Name: automated measurement of pulmonary emphysema and small airway remodeling in cigarette smoke-exposed mice
Uploaded: 2015-01-16T14:00:00.000+00:00
Duration: 10 min 37 s
Description: Watch this Scientific Journal Video about Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice at JoVE.com

我们要感谢弗朗西斯Polverino医学博士，研究员布莱根妇女医院为她对本文的贡献，同时也莫妮卡姚明，BS和凯特韦迪尔，BS他们用小鼠饲养和暴露的小鼠香烟烟雾的援助。 这项工作是由公共卫生服务，美国国家心脏，肺和血液研究所资助HL111835，HL105339，HL114501，乘务员医学研究所格兰特＃CIA123046中，布莱根妇女医院 - 洛夫莱斯呼吸研究所协会，以及剑桥NIHR生物医学支持研究中心。

该协议需〜25周才能完成。协议暴露的小鼠于空气或烟为24周。在烟雾暴露的端部，该协议措施肺功能在小鼠和肺部充气到一个固定压力，固定，并在同一天除去。是需要的研究者嵌入，切割，和染色的肺切片（2-3天），并捕获和分析图像的额外时间（2-4天，取决于动物的数量的研究）。该协议也可以被用于测量在小鼠中与年龄有关的空域放大。 在本协议中所描述的所有程序已经批准的机构动物护理和使用委员会在布莱根妇女医院/哈佛医学院。 1.全身烟雾暴露<ol><li>暴露小鼠吸烟在全身烟雾曝光装置（参见图1）安装在通风橱中。 注:该设备自动加载的研究香烟轮，升ights的香烟，并在侧流烟收集室收集侧流烟，并弹出的香烟。该机结合了侧流烟与主流烟用泵从香烟中提取打造主流和侧流烟的混合物。在混合和稀释室风扇混合与周围空气的烟雾，并驱动烟雾进入曝光室中。 </li><li>地方笼含有小鼠而不除去笼盖（ 图1）在曝光室中。允许小鼠自由地围绕它们的笼子移动，并获得食物和水的烟雾暴露（〜1.75小时）的持续时间。 </li><li>将装满水的香烟室熄灭香烟弹出的下方的水桶。通过泵运行的乙醇（100％），将其连接到设备上。开关设备的微处理器元件，从而启动自动的装载香烟，泵的膨化，并加热香烟二极管灯的上婷线。 </li><li>该设备的负载，灯光和抽10支烟，在一个时间，然后推出用于香烟，取而代之的是一批新的10支香烟，并重复这一过程。每个周期需要9分钟。 </li><li>使其恢复小鼠他们从20支的第一天，40支香烟的第二天，60支香烟的第三天，80支香烟在第四天和100香烟第五天暴露于烟雾抽烟。在适应期，仔细观察小鼠遇险的迹象。 </li><li>经过5天的驯化的，暴露的小鼠每天100香烟，5或6天，每星期6个月。烟雾暴露的这个水平是必要的，以诱导在C57BL / 6野生型小鼠比较适中空域放大（23,24）。暴露对照小鼠到周围室内空气中6个月。烟驯化之前称重小鼠，此后每周以评估烟雾暴露对体重的影响。 </li><li>监测总悬浮颗粒物物（TSPM）在首个60支香烟后曝光室已被熏: <ol><li>称量滤纸，并将其放置到其连接到一个定时采样滤波器和干气计的在线过滤器保持器。定时取样滤波器拉空气通过滤纸和燃气表措施的采样期间的空气流在曝光室。 注:过滤器陷阱颗粒物为20 立方米的暴露室的空气通过过滤器（在干气计测量）通过。 </li><li>计算TPM计数和以前一样和每立方米空气采样（毫克）后，过滤器的重量的变化。理想的TPM的计数是150至200毫克/ 立方米。 </li></ol></li><li>之后所有的香烟都被熏，从曝光室移除笼并观察老鼠困难的症状20分钟。 </li><li>清洁用100％乙醇泵每次使用后，并清洁在机器双周所有端口和杆，以促进空气循环，防止焦油的积聚。 </li></ol> 2.肺功能测试（PFTS）和肺通货膨胀<ol><li>在曝光结束后，通过在200μl盐水的腹膜内途径（USP递送氯胺酮（100毫克/千克），甲苯噻嗪（10毫克/千克），以及乙酰丙嗪（3毫克/公斤）的鸡尾酒麻醉每只小鼠级），并使用兽医软膏对眼睛，以防止它们干燥。等到动物在麻醉的手术平面，评估使用脚趾捏法。 </li><li>剃皮肤前方气管，并用含碘溶液，然后用乙醇消毒的区域。使通过皮肤和皮下组织前使用高压灭菌剪刀气管一个中线切口，并分离sternothyroid肌肉用镊子以暴露气管。 </li><li>传递的丝线缝合后2英寸长的气管，使气管切开的气管与蒸压吨前方面racheal剪刀，插入气管插管（18 G）的进气管切开，并用缝合线将其固定就位。 </li><li>将鼠标连接到Y油管经由气管插管机械呼吸器的适配器，并采用以10ml / kg的潮气量和150次呼吸/分钟的呼吸速度开始机械通气。 注:确保鼠标充分麻醉，以获得精确的测量PFT正是在这个阶段很重要。重剂量的小鼠麻醉剂如果动物没有麻醉的手术平面。所有的PFT演习的整个持续时间为约7.5分钟，因此，它不是通常需要redose小鼠的麻醉剂一旦麻醉的手术平面已经实现。从麻醉诱导安乐死的总时间为〜20分钟。 </li><li>膨胀肺部肺总量（TLC）的3倍的量的历史测量吸气容量（IC）和降低肺不张。接下来，perfor米的单一频率强迫振荡动作（快照150扰动），并评估动态电阻（R），弹性（ERS）和顺应性（CRS）呼吸系统，其次是宽带频率强迫振荡动作（快速总理-3-扰动），并测量中央气道阻力（R N），组织电阻（G）中，组织弹性（H）和比G / N（）。最后，在卷压侧流机动记录quasic-静态顺应性（C ST）。 </li><li>重复这些动作的五倍（或执行其他的措施，直到读数一致获得），每个重复组测量之间膨胀到TLC三次。记录每个参数对各小鼠的平均值。 </li><li>从机械通气机取出小鼠，并用CO 2麻醉，随后通过颈椎脱位安乐死它〔此安乐死方法是经我们的机构动物护理和使用委员会。切开膈肌，运恩胸部在中线，并删除前肋骨揭露肺部。解剖的皮肤和周围的气管皮下组织并传递给气管丝线缝合后的第二2英寸的长度。 </li><li>准备设备肺膨胀（ 图2）: <ol><li>填充的500ml锥形Erlenmeyer烧瓶¾充分用无菌PBS，用橡胶塞密封它，其反转，并暂停其上的环支架（使得磷酸盐缓冲盐水pH7.4的（PBS）中的弯月面为25厘米以上鼠标的心脏）。 </li><li>插入的静脉内给予设置到PBS中，通过橡胶塞烧瓶一端。插入穿过橡胶塞的塑料血清移液管的一个6英寸长的，使得它的开口位于与PBS的弯月面以上，以允许空气取代的PBS离开烧瓶中。 </li><li>打开给一套阀门和运行PBS，虽然该系统冲洗掉空气从给集。 </li></ol></li><li>连接INTRavenous给予组的气管插管，打开阀门，并允许PBS流入肺部通过重力，直到肺完全膨胀。关闭阀门，用手术缝合远端气管插管扎断气管，并取出套管。 </li><li>解除用钳子气管，并切断气管近端结，并解剖结缔组织后气管和肺。小心地取出肺（无切口它们），并放置在肺部中含有10％的盐水，缓冲的福尔马林的管。修复肺O / N在RT，并在第二天，用PBS冲洗两次。 </li><li>嵌入在石蜡肺部，切为5μm厚的切片，然后染色，如下所述用吉尔氏染色部分。 </li></ol> 3.肺气肿<ol><li>石蜡包埋肺切片鳃的染色<ol><li>将载玻片在一个塑料机架孵育它们在70℃烘箱中20-30分钟。 </li><li>德pariffinize幻灯片孵育它们中的每一个的二甲苯四个转变2分钟。 </li><li>再水化载玻片孵育它们中的每一个的100％乙醇的两个转变，接着在各为95％的乙醇的两种变化2-3分钟2-3分钟，然后在PBS中洗两次幻灯片2-3分钟为解决每一个变化。 </li><li>孵育载玻片用于在1 18-48 H:1混合物吉尔的苏木的和改性的哈里斯苏木。 </li><li>洗的幻灯片中的每一个的蒸馏水5变化2分钟，并孵育然后在每个95％乙醇两种变化2-3分钟脱水的幻灯片，随后在每一个的100％2变化2-3分钟乙醇。 </li><li>孵育它们中的每一个的二甲苯四个转变2分钟清除幻灯片。 </li><li>安装幻灯片有明确的安装介质，并添加盖玻片不引入气泡，这会阻碍后续分析。 </li></ol></li><li>随机图像采集: <ol><li>获得黑白图像，TIFF文件S使用显微镜，20 X目标，一个摄像头和软件，可以获取高质量的数字图像。 </li><li>捕获〜20-30图像（×200倍）每只小鼠以随机方式，与失明的实验条件的观测器，避免了肺的充气不足的区域。 </li><li>大盘微滑现场取景器上的幻灯片。实地取景具有包含一系列被标有字母（在垂直方向）的方格的网格和多个（在水平方向），并且每个正方形的横（+），在该中心，并通过一个字母标识和号码（ 例如 ，A1，A2，...... Z25）。 </li><li>使用随机现场生成Excel电子表格中随机选择的图像捕捉一个正方形。要创建一个随机的信，在电子表格中输入EFGHJKLMNPQRSTU单元格B1（研究者可以调整这封信范围涵盖确定一个字母是覆盖在肺部平方的范围内）。接下来，添加公式[= MID（$ B $ 1,1 + INT（RAND（）* LEN（$ B $ 1）]，以单元格C1随机选择一个字母在C1，复制并粘贴到C1 C2，C3，C4等等产生的随机字母列。 </li><li>要创建相邻列中的随机数，输入公式= RANDBETWEEN（5,25）到D1（其中5-25是幻灯片上的典型范围组织）。任选地，调整该范围涵盖确定一个数字，覆盖在肺部的正方形的范围内。 </li><li>复制和粘贴D1到D2，D3，D4等等来产生随机数的旁边，含有随机字母列。因此，每行包含一个对对应于标签上在该领域寻找者的平方随机生成的字母和数字（ 例如 ，E17，H24 ...）。 </li><li>放置在显微镜载玻片上。使用4X显微镜物镜找到在现场取景滑动的相应方（ 例如 ，E17，H24 ...），并把此方在显微镜视野的中心。 </li><li>对准显微镜的中心在选定广场中心领域的"+"。除去现场取景器，专注于使用20X显微镜物镜肺和获取图像的灰度TIF文件。如果肺组织覆盖&lt;微观领域的50％，选择下一个随机生成的广场。重复，直到〜20-30图像捕捉每个动物。保存每个动物的所有图像中，为了使报告的Excel宏来识别的文件标记的动物的标签号码的单个文件夹。 </li></ol></li></ol> 4.形态测量，测量肺气肿该协议使用接穗图像和定制宏分析领空扩大。接穗图像是在Macintosh操作系统上运行原有的NIH图像应用程序的Windows兼容的版本。接穗图像在Windows XP下运行，仍然可以在网上通过Wikiversity.org，其中搜索"接穗图像"将引导用户链接到手动和β4.0.2版本接穗图像。安装和软件操作详见网上补充手动和总结如下。肺泡弦长宏改编自NIH图像中可用的宏。 <ol><li>准备吉尔的接穗进行图像分析染色肺段的TIFF图像: <ol><li>启动接穗图像和加载宏在网上补充表示。选择打开图像明场[1]宏来选择并打开图像TIFF文件。接下来，使用图像编辑工具，以准备图像进行分析。 </li><li>使用油漆刷工具强制图像区域未空域或肺泡壁来对待无论是作为空域或组织。例如，油漆支气管和血管黑，使得它们作为组织进行分析。漆炎性细胞（或灰尘）占据的空间在肺泡白色到它们作为空域分析。 </li><li>通过点击鼠标指针选择画笔的颜色也就是说黑色或白色的LUT窗口的底部。接下来，单击画笔工具。