啮齿动物模型中诱导 ALI。在动物模型中使用LPS代替整个细菌具有优势。它是一种稳定和纯净的化合物,可以储存在冻干形式,直到使用。它是通过TLR4途径进行先天免疫反应的有力兴奋剂,其生物活性易于量化,促进疾病严重程度的滴定,具有良好的可重复性。此外,使用LPS已被证明作为安全模型,诱导急性支气管炎在人类健康志愿...

实验动物模型在基础免疫研究中不可或缺。管理整个细菌或微生物成分已经常用于小动物模型,以诱发局部或全身炎症1。脂多糖(LPS,或细菌内毒素)是一种细胞壁成分和克阴性细菌的表面抗原(例如,肠杆菌、伪多糖,或军团菌)。热稳定和大分子(分子量1-4 x 106 kDa)由脂质莫伊蒂(脂质A)、核心区域(寡糖)和O多糖(或O抗原)组成。脂质A,其疏水性脂肪酸链,锚定分子到细菌膜和调解(在细菌降解)的免疫活性和毒性的LPS。继与LPS结合蛋白(LBP)结合后,LPS:LBP复合物可使位于许多细胞类型的表面的CD14/TLR4/MD2受体复合物结合,通过NF-βB核易位和随后的上升调节诱导强烈的前列腺炎反应细胞因子表达2。
急性肺损伤(ALI)被定义为急性低氧呼吸衰竭与双边肺水肿,在没有心力衰竭3。LPS的气道管理是诱发肺部炎症和ALI4,5,6,7的常见方法。虽然无菌物质不适合研究传染病的药理干预,但机械免疫学问题可以足够精确地回答。LPS 注入气管诱导强大的炎症反应与白细胞入侵, 前列腺炎细胞因子的调节, 和中断的白细胞 - 毛细血管屏障在几个小时到几天内, 取决于 LPS 剂量3, 6,7.
提出的协议描述了通过内切液LPS灌输诱导小鼠的ALI的详细分步程序。该模型已经通过评估细胞因子表达、中性粒细胞细胞入侵和肺泡内白蛋白泄漏进行了验证,如前所述8。

