Name: generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure
Uploaded: 2017-08-25T14:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure at JoVE.com

作者希望对 Mr. 波 (上海市公共卫生临床中心) 在本议定书中的µCT 评估提供技术援助表示感谢。

注意: OE 模型已经生成并用于以前报告的研究 30 、 31 、 32 。所有动物实验均经上海交通大学机构动物护理与使用委员会 (IACUC) 批准. 
 1. 鼠标 <ol> <li> 在受控温度 (20 和 #176; c) 和湿度 (40-60%) 的动物设施的单个通风笼中, 无病原体、7至9周老的雌性 balb/c/c 小鼠。提供12和 #160; h 光和 12 #160; 设备中的暗循环。提供食物和水 ad 随意 . </li>
</ol> 2。臭氧或空气暴露 <ol> <li> 在密封丙烯酸 ( 如 有机玻璃) 框中生成带有电发生器的臭氧。用小风机吹出箱内的空气, 通过排气管连接在箱体内外。使用臭氧探针监测盒子中的臭氧浓度。等到臭氧浓度达到2.5 分之百万分之一 (ppm). 
	注: 臭氧探头可自动开启或关闭臭氧发生器, 并可在 2.5 ppm 时保持盒内的臭氧水平. </li>
	<li> 当臭氧水平达到 2.5 ppm 时, 将小鼠放入盒子中。把老鼠放在盒子里, 每次3小时, 让它们暴露在臭氧中. 
	注: 该盒可在3小时内保持臭氧水平 2.5 ppm, 通过自动开关臭氧发生器打开或关闭和吹出的 CO 2 , 这是由小鼠产生的方块. </li>
	<li> 每周 (每3天) 给两次臭氧暴露 (每次接触持续3小时), 为期7周; 同时将控制小鼠暴露在同一时间内, 并同时进行. </li>
</ol> 3。微计算机断层扫描 <ol> <li> 在7周结束时, 麻醉小鼠腹腔注射 pelltobarbitalum 孢 (1%、0.6-0.8 ml/100 g) 和 #160; 根据个别情况调整剂量以查看鼠标不响应脚趾捏. 和 #160; 监测和保持鼠标在一个稳定的呼吸频率;并确保在程序中不存在自愿的动作. </li>
	<li> 将麻醉小鼠放置在微计算机断层扫描室 (#181; CT). </li>
	<li> 校准和 #181; 使用标准协议和制造商和 #39 的说明进行 CT 检查。设置 X 射线管在50伏和电流在450和 #181; A. 
	注: X 射线和探测器都围绕鼠标旋转. </li>
	<li> 执行和 #181; CT 分析通过获得515投影, 有效像素大小为 0.092 mm, 一个切片的曝光时间为300毫秒, 切片厚度为 0.093 mm. </li>
	<li> 使用软件重建肺部与获得的图像。通过分别设置-750 和-550 Hounsfield 单元的灰度最小值和最大值来调整灰度图像亮度. 
	注: 该软件将自动计算肺实质的体积和 low-attenuation 区 (LAA) 34 , 35 。
	<li> 通过将 LAA 体积除以肺总容积来计算 LAA (LAA%) 的百分比. </li> </ol>
 4. 平板电脑测试 <ol> <li> 在跑步机上为小鼠提供10米/分钟的适应性测试. #160; 注意: 当程序进行时, 电总是关闭的. #160; </li> <li> 管理对小鼠进行疲劳试验。
	<ol> <li> 以10米/分钟的速度将鼠标预热到5分钟 </li>
		<li> 将速度提高到15米/分钟, 10 分钟. </li>
		<li> 增加运动强度: 提高速度5米/分钟, 从20米/分钟开始, 每30分钟, 直到鼠标不能继续运行 36 . </li>
	</ol> </li> <li> 将总运行距离和运行时间分别记录为疲劳距离和疲劳时间. </li> </ol>
 5. 肺功能测量 <ol> <li> 麻醉腹腔注射 pelltobarbitalum 孢 (1%、0.6-0.8 ml/100 g) 的小鼠, 根据个别情况调整剂量, 以查看鼠标是否不响应脚趾捏和 #160; 并等待, 直到老鼠保持自发性呼吸. #160; 监测和保持鼠标在一个稳定的呼吸频率;并确保在程序中不存在自愿的动作. </li>
	<li> 小心地 tracheostomize 老鼠, 并将它们放在与计算机控制的呼吸机相连的身体体积中. 
	注: 通风通过靠近气管导管的下部阀门进行控制。该装置提供了不同的半自动机动, 包括准静态压力容积的机动和快速流动量的机动. </li>
	<li> 通过压力控制通气将平均呼吸频率设置为150呼吸/分钟到麻醉小鼠, 直到每一个呼吸循环都得到一个正常的呼吸模式和完全失效. </li>
