实验得到了 Marga 和基金会, Kerpen, 德国的支持;项目 210-02-16 (MAAA), 项目 210-03-15 (MAAA) 和由德国研究基金会 (DFG;AL1632-02;MAAA), 波恩, 德国;分子医学的中心科隆 (CMMC;大学医院科隆;职业发展计划;MAAA), 科隆财富 (医学学院, 科隆大学;KD)。

所有动物程序都是按照地方政府当局批准的议定书进行的 (土地北威州, 亚利桑那州: 2012. A424), 并符合德国动物福利法和关于动物福利的条例, 用于试验或为其他科学目的。由于肺功能分析可能会影响肺结构, 因此随后的组织学分析, 应在不同的动物中进行 Cdyn 的测定和肺形态计量学的制备和固定。然而, ipGTT 后的 Cdyn 的测量是可能的。由于 ipGTT 期间的压力可能会干扰肺功能测试所需的麻醉, 因此建议在 ipGTT 后大约2周的恢复期, 允许小鼠从体重损失和血液参数的变化中恢复12。
1. 腹腔葡萄糖耐受试验的准备 (ipGTT)
注: 禁食12小时后, 完整的 ipGTT 需要大约2小时。
<ol><li>由于压力对血糖的影响很大, 所以要保证小鼠的适应和科学家的训练。</li>
	<li>在安静和无应力条件下将动物转移到实验区。</li>
	<li>考虑使用 hypercaloric 饮食诱发小鼠肥胖。有关进一步建议, 请参阅讨论部分。</li>
	<li>快速动物在一夜之间12小时, 不限制获得水。第二天, 12 小时禁食后, 根据制造商的协议准备血糖表 (参见材料表), 方法是在测试条端口插入一个新的测试条。</li>
	<li>使用无菌剪刀切割尾部尖端, 同时轻轻地将鼠标保留在尾部, 并立即通过将自由流动的血滴 (最小样本大小0.5 µL) 应用于血糖表的试纸来测量禁食的血糖。 注: 在血液样本充分应用后, 在屏幕上开始倒计时计时器。4秒后, 测试结果显示在屏幕上。</li>
	<li>之后, 使用颜色标记分别对动物进行称量和标记。</li>
	<li>管理2克葡萄糖/千克体重通过腹腔注射。确保注射量为 0.1 mL/10 g 体重 (27 克针和 1 cc 注射器)。</li>
	<li>随后, 通过在新的试纸上应用一滴自由流动的血液来测量15、30、60和120分钟后的血糖。 注: 通过对尾部尖病房的温和按摩, 可以增加血流量。如果尾部伤口 encrusts, 用0.9% 氯化钠溶液浸泡的消毒拭子清洗。</li>
	<li>允许动物在他们的家庭笼子里休息, 在测量之间可以无限制地获得水。</li>
</ol>2. 肺功能分析测量 cDyn
注: 对于不受干扰的测定和 cDyn, 小鼠需要在深部麻醉下通风。无压力的动物处理和正确的麻醉监测是必不可少的。有关使用无菌技术的一般说明, 请 Hoogstraten-米勒et查看文章。15
<ol><li>在每组实验之前校准 plethysmograph, 并在软件中准备学习设置 (请参阅材料表)。</li>
	<li>手术前, 深麻醉动物通过腹腔注射甲苯噻嗪 (10 毫克/千克体重) 和氯胺酮 (100 毫克/千克体重) (27 克针和 1 cc 注射器)。确保注射量为每体重 0.1 mL/10 克。 注意: 由于氯胺酮对小鼠有适当的镇痛作用, 因此不需要额外的止痛治疗。侵袭性气管导管/plethysmograph 程序大约需要5-7 分钟, 然后才能开始数据采集。</li>
	<li>将鼠标放在加热垫上的仰卧位, 以保持体温。</li>
	<li>用药膏捂住眼睛, 以防麻醉时干燥。</li>
	<li>用脚趾捏-反应不断监测麻醉深度。 注: 额外的麻醉管理可能是必要的, 以维持手术平面的麻醉。</li>
	<li>用70% 乙醇滋润甲状腺区域的皮草。</li>
	<li>在胸骨的颈静脉切口和 mentum 的块茎 symphyses 之间, 小心地切开中线的皮肤1厘米, 用镊子将其抬起, 用钝剪刀在目视检查下修剪皮肤 (图 1A)。</li>
	<li>可视化皮下脂肪组织和甲状腺。</li>
	<li>通过仔细的钝器将甲状腺裂片与 sternothyroid 和 sternothyroid 肌肉的解剖分开, 使气管暴露出来 (图 1B)。小心不要伤害任何血管和导致出血, 因为这可能会对心血管系统造成不良影响, 最终对测量。</li>
	<li>随后, 用钝钳在气管和食道之间通过4-0 编织手术缝合。用微剪刀仔细切开气管软骨与喉部之间的气管。</li>