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			<td height="21" style="height:21px;width:653px;">1 ml syringes</td>
			<td style="width:289px;">BD, Franklin Lakes, NJ, USA</td>
			<td style="width:104px;">300013</td>
			<td style="width:143px;"></td>
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			<td height="21" style="height:21px;">10 ml syringes</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>309110</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-CD115 (c-fms) APC</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>17-1152-80</td>
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			<td height="21" style="height:21px;">Anti-CD11b (M1/70) - FITC</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>11-0112-81</td>
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			<td height="21" style="height:21px;">Anti-CD45 (30-F11) - eF450</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>48-0451-82</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-F4/80 (BM-8) - PE Cy7</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>25-4801-82</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-Gr1 (RB6-8C5)</td>
			<td>BD Biosciences, Franklin Lakes, NJ, USA</td>
			<td>552093</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-Ly6C (HK1.4) PerCP-Cy5.5</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>45-5932-82</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-Ly6G (1A8) APC/Cy7</td>
			<td>Bio Legend, San Diego, CA</td>
			<td>127623</td>
			<td></td>
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			<td height="21" style="height:21px;">Buprenorphine hydrochloride</td>
			<td>Indivior UK Limited, Berkshire, UK</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">C57BL/6 mice, female, 10 - 12 weeks old</td>
			<td>Charles River, Wilmongton, MA, USA</td>
			<td></td>
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			<td height="21" style="height:21px;">CaliBRITE APC-beads (6&#181;m)</td>
			<td>BD Biosciences, Franklin Lakes, NJ, USA</td>
			<td>340487</td>
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			<td height="21" style="height:21px;width:653px;">Canula 23 gauge 1&#39;&#39;</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>300800</td>
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			<td height="21" style="height:21px;width:653px;">Canula 26 gauge 1/2&#39;&#39;</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>303800</td>
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			<td height="21" style="height:21px;">Cell strainer 70 &#181;m</td>
			<td>BD Biosciences, Franklin Lakes, NJ, USA</td>
			<td>352350</td>
			<td></td>
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			<td height="21" style="height:21px;">Collagenase Type I</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>1148089</td>
			<td></td>
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			<td height="21" style="height:21px;">Deoxyribonuclease II</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>D8764&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS), sterile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>D8662</td>
			<td></td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS), without calcium chloride and magnesium chloride, sterile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>D8537</td>
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			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA) solution</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>E7889</td>
			<td></td>
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			<td height="21" style="height:21px;">FACS tubes, 5 ml</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>551579</td>
			<td></td>
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			<td height="21" style="height:21px;">Fetal calf serum (FCS)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>F2442</td>
			<td></td>
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			<td height="21" style="height:21px;">Forceps</td>
			<td>Fine Science Tools, Heidelberg, Germany</td>
			<td>11049-10</td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Baxter, Unterschlei&#223;heim, Germany</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Ketamine hydrochloride</td>
			<td>Serumwerk Bernburg, Bernburg, Germany</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Lipopolysaccharides (LPS) from Escherichia coli O111:B4</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>L2630</td>
			<td></td>
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			<td height="21" style="height:21px;">LIVE/DEAD Fixable Dead Cell Green Kit</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>L23101</td>
			<td></td>
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			<td height="21" style="height:21px;">Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&#8482;), Clone 2.4G2</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>553141</td>
			<td></td>
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			<td height="21" style="height:21px;">Red blood cell lysis buffer</td>
			<td>Thermo Fisher, Waltham, MA, USA</td>
			<td>00-4333-57</td>
			<td></td>
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			<td height="21" style="height:21px;">RPMI-1640, with L-glutamine and sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>R8758</td>
			<td></td>
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			<td height="21" style="height:21px;">Scissors</td>
			<td>Fine Science Tools, Heidelberg, Germany</td>
			<td>14060-09</td>
			<td></td>
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			<td height="21" style="height:21px;">Sodium azide (NaN3)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>S2002</td>
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			<td height="21" style="height:21px;">Spring scissors</td>
			<td>Fine Science Tools, Heidelberg, Germany</td>
			<td>15018-10</td>
			<td></td>
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			<td height="21" style="height:21px;">Tissue forceps</td>
			<td>Fine Science Tools, Heidelberg, Germany</td>
			<td>11021-12</td>
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			<td height="21" style="height:21px;width:653px;">Tubes</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30125150</td>
			<td></td>
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			<td height="21" style="height:21px;">Venous catheter, 22 gauge</td>
			<td>B.Braun, Melsungen, Germany</td>
			<td>4268091B</td>
			<td></td>
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			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td>Serumwerk Bernburg, Bernburg, Germany</td>
			<td></td>
			<td></td>
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inducing acute lung injury in mice by direct intratracheal lipopolysaccharide instillation

脂多糖(LPS)气道管理是研究小动物模型中肺部炎症和急性肺损伤(ALI)的常用方法。已经描述了各种方法,例如吸入气溶胶LPS以及鼻腔或内气浸灌输。提出的方案描述了通过直接宫内LPS灌输诱导小鼠ALI的详细分步程序,并执行对血液样本、支气管性(BAL)液体和肺组织的FACS分析。腹管内镇化后,气管暴露,LPS通过22G静脉导管施用。根据使用的LPS剂量,在数到几天内诱导一种强健且可重复的炎症反应,其内为白细胞入侵、前列腺炎细胞因子的增调和白细胞屏障的中断。协议详细介绍了采集血液样本、BAL液体和肺采集以及FACS分析的处理。虽然无菌LPS的使用不适合研究传染病的药理干预,但所述方法提供最小的侵入性、简单的处理和良好的可重复性来回答机械免疫学问题。此外,剂量滴定以及使用替代LPS制剂或小鼠菌株允许调节临床效果,这可能表现出不同程度的ALI严重程度或早期与晚发病症状。

LPS灌输后,通过评估细胞因子表达、中性粒细胞渗透和肺毛细血管屏障破坏24小时和72小时后,通过评估小鼠诱导ALI的方法得到了验证。PBS注射的动物充当控制。内切术LPS管理诱导一个强大的肺前列腺炎反应。与对照动物相比,肺组织中TNF-α的表达明显增强,达到持续和超过50倍的增幅[RQ(TNF-+/18s);24小时:53.7(SD = 11.6);72小时:55.0(SD = 20.6);p <0.05)](图3A)。白细胞侵入组织和藻?...