	<li> 使用体积中产生的负压力对设备执行准静态压力-容积机动. </li>
	<li> 在准静态压力-容积回路内执行快速流动容积机动, 以记录 FVC 和 FEV。将肺部充气到 +30 厘米 h 2 O, 然后立即将其连接至高度负压, 以强制执行有效期, 直到剩余体积在-30 厘米 h 2 o. 记录 FEV 在第一25、50和75毫秒中的呼气 (FEV 25 , 分别 FEV 50 和 FEV 75 。拒绝次优演习。对于每一个测试, 每个单一的鼠标, 进行至少三可接受的演习, 以获得一个可靠的平均所有数值参数. </li>
</ol> 6。BALF 集合 <ol> <li> 使用 pelltobarbitalum 孢 (1%、1.8-2.4 ml/100 g) 和 #160 进行晚期麻醉; 根据个别情况调整剂量, 以观察鼠标不响应脚趾捏和失去呼吸), #160; 灌洗2毫升 PBS 通过一个1毫米口径的气管插管, 然后检索 BALF 10 . </li>
	<li> 将检索到的灌洗等分, 并将其离心4和 #176; C 和 250 x g 为10分钟. </li>
	<li> 收集上清液以便立即使用, 并将剩余物存储在-80 和 #176; C 或液氮. </li>
	<li> 使用例计算单元总数. </li>
	<li> 重在 PBS 中的细胞颗粒, 然后旋转 (1400 x g, 6 分钟) 250 和 #181; L 悬浮细胞在幻灯片上使用滑动微调离心机. </li>
	<li> 根据制造商和 #39 的协议将赖特染色应用于幻灯片上的单元格. </li>
	<li> 每只鼠标计数200单元格; 根据标准形态学, 在400X 放大倍数下, 将细胞识别为巨细胞或嗜中性白血球; 并计算它们的数量. </li>
</ol> 7。心脏血液取样 <ol> <li> 通过心脏穿刺收集血液, 将其加载到1.5 毫升的管中, 并将其放在冰上30分钟. </li>
	<li> 在 2000 x g 和4和 #176 上离心血样5分钟; C. </li>
	<li> 将上清液 (血清) 转移到新管上, 并将其存储在-80 和 #176; C 或液氮. </li>
	<li> 为 IL-1 和 #946、IL-10、TNF 和 #945 制备血清; 使用各自的 ELISA 试剂盒进行检测测试. </li>
</ol> 8。肺形态计量学分析 <ol> <li> 从老鼠身上解剖肺部和气管。
	<ol> <li> 在牺牲后立即将每个被安乐死的鼠标置于手术板上. </li>
		<li> 解剖阔和前气管肌肉, 以可视化和进入气管环. </li>
		<li> 打开胸腔。解剖肺部和气管, 但不要把心脏和肺部分开. </li>
	</ol>
</li> <li> 通过 PE90 聚乙烯管将气管导管连接到含有4% 甲醛的注射器. 
	警告: 甲醛是有毒的。戴上手套和安全眼镜, 并使用通风罩内的解决方案. </li>
	<li> 用4% 甲醛 (10 滴, ~ 200 和 #181; L) 通过气管导管完全充气肺部。在通货膨胀完成后取出心脏. </li>
	<li> 保持15和 #160 的肺部; ml 管包含10毫升的4% 甲醛, 至少4小时. </li>
	<li> 将肺部嵌入石蜡中。通过石蜡切片和旋转切片获得5和 #181; m 段。在切片过程中, 暴露支气管树区内肺组织的最大表面积. </li>
	<li> 用于形态分析, 在切片上进行苏木精和曙红 (H 和 #38; E) 染色. </li>
	<li> 用明亮的场直立显微镜 (物镜, 20X; 曝光时间, 1.667 毫秒) 来图像剖面. </li>
	<li> 有两名调查人员对治疗方案视而不见, 独立计算组织学部分。使用平均线性截距 (L m ) 作为测量 inter-alveolar 间隔壁距离的参数。使用以下步骤确定 L m : <ol> <li> 打开 Photoshop 中各节的图像, 并在图像上绘制一个手提五550和 #181; m 和 #160; 长行. </li>
		<li> 计算网格线上的肺泡数. </li>
		<li> 通过将网格线的长度除以肺泡的数目来计算 L m 。对于量化, 每只鼠标图像五节。获取每个部分的十图像 (每个字段一个图像) 并随机评估。在野外选择的过程中, 通过在前方或另一个方向移动一个磁场来避免气道和容器的电场. 
		注: 数据显示为平均值和 #177; S.E.M。对 air-exposed 小鼠和臭氧暴露小鼠进行了 un-paired t 检验。每组三只动物被用来计算显著的差异。p 值的和 #60; 0.05 被认为是重要的. </li>
	</ol>
	</li>
</ol>