	<li>使用气管插管 (0.04 英寸/1.02 毫米直径) 在视觉控制下 (图 1C)。修复管通过结扎与手术缝合, 以避免任何泄漏的系统。</li>
	<li>接下来, 将动物移动到身体室的加热床上, 将气管管连接到面板 (图 1D), 然后按控制器前面板上的通风按钮 (图 1E) 打开通风。</li>
	<li>通过观察胸部运动作保和通气率来测量通气量。确认气管导管的正确放置, 确保胸腔两侧同时移动。</li>
	<li>监视计算机屏幕上的压力信号 (图 1F)。确保通风曲线均匀。如果情况并非如此, 请将动物分离并检查手术侧。当心血或粘液堵塞气管管。 注意: 对于体重为20-25 克的成年动物, 根据制造商的建议, 建议使用图 2中所示的通风机设置。</li>
	<li>为了控制通气过程中的反式肺压力的变化, 在接近肺部水平的深度处插入食道管 (0.04 英寸/1.02 毫米直径)。放置管道时, 注意屏幕。放置在屏幕上可以看到最大的压力偏转和最小的心脏工件的管。</li>
	<li>手术后, 准备动物的测量。注入麻醉通过腹腔注射氯胺酮 (100 毫克/千克体重) 使用27克针和 1 cc 注射器。确保注射量为每体重 0.1 mL/10 克。 注: 评估支气管 hyperreagibility, nebulize 乙酰甲胆碱, 一个非选择性毒蕈受体激动剂的副交感神经系统, 这诱导支气管收缩。数据采集是在四不同阶段执行的 (图 3)。</li>
	<li>根据 manufacturer´s 协议启动数据采集。 注意: 软件会自动引导用户完成采集过程。</li>
	<li>应用10µL 的 PBS (车辆) 在喷雾器, 并开始雾化后5分钟的驯化。接下来, 请遵循3分钟的响应阶段, 其中的 Res (cmH2o/毫升/秒) 和 cDyn (mL/cmH2O) 被测量。最后, 在下一个雾化之前, 为动物提供3分钟的恢复阶段。</li>
	<li>按照软件逐步应用10µL 增加浓度的乙酰甲胆碱 (2.5 µg/10 µL, 6.25 µg/10 µL, 12.5 µg/10 µL) 在通风机上。</li>
	<li>一旦所有的测量已经执行和记录, 牺牲的动物宫颈脱位。</li>
</ol>3. 肺隔离法定量分析成年小鼠的形态计量学
<ol><li>深麻醉动物通过腹腔注射甲苯噻嗪 (10 毫克/千克体重) 和氯胺酮 (100 毫克/千克体重) (27 克针和 1 cc 注射器)。注射量应为每体重 0.1 mL/10 克。 注: 在达到手术耐受状态后, 术后的准备需要大约5分钟, 其次是器官灌注和30分钟的固定。</li>
	<li>一旦动物达到了手术耐受的状态 (负趾捏-反应), 用70% 乙醇消毒动物, 用手术胶带将动物固定在垫上。</li>
	<li>通过心脏穿刺和流血牺牲动物。简单地, 用钝剪刀在皮肤和腹膜切开腹部用内侧切口。</li>
	<li>找到肝脏的隔膜头病房, 并仔细地将肝脏与隔膜分开。</li>
	<li>用钝剪刀在隔膜上做一个小切口, 用20克针附着在2毫升的注射器上, 使心脏的左心室有斑点。慢慢地抽血化验动物。 注意: 慢和仔细的放血是重要的, 以防止心室崩溃由于负压, 抑制不受干扰的血液流动。</li>
	<li>用弯曲的钝剪刀在肋骨的整个长度上通过胸骨旁切口轻轻地打开胸腔来解剖肺。</li>
	<li>然后, 抬起肋骨笼露出胸腔 (图 3C)。摘除胸腺以观察心脏和肺部。 注: 右心室可选择性注射, 其次为带有冰寒 PBS 的肺血管系统灌注, 然后用固定液 (例如, 4% (质量/体积) 多聚甲醛 (粉煤灰)] 是可能的。应注意的是, 有一个增加的风险, 破裂牙槽 septae 和负面影响肺结构使用这种方法。</li>
	<li>先仔细取出心脏, 解剖肺。</li>
	<li>随后, 用钝钳在气管和食道之间通过4-0 编织手术缝合。</li>
	<li>接下来, 仔细切割气管软骨与喉部之间的气管, 用静脉注射套管 (26 克) 插管, 并用固定剂 (例如, 4% (质量) 在20厘米 H2O 的恒定压力下, 充气肺部。/容积) 的粉煤灰]。</li>
	<li>对于粉煤灰固定, 在室温下将定影液保持在30分钟。之后, 结扎气管, 取出套管。然后, 小心地在不损害组织的情况下对肺进行切除, 并将其贮存在4摄氏度的固定剂中过夜。 注: 另外, 根据 ATS/ETS 共识纸 2.5% GA 缓冲 OsO4, 尿溶液用于适当的组织稳定。要进一步组织准备, 请参阅夏et 等的协商一致文件。14
</li>
</ol>