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Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

这里介绍的是一个逐步的过程,通过直接的内切内脂多糖灌输诱导小鼠急性肺损伤,并执行FACS分析血液样本,支气管洗浴液和肺组织。微创性、简单处理、良好的可重复性和疾病严重程度的滴定是这种方法的优点。

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. ...

可靠的动物模型是基础研究的关键组成部分。我们的 murine 方法允许回答关于哺乳动物急性肺损伤发育的力学和动力学的免疫学问题。微创性、简单处理以及良好的可重复性是该技术的主要优点。此外，通过剂量滴定，我们可以调节临床效果。该技术反映了严重肺损伤患者的临床状况，并允许研究潜在的机制。在零下20摄氏度下，以每毫升5毫克的浓度将脂质多糖（LPS）储存在等物中。对于腹内注入，将无菌磷酸盐缓冲盐水中解冻的LPS稀释至每毫升2000微克的最终浓度。将 22 量的静脉导管切割到 20 毫米的长度。将鼠标放在温度控制表上的易发位置，以保持 37 摄氏度的体温。在文本协议中描述的麻醉后，应用无菌眼科润滑剂，以防止麻醉下角膜干燥。抬起头部和钩切口，在水平杆上放置约五厘米以上的水平杆，而前爪保持与桌子的密切接触。相对于桌子以 90 度角超长颈部。在喉部上方的皮肤上放置一个冷光源，以帮助可视化声带，并瞄准气管。用钳子握住舌头，拉直喉咙，以便于插管。正确可视化和识别喉部对于确保导管正确的腹内位置至关重要。外部冷光源有助于此步骤。将导管插入气管约 10 毫米。确保插入不会太深，因为这将导致液体单方面注入到右或左主支气管中。如果喉部发生阻力，请收回导管几毫米，然后再前进。现在，使用移液器注射LPS稀释的MPBS。注入的体积取决于鼠标的体重。连接注射器并添加 50 微升空气，以确保在肺部分布完整的液体体积。慢慢取出导管。将鼠标的上半身保持直立位置 30 秒钟，以避免气管中液体泄漏。用胶带将安乐死小鼠放在手术台上，并很快用70%乙醇对腹部的毛皮进行消毒。用剪刀和钳子小心地打开中线中的腹腔。取出部分肠道，以达到进入腹主动脉椎柱的维纳卡瓦下部的权利。找到肾静脉，并插入一个弯曲的，23规格的管，连接到一毫升注射器到静脉卡瓦下，直接在静脉汇合下方。吸气250微升的血液，并转移到1.5毫升管充满了20微升0.5摩尔EDTA溶液。轻轻摇动以方便 EDTA 混合，将管子放在冰上。对于支气管，准备三个一毫升注射器，每个注射器有0.5毫升的无菌PBS和0.1毫升的空气。用70%乙醇消毒喉咙的皮毛，用剪刀和钳子小心地暴露气管。调动气管，并环绕缝合线。用微剪刀刺穿气管，插入一个22规格的静脉导管，切割到20毫米的长度。用缝合固定导管，并注入0.5毫升无菌PBS和0.1毫升空气。60 秒后吸气液体。用额外的两个注射器重复该过程，并在冰的 15 毫升管中收集整个吸气。小心地用剪刀和钳子打开胸部，以收获肺部。沿着成本边线切割隔膜，用两个侧切口切开肋骨。小心避免刺穿肺部。颅骨抬起胸骨并固定或拆下。准备两个 10 毫升注射器，带 37 摄氏度的高温 PBS。在左心室做一个小切口。用 26 量的管刺右心室。然后，用预热的PBS冲洗肺循环。注意在手术过程中肺部变苍白。取出肺的右叶，切成两半。将它们冷冻在液氮中，然后长期储存在零下80摄氏度，以进一步进行基因表达和蛋白质分析。通过剪刀和钳子切碎组织，取出整个左肺并将其均匀化在48井的盘子中。在37摄氏度的消化缓冲液中孵育组织60分钟。通过小心地上下移液，进一步进行均质化。将血液样本转移到五毫升的FACS管中，将血液与两毫升红血球解液缓冲液轻轻混合。将管子放在冰上，两分钟后通过加入两毫升的冰冷PBS来终止反应。将样品在400倍g下离心5分钟，然后丢弃上一代。根据前面描述的协议，将细胞颗粒与 60 微升的 FACS 缓冲液重新填充，并处理后续 FACS 染色的过程。在 400 倍 g 下将 BAL 液体离心五分钟。吸上清液并冷冻在液氮中，然后长期储存在零下80摄氏度，进行进一步的蛋白质分析。在用两毫升冷 FACS 缓冲液重新悬浮 BAL 细胞颗粒后，使用 100 微米网状过滤器将悬浮液转移到五毫升 FACS 管中，以抑制头发。再次离心样品后，使用 60 微升的 FACS 缓冲液重新填充颗粒，并按照前面描述的协议进行后续 FACS 染色。