Z.W.S. 和. 是目前的雇员和细胞生物医学组 (纳斯达克: CBMG) 的股票期权持有人。其他作者宣称他们没有竞争利益。

在这项研究中, 我们提出了一个可靠的方法来生成一个新的 COPD 模型。与其他模型 (即LPS 或 PPE 模型) 相比, 此 OE 模型重述 COPD 患者的病理过程。由于香烟烟雾是导致人患 copd 的主要危险物质40, CS 模型仍然是最受欢迎的 copd 模型41,42。然而, CS 模型需要 3-到12月的 r-和 #38;D 期新药。与 CS 模型相比, 目前的 OE 模型将生成周期缩短为6-8 周。我们在以前的研究中观察到臭氧暴露在雄性小鼠6周后的肺气肿30,31,32。在本研究中, 我们将 oe 协议应用于雌性小鼠7周, 成功地生成了雌性 oe 模型。因为据报道, 在一些国家, copd 的死亡率已经下降, 但在妇女中增加了33, 因此有必要研究女性 copd 患者的致病机制和治疗方法。我们知道, 上述的 COPD 模型 (即, LPS, PPE 和 CS) 可以在男性和女性动物的工作,43。这项工作的目的是提出一个额外的 copd 模型, 既重述病理过程的 copd 患者, 并要求一个非常短的世代期。
生成此模型的关键步骤是将小鼠暴露于臭氧 (2.5 ppm 的水平) 每周两次 (每3天一次), 为期6-8 周 (在本研究中, 我们使用7周, 虽然我们没有尝试剂量上升)。通过控制暴露频率和臭氧浓度的关键参数, 我们成功地再现了男性 C57BL/6 小鼠的肺气肿31,32, 雄性 balb/c/c 小鼠30, 和雌性 balb/c/c 小鼠 (当前研究)。肺气肿表型由臭氧暴露引起, 气流限制和肺实质破坏类似于 COPD 患者所见的变化44,45.
这项研究仍有局限性。例如, 由于人类慢性阻塞性肺病的发病机制与肺部的解剖有关, 因此应在具有类似于人类的肺部解剖结构的动物中产生一种理想的 copd 模型。与大动物相比, 小动物比人拥有更不广泛的气道分支46。我们必须承认, 在大型动物中生成 COPD 模型会更有意义。然而, 在动物中建立 large-scale 模型是非常困难的。这个模型的另一个局限性是它的临床相关性。虽然它确定臭氧能与呼吸道反应和损伤肺组织19,22, 虽然有证据表明, 一些 COPD 患者的症状恶化后暴露在臭氧28,臭氧不是 COPD 患者的主要病因。然而, 我们仍然建议和使用这个 OE 模型, 因为臭氧和 CS 造成的损害呼吸系统通过诱导炎症和导致氧化应激26,27。因此, 一种能在 OE 模型中治疗 copd 的新药, 大概也可以在 CS 模型中工作, 因此可能为 copd 患者开发。
OE 模型的应用不限于对 COPD 分子和细胞机制的破译。我们最近的两篇论文还研究了 n-乙酰半胱氨酸 (NAC)31和 NaHS32 (一个外源性的 H2S) 治疗 COPD 通过外源管理的两种药物的 OE 模型。在第一项研究中, 我们发现了一个逆转气道超反应和减少气道平滑肌质量后, 管理的 NAC。这些影响可能表明, 在 COPD 患者的潜在临床好处的基础31。在第二次研究的 OE 模型, 我们发现, 外源性 NaHS 的管理逆转肺部炎症和部分逆转肺气肿的特点。因此, 通过这个 OE 模型, 我们在一项初步研究中证明, NaHS 可以成为 COPD 患者的潜在药物候选者,32。因此, OE 模型在 COPD 的机制研究和药物筛选方面具有潜在的应用价值。