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作者没有什么可透露的。

本报告为三种不同方法分析肥胖对葡萄糖代谢和肺部结局的影响提供三项议定书。首先, 葡萄糖耐受性试验提供了分析细胞内葡萄糖吸收的机会, 并可以表明胰岛素抵抗。第二, 全身 plethysmography 是一种测量肺功能的技术, 从而有助于测试新疗法的疗效。第三, 标准化的固定协议是定量形态学分析的关键, 以评估肥胖对结构变化的影响。
饮食诱发肥胖在动物研究中的作用
对于现有方法和未来应用的意义: 模仿人类的饮食习惯, 导致肥胖, 饮食诱发肥胖 (迪奥) 模型被广泛应用于啮齿动物。与模拟代谢性疾病的转基因小鼠相比, 迪奥模型能够分析病因学、病理学和未来治疗方案。最近由 Heydemann et进行的一项审查, 概述了糖尿病研究中不同的小鼠高脂饮食模型16。营养成分的具体修改提供了未来研究的可能性, 微观和宏观营养素对肥胖分子机制的影响, 从而对肺的结构和功能。
修改和疑难解答: 各种报告显示, 肥胖可由膳食营养素组成的不同变化引起16, 这些知识反过来又导致了近年来各种饮食的发展, 例如,高脂肪或碳水化合物含量或两种饮食组合的饮食, 也就是所谓的西式饮食16。最后, 对饮食的选择取决于研究的问题和研究的目的。除了控制这些重要因素外, 适当控制饮食的选择是至关重要的;否则, 对结果的解释将受到限制。然而, 表型不仅是由饮食类型引起的, 而且也是喂养期、性别和小鼠应变的结果17。
技术的局限性: 在饮食的各种营养成分旁边, 动物和背景菌株的反应可以解释肥胖诱导的结果和表型。例如, 它已经表明, BALB 小鼠比 C57Bl/6 小鼠更容易患肝脂肪变性18。轻微的遗传变异,例如, 由于遗传漂移 (C57Bl/6J 与 C57Bl/6N), 也可能影响肥胖的易感性19。这突出了技术的局限性, 与基因修饰小鼠的不同基因背景菌株, 因为即使是菌株和供应商的小鼠获得可能影响的结果。
《议定书》内的关键步骤: 为了可靠、可重复和比较的结果, 实验组和控制小组之间, 最好窝应用于产生控制和实验小鼠, 环境因素和压力应避免, 和控制小鼠应与实验鼠并行研究12。
腹腔葡萄糖耐受试验 (ipGTT)
对现有方法和未来应用的意义: 为了确定迪奥对小鼠代谢的影响, 存在不同的筛选试验。建立了小鼠代谢分型中心 (MMPC) 联盟, 提出了评估小鼠代谢表型的标准方法, 并公布了描述和执行葡萄糖代谢试验的标准操作程序。鼠标中的稳态12。GTT 是衡量人体细胞在摄取适量糖后能够摄取葡萄糖的一种全球方法, 这反过来又预示着胰岛素分泌和胰岛素效应。血清胰岛素水平的差异往往说明糖耐量的改变。因此, GTT 已成为最广泛使用的生理测试, 以描述小鼠模型的糖尿病和肥胖。由于 ipGTT 是一种快速简便的方法, 因此可以作为一种标准化的方法来分析饮食和/或治疗对新陈代谢的影响。
修改和疑难解答: GTT 是例行执行后, 一夜之间的快速均衡血糖水平的小鼠20。由于空腹时间对所研究的参数有很强的影响, 因此对结果的比较和解释具有重要意义, 因此禁食期应适应小鼠的年龄和体重。考虑到老鼠是夜行动物, 每天摄入的食物总摄取量的2/3 在一夜之间消耗, 隔夜禁食引发分解状态。因此, 起始时间点和禁食期间对结果有高度相关的影响, 因此这些参数应标准化12。此外, 长期禁食耗尽糖原储存, 从而减少基线血糖的变异性。最后, 由于胰岛素刺激的葡萄糖在小鼠体内的应用在长时间禁食后得到增强21, 建议5至6小时禁食时间来评估胰岛素的作用;然而, 隔夜禁食足以测试葡萄糖利用率12,20,22。葡萄糖通常被管理通过ip 注射和葡萄糖的剂量调整到体重 (通常1或2克/千克体重)20,22。在迪奥模型中, 体重增加, 主要是由于脂肪质量较高;然而, 葡萄糖摄取主要发生在肌肉、大脑和肝脏中。由于这些组织的数量一般不会被迪奥改变, 肥胖小鼠的葡萄糖摄入量不成比例地高, 与精益小鼠相比, 这反过来会影响数据的解释, 导致误诊的葡萄糖不耐受22.因此, 禁食期需要规范化, 在解释 GTT 结果时需要考虑身体成分23。因此, 计算机断层成像 (CT) 或磁共振成像 (MRI) 扫描是适用的。例如, 手持式全血葡萄糖监测仪 (参见材料表) 可以获得葡萄糖测量, 而这些显示器比血糖分析仪更常用。由于少量血液容量需要-典型地5µL 或较少-他们比血浆分析仪更加实用。