现在，使用100微米网状过滤器将消化的左肺组织转移到5毫升的FACS管中，以提取团块。通过添加两毫升冰冷的 FACS 缓冲液来终止消化过程，然后以 400 倍 g 将样品离心 5 分钟。丢弃上流剂，再用 60 微升的 FACS 缓冲液重新填充颗粒，并按照前面描述的协议进行后续 FACS 染色。对于 FACS 分析，在 5 毫升管中的 60 微升细胞中加入 20 微升 CD16/CD32 抗体阻断溶液。在4摄氏度下孵育细胞15分钟，以阻止免疫球蛋白与Fc受体的非特异性结合。同时，准备与FACS缓冲液和抗体的主混合，如文本协议中所述。堵塞后，不要清洗细胞。每个样品添加 20 微升抗体母体混合，获得 100 微升的最终体积。在黑暗中，在4摄氏度下孵育样品20分钟。与以前一样，用一毫升的 FACS 缓冲液和离心机清洗每个样品。丢弃上一提液，用 FACS 缓冲液将颗粒重新暂停至适当的细胞浓度，以进行 FACS 测量。最后，在每个样品中添加固定数量的市售氟铬耦合校准珠，以确定绝对细胞数。使用血液、BAL 和组织细胞的文本协议中显示的浇注策略进行流式细胞测量。24以及72小时后，在脑内灌输，TNF-alpha在肺组织中的表达明显上升，达到持续和超过50倍的增长相比，对照动物。Leucocyte入侵组织和肺活量是急性肺损伤发展的标志和特征。FACS分析显示，嗜中性粒细胞大量渗入肺间，与24小时后对联细胞相比，绝对细胞数量增加了近9倍。绝对中性粒细胞计数在72小时后略有下降，但是，与对联相比，因子增加。与间质中性粒细胞渗透一致，MMP-9在全肺组织中的表达在总观察期内同样显著增加。嗜中性粒细胞不仅在肺组织中增加，而且在BAL液体中增加。与对照动物相比，折叠增加比肺组织更明显。肺水肿由于肺功能障碍的严重损伤是急性肺损伤发育的病理。ELISA对BAL流体中的白化粉含量进行分析后，在LPS注入后24小时72小时内，屏障功能显著丧失。正确的导管放置对于LPS的双边灌输至关重要。通常，呼吸模式的变化，如咳嗽或喘气，表明正确的胃内注入液体。在诱导急性肺损伤后，可以执行一些后续步骤，如 FACS 分析、PCR、蛋白质检测，以回答相应的研究问题。

Research

JoVE Journal

Medicine

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

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

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

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 ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

在诱导小鼠心肌梗死的微创手术过程

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

插管介导气管内（IMIT）灌输：一种无创，龙专用输送系统

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

灌输和固定方法有用的小鼠肺癌研究

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

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

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

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

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

聚丙烯酸/纳米水的气管内灌注积液模型

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

通过四氯化碳诱导大鼠急性肝损伤 （CCl4） 通过奥罗胃管暴露

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

期新生儿大鼠干细胞的内切内培养

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...