据估计, COPD, 包括肺气肿和慢性支气管炎, 可能是世界上的第三个主要死因在 2020年1,2。在未来40年3中, 在40岁以上的人口中, COPD 的潜在发病率估计为12.7% 男性, 8.3% 为女性。目前尚无药物可逆转 COPD 患者的渐进性恶化4。可靠的 COPD 动物模型不仅要求对疾病的病理过程进行模拟, 而且需要较短的周期。目前的 COPD 模型, 包括 LPS 或 PPE 诱发的模型, 可以诱发肺气肿样症状5,6。一个单一的管理或一个星期的挑战, LPS 或 PPE 的小鼠或大鼠结果在支气管肺泡灌洗液 (BALF) 的显着中性, 增加炎调解人 (例如, TNF-α和 IL-1β) 在 BALF 或血清, 产生肺实质破坏-扩大空气空间, 并限制气流5,6,7,8,9,10。然而, LPS 或 PPE 不是人类慢性阻塞性肺病的原因, 因此不模仿病理过程11。CS 诱导模型产生持续气流限制, 肺实质破坏, 并减少功能锻炼能力。但是, 传统的 CS 协议至少需要3月才能生成 COPD 模型12,13,14,15。因此, 重要的是要产生一个新的, 更有效的动物模型, 满足这两个要求。
最近, 除了吸烟, 空气污染和职业暴露已成为更常见的原因 COPD16,17,18。臭氧作为主要污染物之一 (虽然不是空气污染的主要成分), 可以直接与呼吸道发生反应, 损害儿童和青年成人的肺组织19,20,21 ,22,23,24,25。臭氧, 以及其他刺激, 包括 LPS, PPE, 和 CS, 参与了严重的生化途径的肺氧化应激和 DNA 损伤, 并与启动和促进 COPD26,27。另一个因素是, 一些 COPD 患者的症状在暴露于臭氧后恶化, 表明臭氧会扰乱肺部功能18,28,29。因此, 我们产生了一个新的 COPD 模型, 反复暴露小鼠到高浓度的臭氧7周;这导致气流缺陷和肺部实质损伤类似于以前的调查30,31,32。我们在本研究中推广了对雌性小鼠的 OE 协议, 并成功地再现了我们以前研究的雄性小鼠的肺气肿30,31,32。由于 copd 死亡率在男性减少, 但在许多国家的妇女增加了33, 需要对女性 copd 模型进行研究, 以探讨慢性阻塞性肺病患者的发病机制和治疗方法。OE 模型对所有性别的适用性进一步支持它作为 COPD 模型的使用。