技术的局限性: 禁食的小鼠表现出对禁食持续时间的依赖性, 并显示体重、体温、血容量、心率以及血清参数的变化, 如游离脂肪酸水平和酮体的显著损失24. 这个新陈代谢的重音由事实触发, 老鼠的住房温度在动物设施被规范化到大约23°c, 并且在他们的热中性温度之下30°c25。长时间禁食在 subthermo 中性温度可能导致麻木, 特点是新陈代谢率的减退26,27。
《议定书》内的关键步骤: 如上所述, 为了在实验组和对照小组之间取得可靠结果, 最好在研究和禁食期间使用窝, 时间点需要标准化。
肺功能分析
在这项协议中, plethysmograph 直接测量的压力变化是驱动呼吸, 并由此产生的流动进出气道。流量由位于 plethysmograph 壁的 pneumotachograph 测量。为排除胸壁的阻力, 测量气道开口压力 (口压) 和 transpulmonary (食管管) 压力, 计算气道阻力和动态顺应性。动态符合性是通过通过计算的, 其最小和最大肺体积的差异通过流进行划分。
对现有方法和未来应用的意义: Plethysmography 是分析肺力学性质的标准程序, 因此可以用于今后的研究, 分析肺部病理和治疗方案。为了比较群体和研究, 标准化的方法是必不可少的。
修改和故障排除: 一般情况下, 该技术可分为无限制和约束的全身 plethysmography。无限制的方法确定增强的停顿 (金边) 和允许分析正常呼吸模式, 而克制, 侵入方法直接测量压力, 流量, 或体积。肺力学性能由耐药性和 elastance 决定;当阻力计算为压力与流量的比值时, elastance 反映压力与体积的比值28。相比之下, 无拘无束的全身 plethysmography 只是测量 plethysmograph 内部的压力, 因此计算阻力和 elastance 是不可能的。在 2007年, 伦德布艾得et . 已经指出, 金边不是测量气道阻力的正确参数, 而是表示呼吸模式29的非特异性反射。因此, 为了正确估计肺力学, 侵入性 plethysmography 是不可缺少的29,30,31。
由于呼吸参数取决于小鼠的年龄和大小, 需要调整通风参数。例如, 潮汐体积与体重有关, 应设置为10µL/克体重, 呼吸频率为每分钟120-250 呼吸。在调整这些参数时, 调查员应考虑到平均潮汐量与呼吸频率的32呈反比关系。由于自发呼吸影响压力, 流量和体积, 必须监测麻醉的深度和通风曲线必须不断观察。然而, 麻醉本身可以直接对肺功能造成负面影响。例如, 异丙酚和氯胺酮对诱导气道收缩的部分保护作用与硫喷妥钠33相比。此外, 临床研究表明氯胺酮具有抗胆碱能的作用, 可作为严重哮喘的潜在支气管扩张剂34。吸入麻醉剂, 如异氟醚与疼痛治疗结合, 被认为是可控的替代注射麻醉;然而, 挥发性麻醉剂后呼吸道刺激报告, 因此排除吸入麻醉药作为替代35。
技术局限性: 采用限制性侵入法测量肺机械性能是由于必要的气管切开, 最后的手术, 从而限制了研究的单点分析, 没有选择的方法来调查疾病进展.为了降低侵袭程度, 可以对有意识动物的转移阻抗进行测量, 从而实现纵向研究。然而, 当测量非气管切开动物的呼吸力学时, 鼻子的阻力有助于总的呼吸阻力, 从而使测量复杂化后, 乙酰甲胆碱挑衅13。
协议中的关键步骤: 在这里, 我们只展示了一种侵袭性肺功能的方法。由于有几种已建立的侵袭性肺功能方法存在, 研究中的标准化和在出版物中使用的方法、团体和麻醉制度的详细描述是必要的比较研究。
肺切除术形态计量学分析
对现有方法和未来应用的意义: 定量形态计量学分析可用于研究肥胖对肺结构 (支气管和肺泡) 的影响, 以解释侵入性的结果。plethysmography, 并研究可能的治疗方案, 肺结局。组织学评估的数据可能会因固定剂和使用的固色程序而有所不同。由于肥胖对肺泡和支气管结构、细胞外基质和细胞组成有一定的影响, 因此有必要在今后的研究中确定合适的技术。对于无偏见的定量形态计量学, 应按照美国胸社会 (ATS)/欧洲呼吸系统学会的标准, 对肺结构进行定量评估, 以进行组织处理14.
修改和疑难解答: 在 2010年, 夏et . 发表了一份官方声明, 说明安非他明类兴奋剂/再培训标准对肺结构进行定量评估, 在肺隔离和固定之前应考虑到这一点。14. 在气管灌注护,原位固定, 固定容积固定, 或真空膨胀可以执行充气肺组织36。通货膨胀, 从而扩大空域, 取决于固定程序和在固定过程中施加的压力等级。例如, 高压会导致肺泡壁破裂, 从而影响结果。与通气参数相似, 理想的固定参数取决于小鼠的年龄、大小和表型。固定可以通过化学物质或物理手段, 包括化学剂和/或冷冻保存来实现。气管内注入适当的护模仿组织膨胀在呼吸中, 反映在体内条件, 因此被广泛使用的37。20-25 厘米以上的最高点的肺建议足够的压力, 使用宽和短油管, 以允许快速和均匀穿透14。固定的一个主要目标是防止变性过程和保存细胞和组织在一个 "类似生命的状态", 同时保持肺实质的建筑完整性。此外, 组织必须保持其反应性的抗体, 污渍和核酸探针。除正常酵母的效果外, 还必须防止组织处理的不良影响, 包括热蜡、切片和脱蜡等的渗透。固定剂的选择以及组织的进一步处理, 例如通过嵌入, 可能导致组织收缩, 肿胀和硬化的各种组成部分, 并导致工件, 如增加的自体荧光38。例如, 固定在10% 缓冲福尔马林和进一步处理可能导致收缩 20%-30% 相对于最初的容量39。因此, 夏et在其2010共识文件中建议使用2.5% 戊二醛缓冲锇毒气和醋酸铀, 以避免组织萎缩。在该固定试剂的气道灌注后, 肺体积、内部结构、组织精细结构和细胞结构保持在14。蛋白质变性和交联形成是组织内固定的两大重要机制。脱水化合物或絮凝剂, 如醇或丙酮, 会导致蛋白质变性, 从而导致三级蛋白结构的改变, 从而破坏疏水性的捆绑。非凝胶固定剂, 如多聚甲醛或戊二醛化学反应与蛋白质, 形成分子间和分子内的交叉联系。由于几种染色程序, 如苏木精和伊红染色, 取决于分子间的相互作用, 染色结果可能较差, 取决于固定剂。免疫组化的抗原检索方法表明, 某些固定的反应是可逆的, 特别是甲醛40。
技术的局限性: 由于护气管内灌注去除了肺泡表面衬里, 所以可以对例如,肺泡微结构、粘液积聚或炎症细胞迁移的解释进行操作。41,42,43。
《议定书》内的关键步骤: 在这里, 我们显示4% 粉煤灰的气管灌注是一种广泛的方法, 可以想象肥胖对肺部结局的影响。如上所述, 必须根据研究问题修改该议定书, 按照安非他明类兴奋剂/方案建议14。
除了前面提到的参数, 压力是影响研究结果的主要因素。因此, 训练老鼠和科学家是必不可少的。小鼠应适应抑制, 应转移到实验区在安静的条件下44。压力影响新陈代谢,例如由于应激激素释放, 释放的葡萄糖水平增加, 这一效应可以被误解为糖耐量受损。应激激素释放也改变麻醉药物的易感性, 具体来说, 麻醉药剂量增加, 达到手术耐受的时间延长。如前所述, 麻醉剂数量的增加会影响支气管收缩, 而压力本身也会引起支气管扩张。
总之, 本文提供了三种方法来评估肥胖和代谢对小鼠肺结构和功能的影响。所有被提及的方法可以转移到其他疾病模型和啮齿动物种类, 例如肥胖由于基因修改或老鼠模型。这些技术的应用有助于在特定基因消融的迪奥模型中定义新的分子机制, 或测试新的治疗方法, 以治疗/预防肥胖对慢性肺病的不良影响。