<table><tbody><tr><td>BALB/c mice</td><td>Slac Laboratory Animal,Shanghai, China</td><td>N/A</td><td>7-to-9-week-old female BALB/c mice were used in this study.</td></tr><tr><td>Individual ventilated cages</td><td>Suhang, Shanghai, China</td><td>Model Number: MU64S7</td><td>The cages were used for housing mice in the animal facility.</td></tr><tr><td>Sealing perspex-box</td><td>Suhang, Shanghai, China</td><td>N/A</td><td>The box was used&amp;nbsp; to contain the ozone generator. Mice were exposed to ozone within the box.</td></tr><tr><td>Electric generator</td><td>Sander Ozoniser, Uetze-Eltze, Germany</td><td>Model 500&amp;nbsp;</td><td>The device was used for generating ozone.</td></tr><tr><td>Ozone probe</td><td>ATi Technologies, Ashton-U-Lyne, Greater Manchester, UK</td><td>Ozone 300</td><td>The device was used for monitoring and controlling the generation of ozone.</td></tr><tr><td>Pelltobarbitalum natricum</td><td>Sigma, St. Louis, MO, USA</td><td>P3761</td><td>Mice were anesthetized by intraperitoneal injection of pelltobarbitalum natricum.</td></tr><tr><td>Micro-Computed Tomography</td><td>GE Healthcare, London, ON, Canada</td><td>RS0800639-0075</td><td>This device was used for acquiring images of the lung.</td></tr><tr><td>Micro-view 2.01 ABA software</td><td>GE Healthcare, London, ON, Canada</td><td>Micro-view 2.01&amp;nbsp;</td><td>This device was used for reconstruct the lung and analyze volume, LAA of the lung.</td></tr><tr><td>Treadmill machine&amp;nbsp;</td><td>Duanshi, Hangzhou, Zhejiang, China</td><td>DSPT-208</td><td>This machine was usd for fatigue test.</td></tr><tr><td>Body plethysmograph</td><td>eSpira&amp;trade; Forced Manoeuvres System, EMMS, Edinburgh, UK</td><td>Forced Manoeuvres System</td><td>This device was used to test spirometry pulmonary function.</td></tr><tr><td>Ventilator</td><td>eSpira&amp;trade; Forced Manoeuvres System, EMMS, Edinburgh, UK</td><td>Forced Manoeuvres System</td><td>This device was used to test spirometry pulmonary function.</td></tr><tr><td>Slide spinner centrifuge</td><td>Denville Scientific, Holliston, MA, USA</td><td>C1183&amp;nbsp;</td><td>It was used to spin BALF cells onto slides.</td></tr><tr><td>Wright Staining</td><td>Hanhong, Shanghai, China</td><td>RE04000054</td><td>&amp;nbsp;It was used to staining macrophages, neutrophils in the suspended BALF.</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific, Horsham, PA, USA</td><td>4000</td><td>It was used to count cells.</td></tr><tr><td>IL-1&amp;beta;</td><td>Abcam, Cambridge, MA, USA</td><td>ab100704</td><td>They were used to test the respective factors in serum.</td></tr><tr><td>IL-10</td><td>Abcam, Cambridge, MA, USA</td><td>ab46103</td><td>They were used to test the respective factors in serum.</td></tr><tr><td>TNF-&amp;alpha;</td><td>Abcam, Cambridge, MA, USA</td><td>ab100747</td><td>They were used to test the respective factors in serum.</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma, St. Louis, MO, USA</td><td>P6148</td><td>The lung was inflated by 4% paraformaldehyde.</td></tr><tr><td>Paraffin</td><td>Hualing, Shanghai, China</td><td>56#</td><td>It was used to embed the lung.</td></tr><tr><td>Rotary Microtome</td><td>Leica, Wetzlar,&amp;nbsp; Hesse, Germany</td><td>RM2255</td><td>It was used for sectioning the lung.</td></tr><tr><td>Hgaematoxylin and Eosin (H&amp;amp;E) staining solution</td><td>Solarbio, Beijing, China</td><td>G1120</td><td>H&amp;amp;E staining was done for morphometric analysis.</td></tr><tr><td>Upright bright field microscope</td><td>Olympus, Center Valley, PA, USA</td><td>CX41</td><td>It was used to image the H&amp;amp;E staining slides.</td></tr><tr><td>Adobe Photoshop 12</td><td>Adobe, San Jose, CA, USA</td><td>Adobe Photoshop 12</td><td>It was used to count the number of alveoli on the H&amp;amp;E stained images.</td></tr><tr><td>GraphPad prism 5</td><td>Graphpad Software Inc., San Diego, CA</td><td>GraphPad prism 5</td><td>It was used for data analysis and production of figures.</td></tr></tbody></table>

generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure

慢性阻塞性肺疾病 (COPD) 的特点是持续气流限制和肺实质破坏。它在老龄人口中的发病率很高。目前常规的 COPD 治疗主要集中在症状修饰药物;因此, 迫切需要开发新的治疗方法。合格的慢性阻塞性肺病动物模型有助于确定基本的机制, 并可用于新的药物筛选。目前的 copd 模型, 如脂多糖 (LPS) 或猪胰弹性蛋白酶 (PPE) 诱导肺气肿模型, 产生 copd 样病变的肺部和呼吸道, 但没有其他类似的发病机制的人 copd。香烟烟雾 (CS) 诱导模型仍然是最流行的之一, 因为它不仅模拟 copd 样病变的呼吸系统, 但它也基于其中一个主要的危险物质, 导致 copd 的人。然而, CS 诱导模型的耗时和 labor-intensive 的方面极大地限制了它在新药筛选中的应用。在这项研究中, 我们成功地产生了一个新的 COPD 模型, 暴露小鼠到高水平的臭氧。该模型显示如下: 1) 减少了强迫呼气体积 25, 50, 75/强迫肺活量 (FEV25/FVC, FEV50/FVC, 和 FEV75/FVC), 表明肺功能恶化;2) 肺肺泡肿大, 肺实质破坏;3) 减少疲劳时间和距离;和 4) 炎症增加。综合起来, 这些数据表明, 臭氧暴露模型是一种与人类相似的可靠动物模型, 因为臭氧过度曝光是 COPD 的病因之一。此外, 根据我们以前的工作, 它只花了 6-8 周, 创建一个 OE 模型, 而它需要 3-12 月, 以诱导卷烟烟雾模型, 表明 oe 模型可能是一个很好的选择 COPD 的研究。