根据世界卫生组织 (卫生组织) 的数据, 在 2008年, 超过14亿个年龄在20岁以上的成年人超重, 身体质量指数 (BMI) 大于或等于 25;此外, 2亿多名男子和近3亿名妇女肥胖 (BMI≥30)1。肥胖和代谢综合征是多种疾病的主要危险因素。肥胖和随之增加的白脂肪组织质量与2型糖尿病有密切关系2,3, 心血管疾病包括冠心病 (CHD), 心力衰竭 (HF), 心房颤动4和骨关节炎5, 它们在呼吸道疾病发病机制中的功能作用仍然不甚了解。然而, 流行病学研究表明, 肥胖与慢性呼吸道疾病密切相关, 包括劳力呼吸困难、阻塞性睡眠呼吸暂停综合征 (OSAS)、肥胖低通气综合征 (职业职业治疗)、慢性阻塞性肺病 (COPD), 肺栓塞, 吸入肺炎和支气管哮喘6,7,8,9。将肥胖和摄动代谢、例如、胰岛素抵抗和 II 型糖尿病与慢性肺病发病机制联系起来的潜在机制, 不仅包括对通气的体重增加的机械和物理后果, 而且还诱发慢性亚急性炎症状态10,11。过去十年中肥胖和肺部疾病的增加, 加上缺乏有效的预防策略和治疗方法, 突显出需要调查分子机制, 以确定管理肥胖相关肺的新途径。疾病.
在这里, 我们描述了三标准测试, 这是研究肥胖及其对小鼠肺结构和功能的影响的重要基础知识: (1) 腹腔葡萄糖耐受 (ipGTT) (2) 气道阻力 (Res) 和呼吸的测量系统遵从性 (Cdyn);(3) 肺的制备和固定, 供随后的定量组织学评估。ipGTT 是一个强有力的筛选测试, 以测量葡萄糖摄取量, 从而影响肥胖对新陈代谢。这种方法的简单性使得标准化很好, 因此实验室之间的结果可比性。更复杂的方法, 如高血糖钳或研究孤立胰岛, 可用于详细分析代谢表型12。在这里, 我们评估葡萄糖耐受性, 以确定肥胖相关的系统性和代谢紊乱的状态, 作为进一步研究肺部结局的基础。为了评估肥胖和代谢紊乱对肺功能的影响, 我们测量了气道阻力 (Res) 和呼吸系统依从性 (Cdyn)。为描述肺部疾病, 有无节制的和克制的方法评估肺功能。无拘无束的 plethysmography 在自由运动的动物模仿自然状态, 反映呼吸模式;与此相反, 侵入性方法, 如输入阻抗测量的 cDyn 在深麻醉小鼠评估动态肺力学, 是更准确的13。由于慢性呼吸道疾病是由肺组织的组织学改变反映出来的, 因此适当的肺部固定术是迫在眉睫的。组织固定和制备方法的选择取决于肺的隔间, 例如传导气管或肺实质14。在这里, 我们描述了一种方法, 允许定性和定量评估传导气道, 以研究肥胖对哮喘发展的影响。