每个组的3D µCT 图像的示例显示在图 1a中。臭氧暴露的小鼠的总肺容积明显较大 (图 1a 和 b) 和 LAA% (图 1c) 比 air-exposed 控制小鼠。六周的臭氧暴露后, 肺容积和 LAA% 保持升高31,32。肺容积和 LAA% 的增加代表肺气肿的表型。肺肺泡肿大的例子在图 2a中说明了肺气肿的形成。在臭氧暴露的小鼠 (图 2b) 中观察到平均线性截距 (Lm) 的增加, 证实了臭氧暴露后肺实质破坏的发生。
肺功能测量的气流率参数, 显示为 FEV25/FVC, FEV50/FVC, 和 FEV75/FVC。结果表明, 臭氧暴露小鼠的所有参数 (图 3a-c) 均与 COPD 患者的典型肺功能缺损 (4,37一致。为了进一步评估 OE 模型, 我们进行了一个运动耐受性测试, 以取代6分钟步行测试 (6MWT)38,39, 这是常用的评估功能锻炼能力的变化在 COPD 患者。臭氧暴露显著降低了疲劳时间和疲劳距离 (图 4a 和 b)。
为解决慢性阻塞性肺病在 OE 模型中的发病机制, 从小鼠 BALFs 中计算出巨噬细胞和中性粒细胞;在小鼠血清中检测到炎细胞因子 (IL-1β和 TNF-α) 和抗炎细胞因子 (IL-10)。臭氧暴露的小鼠表现出显著的炎症细胞的增加, 包括巨噬和中性粒细胞 (图 5a-c), 以及显著减少的 IL-10 和增加 IL-1β和 TNF (图 6a-c).所有的数据表明, OE 模型扼要人 COPD 样症状。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56095/56095fig1.jpg"> 
图 1。臭氧暴露增加了µCT 检测到的肺容积和 LAA%.(a) 有代表性的3D 图像显示 air-or 臭氧暴露的小鼠在各自暴露后7周的肺部。(b) 两组的个别肺容量是从3D 影像中抽取, 以供统计。臭氧暴露的小鼠肺总容积明显增加。(c) 两个群体的个人和平均 LAA%。臭氧暴露的小鼠的 LAA% 明显增加。红色表示 LAA (体, 密度为 2,550-2, 700 Hounsfield 单位)。以红色显示的气管和支气管在这个数字被去除了计算肺 LAA。数据显示为平均± S.E.M. p 和 #60; 0.01, *** 和 #60; 0.001。<a href="//ecsource.jove.com/files/ftp_upload/56095/56095fig1large.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/56095/56095fig2.jpg"> 
图 2。臭氧暴露增加了 Lm.(a) 在 air-exposed 或臭氧暴露小鼠的 he 染色切片中有代表性的肺肺泡间隙显微。(b) 从两组的肺部分对 Lm 的个人价值进行了量化统计。在臭氧暴露的小鼠中观察到 Lm 的增加。数据显示为平均± S.E.M. P 和 #60; 0.01。<a href="//ecsource.jove.com/files/ftp_upload/56095/56095fig2large.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/56095/56095fig3.jpg"> 
图 3.臭氧暴露降低肺功能.(a-c)对于 air-exposed 和臭氧暴露的小鼠, 在前 25, 50, 和75毫秒的快速过期 (FEV25, FEV50, FEV75分别) 的个人 FEV, 以及 FVC, 都记录。分别计算了 FEV25、FEV50和 FEV75到 FVC 的百分比。FEV25/FVC、FEV50/FVC 和 FEV75/FVC 在臭氧暴露的小鼠中均明显减少。数据显示为平均± S.E.M. * p 和 #60; 0.05, ** 和 #60; 0.01。<a href="//ecsource.jove.com/files/ftp_upload/56095/56095fig3large.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/56095/56095fig4.jpg"> 
图 4.臭氧暴露会降低疲劳时间和疲劳距离。(a) 记录了 air-or 臭氧暴露小鼠的个体运行时间。臭氧暴露小鼠的疲劳时间明显减少。(b) 记录了两组的个别跑距离。臭氧暴露的小鼠的疲劳距离明显下降。数据显示为平均± S.E.M. * P 和 #60; 0.05。<a href="//ecsource.jove.com/files/ftp_upload/56095/56095fig4large.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/56095/56095fig5.jpg"> 
图 5.BALF 计数的炎症细胞.(a) air-or 臭氧暴露小鼠的总细胞总数 (总数) 的个体和平均数。(b) 两组中的巨噬细胞 (MAC) 的个体和平均数目。(c) 两组中性粒细胞 (新) 的个体和平均数目。数据显示为平均± S.E.M. * p 和 #60; 0.05, ** 和 #60; 0.01。<a href="//ecsource.jove.com/files/ftp_upload/56095/56095fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
ithin 页 = "1" ><img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56095/56095fig6.jpg"> 
图 6.血清炎症和抗炎细胞因子检测.(a) air-or 臭氧暴露小鼠的 IL-10 量的个体值和平均值。(b) 两组 IL-1β的个人和平均价值。(c) 两组中 TNF-α量的个体值和平均值。数据显示为平均± S.E.M. * P 和 #60; 0.05。<a href="//ecsource.jove.com/files/ftp_upload/56095/56095fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure at JoVE.com

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

这项研究描述了一个新的慢性阻塞性肺疾病 (COPD) 动物模型的成功生成, 反复暴露小鼠到高浓度的臭氧。

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

generation-chronic-obstructive-pulmonary-disease-model-mice-repeated

Nanyang Technological University

Research

JoVE Journal

Medicine

10.9K 次观看.  Cellular Biomedicine Group, Shanghai. 这项研究描述了一个新的慢性阻塞性肺疾病 (COPD) 动物模型的成功生成, 反复暴露小鼠到高浓度的臭氧。

视频: Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

科研

免疫与感染

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

医学

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.

在暴露于臭氧的小鼠的弹性周期中的肺微 rna 分析

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

使用强迫振动技术在小鼠呼吸系统力学评价

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

生物学

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

使用全身吸入系统在小鼠中暴露香烟烟雾

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

小鼠肺气肿评价持续压力下的肺固定

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

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 ...

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

通过反复吸入结晶二氧化硅粉尘建立的矽肺小鼠模型

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

<meta charset="utf8"></head><body>鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

鼻腔博来霉素雾化诱导的肺纤维化小鼠模型

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...