<table>
	<tbody>
		<tr>
			<td>GlucoMen LX</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>38969</td>
			<td>blood glucose meter</td>
		</tr>
		<tr>
			<td>GlucoMen LX Sensor</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>39765</td>
			<td>Test stripes</td>
		</tr>
		<tr>
			<td>Glucose 20%</td>
			<td>B. Braun, Melsung, Germany</td>
			<td>2356746</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe Software</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-002</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe RC Single Site Mouse Table</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-001</td>
			<td></td>
		</tr>
		<tr>
			<td>FPRC Controller</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1075-001</td>
			<td></td>
		</tr>
		<tr>
			<td>FPRC Aerosol Block</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1106-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerogen neb head-5.2-4um</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-2306-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST, British Columbia, Canada</td>
			<td>11065-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>14105-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Perma-Hand 4-0</td>
			<td>Ethicon, Puerto Rico, USA</td>
			<td>736H</td>
			<td>Surgical suture</td>
		</tr>
		<tr>
			<td>Roti-Histofix 4%</td>
			<td>Roth</td>
			<td>P087.1</td>
			<td>4% Paraformaldehyd</td>
		</tr>
		<tr>
			<td>Ketaset</td>
			<td>Zoetis, Berlin, Germany</td>
			<td>10013389</td>
			<td>Ketamine</td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>770081</td>
			<td>Xylazine</td>
		</tr>
	</tbody>
</table>

intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes

肥胖和呼吸系统疾病是主要的健康问题。肥胖正在成为一个新兴的流行病, 预计到2030年世界各地有超过10亿的肥胖人口, 这就代表了社会经济负担的增加。同时, 肥胖相关的并发症, 包括糖尿病以及心脏和慢性肺病, 都在不断上升。虽然肥胖与哮喘加重的风险、呼吸道症状恶化和控制不力有关, 但肥胖和摄动代谢在慢性肺病发病中的功能作用往往被低估,而潜在的分子机制仍然难以捉摸。本文旨在探讨肥胖对代谢的影响以及肺结构和功能的评估方法。在这里, 我们描述了三技术的老鼠研究: (1) 评估腹腔葡萄糖耐受 (ipGTT), 以分析肥胖对葡萄糖代谢的影响;(2) 测量气道阻力 (Res) 和呼吸系统依从性 (Cdyn), 分析肥胖对肺功能的影响;(3) 肺的制备和固定, 供随后的定量组织学评估。肥胖相关的肺部疾病可能是多因素的, 其产生于全身炎症和代谢失调, 可能对肺功能和治疗反应产生不利影响。因此, 研究分子机制和新疗法效果的标准化方法是必不可少的。

腹腔葡萄糖耐受试验 (ipGTT) (图 4)、肺功能测试 (图 5) 的代表性结果, 以及说明苏木精和红红染色肺部的代表性图像 (图 6)。
ipGTT 7 周高脂饮食 (风) 后, 在肥胖小鼠 (蓝色) 中进行。标准膳食喂养的老鼠作为控制 (黑色)。肥胖小鼠在腹腔葡萄糖注射液后显示血糖水平增加15和30分钟, 表明细胞葡萄糖摄取受损 (图 4)。
为了研究肥胖对肺功能的影响, 在7周的高脂饮食 (风) 后, 对肥胖小鼠 (蓝色) 进行了侵袭性肺功能分析。与控制小鼠 (黑色) 相比, 肥胖小鼠的气道阻力增加了1.5 倍 (图 5)。
为观察护气管内灌注加压固定对肺实质的影响, 显示了苏木精和红红染色肺切片的代表性图像 (图 6)。压力太小导致多个非膨胀区域、厚牙槽 septae 和多边形形肺泡 (A), 而过多的压力会导致过度膨胀的肺气肿样区域, 并破坏肺泡 septae (C)。应用适当的压力在肺固定导致一个完全地充气肺与圆形肺泡 (B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56685/56685fig1.jpg"> 图 1: 侵袭性肺功能的示意图表示形式.(-c)气管切开的步骤。(D)动物与 plethysmograph 面板的连接。(E)用于侵袭性肺功能的硬件设置。(F)数据获取的截图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56685/56685fig1large.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/56685/56685fig2.jpg"> 图 2: 评估支气管 hyperreagibility 的数据采集方案.数据采集包括初始驯化期 (5 分钟), 其次是三十年代的物质雾化, 3 分钟的反应阶段, 3 分钟的恢复阶段前吸入的物质浓度。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56685/56685fig2large.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/56685/56685fig3.jpg"> 图 3: 准备步骤的示意图表示形式。(A-d)气管切开的步骤。(A)皮肤切口。(B)胸腔的所在地。删除肋骨笼后的(C)视图。删除心脏后的(D)视图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56685/56685fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56685/56685fig4.jpg"> 图 4: 代表性的腹腔葡萄糖耐受试验 (ipGTT).C57Bl/6N 小鼠喂食高脂肪饮食6-8 周;控制小鼠接受标准饮食。n = 3;平均电子扫描电镜;进行了统计学分析, 分别进行了双路方差检验和 Bonferroni 前后。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56685/56685fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56685/56685fig5.jpg"> 图 5: 代表性肺功能测试.C57Bl/6N 小鼠喂食高脂肪饮食6-8 周;控制小鼠接受标准饮食。n = 3;平均电子扫描电镜;进行了统计学分析, 分别进行了双路方差检验和 Bonferroni 前后。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56685/56685fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56685/56685fig6.jpg"> 图 6: 说明苏木精 & 红染肺部的代表性图像.三不同等级的气管内通货膨胀: (A) 压力太小 (B) 适当的压力, 并且 (C) 压力太大。uia;倒塌区域, 内部审计;过度膨胀的区域;图像是在20X 放大倍数下拍摄的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56685/56685fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>通风机设置</td>
			<td>Spalte1</td>
		</tr>
<tr>
<td>最大.描边音量</td>
			<td>0.25ml</td>
		</tr>
<tr>
<td>最大.口压</td>
			<td>30cmH2O</td>
		</tr>
<tr>
<td>深度通货膨胀最大。卷</td>
			<td>0.5 毫升</td>
		</tr>
<tr>
<td>深度通货膨胀最大。压力</td>
			<td>30cmH2O</td>
		</tr>
<tr>
<td>率</td>
			<td>160呼吸每分钟</td>
		</tr>
</tbody></table>表 1: 成人鼠标的通风机设置.对于较小的动物, 需要调整通风参数。

Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

肥胖的发病率在上升, 并增加了慢性肺病的风险。为了建立基本机制和预防战略, 需要有明确定义的动物模型。在这里, 我们提供三种方法 (葡萄糖耐受性测试, 身体 plethysmography 和肺固定), 以研究肥胖对肺结局的影响小鼠。

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

intraperitoneal-glucose-tolerance-test-measurement-lung-function

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.0K 次观看.  University of Cologne. 肥胖的发病率在上升, 并增加了慢性肺病的风险。为了建立基本机制和预防战略, 需要有明确定义的动物模型。在这里, 我们提供三种方法 (葡萄糖耐受性测试, 身体 plethysmography 和肺固定), 以研究肥胖对肺结局的影响小鼠。

视频: Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

科研

免疫与感染

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡:隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.