要改变笔刷大小，双击画笔工具，并输入一个合适的画笔大小。单击并拖动鼠标在图像绘制结构所选择的颜色（见上面＃1）。 </li></ol></li><li>测量空域平均弦长<ol><li>选择宏弦长空气[2]来测量空域弦长。 </li><li>阈图像通过第一，点击靠近图像中心的鼠标然后拖动鼠标向上或向下调整阈值（一个介于0和255，其被显示在信息窗口）。点击鼠标第二次接受阈值。调整阈值，以使肺泡壁的厚度相同，在原始的图像。 注:至关重要的是，研究者不阈值之下，从而创建在肺泡壁断裂中不将产生与原始图像存在虚高的弦长值。 </li><li>该程序会自动删除单个像素（8白色像素包围单一的黑色像素）。 </li><li> EM&gt; 4.2.4。观察提示用户重新阈二进制图象的一个窗口，继续宏，或者取消该宏。再次门槛，回答Y的提示，并选择确定按钮。 </li><li> EM&gt; 4.2.5。可视化的水平和垂直网格窗口与线5像素相隔宏创建。该程序测量的重叠空域水平线和垂直线的长度。 </li><li>保存在任何文件夹中的文件，但不要更改默认名称，否则Excel报表宏将找不到文件（格式为空域弦长附加"CLa.txt"的形象的名字。 注意:如果程序不进行测量时，阈值可能太低（必须&gt; 1）。如果发生这种情况，研究人员重新阈值使用更高值的图像。 </li><li> EM&gt; 4.2.7。对于肺泡区测量（除了弦长），运行额外[4]和[5]的宏以及。 </li><li> EM&gt; 4.2.8。继续进行，直到所有的图像进行了分析。 </li></ol></li><li>分析使用Excel报表宏结果<ol><li>打开Excel报表20x.xls。手动启用宏如有必要，取决于Excel版本的默认安全设置被使用。 </li><li>观察宏窗口宏的列表（见表1）。 注:CL_Air_1报告空域的单一的动物（文件夹）的弦长。 CL_Air_Multi报告空域多个动物（文件夹）的弦长。 AP_No_Edge_1报告肺泡面积没有一个单一的动物（文件夹）的边缘接触.AP_No_Edge_Multi没有多个动物（文件夹）的边缘接触报告肺泡面积。 AP_With_Edge_1报告肺泡与一个单一的动物（文件夹）边缘的接触面积。 AP_With_Edge_Multi与边缘Ç报告肺泡面积ontacts多个文件夹。 </li><li>选择CL_Air_Multi宏报告肺泡弦长测量对应于来自多个小鼠的图像的多个文件夹。该计划报告所有选定文件夹中的_CLa.txt文件。通过将其移动到当前文件夹的子文件夹或重命名_CLa部分省略_CLa.txt文件（如需要）。 </li><li>从文件窗口中，通过突出显示该文件夹，然后选择OK（该程序不支持多选在同一时间），一次选择一个文件夹。由于每个文件夹中选择，观察它在工作表上。选择"取消"或关闭文件窗口以便继续。 注:根据Excel的版本中，研究人员可能被选中的每个文件夹后，导航回到一级文件夹。 </li><li>观察一个单独的工作表为每个文件夹，显示出统计后统计弦长数据的所有_CLa.txt文件结合每个_CLa.txt文件。 </li><l我&gt;一个单独的工作表"的所有CL"包含了所有的所有工作表中的数据。 </li><li>重命名并保存电子表格。默认的文件名是追加_CLa.xls父文件夹的名称。选择另一个宏之前关闭工作表。 </li></ol></li></ol> 5.小气道重塑<ol><li>染色和图像采集<ol><li>染色使用商业试剂盒的肺切片与Masson三色染色，并按照制造商的说明。 </li><li>两肺，可完全被容纳（包括蓝色层，它是ECM蛋白的层，其被沉积在气道外）用20X物镜的显微镜的图像区域内捕获所有气道的图像。 注:较大的气道不与ECM蛋白的CS暴露小鼠增加沉积有关。 </li><li>保存彩色图像为JPEG文件。 </li></ol></li><li>图像分析:打开图像文件在图像分析软件程序。 <ol><li>打开并命名记录测量日志文件。 </li><li>选择画线工具。绘制4线交叉的气道的内腔（内直径）来测量气道的尺寸和只包括那些具有气道中的分析所需的大小。接着，绘制12等距线（在对应于一个时钟的数目的气道的位置）从外膜层邻接的气道出到蓝染色的区域的边缘的边缘延伸的包围气道以测量厚度气道外沉积ECM蛋白的层（ 图5）。避免测量那里的气道与其他气道或容器相互作用的区域。 </li><li>第一个记录中的所有记录文件的内直径线，然后记录12行，用于测量周围的气道蓝色的ECM层的厚度。 </li><li>关闭图像，然后打开一个图像。 </li> &lt;李&gt;重复这些步骤，直到动物所有图像都被记录到一个文件夹中。 </li><li>在开始的，其中包含从下捕捉动物的肺切片图像的下一个文件夹中的步骤5.2。从所有的实验动物的完成所有气道中的肺切片的分析之后退出程序。 </li><li>进入4个内部直径测量和记录每个气道每只小鼠中的数据日志文件到Excel电子表格12的ECM蛋白层的厚度测量。平均直径和ECM蛋白层厚度测量每只动物。 </li><li>转换测量从像素到微米。 </li><li>集团根据其内径尺寸的气道（ 例如 ，300-399微米，400-499微米等）的气道周围具有类似大小的空气与烟雾暴露的小鼠和比较ECM蛋白层厚度测量（ 例如 ，气道具有直径为300-899微米; <strong&gt;的图5）。 </li><li>如果需要的话，免疫染色的肺切片为单个蛋白质（包括胶原质和基底膜蛋白），并执行类似的分析以量化使用这种方法的目的蛋白质的沉积。 </li></ol></li></ol>

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We have no conflicts of interest to disclose.

Most researcher use mice to model the main chronic lung pathologies and abnormal lung physiology in COPD (airspace enlargement, SAR, and increases in lung compliance) present in the human disease. A comprehensive approach to assess the effect of molecules of interest on both emphysema development and SAR is needed in mice in order to comprehensively assess the activities of molecules of interest in these chronic disease processes. 
There are several critical steps in this protocol. First, dur...

利用动物模型，研究慢性阻塞性肺病是具有挑战性的，因为没有模式可以完美地复制人类疾病（2）的所有功能。大多数研究者用老鼠COPD模型，因为在他们的肺的生理，病理，遗传，代谢和小鼠和人类之间的相似之处。此外，老鼠是相对便宜的学习，和6个月的CS曝光（5,7-9）内均肺气肿和小气道重塑发展。 香烟烟雾诱导的慢性阻塞性肺病 :有几种方法可诱导的COPD小鼠。大多数研究人员暴露小鼠的CS，这是人类慢性阻塞性肺病的主要的致病因素。 CS暴露6个月导致肺气肿和小气道重塑（SAR）的小鼠的发展，但根据不同的小鼠品系研究这种疾病的被诱导的严重程度而变化。例如，NZWLacZ小鼠抗CS诱发肺气肿的发展而AKR / J小鼠是extremelŸ敏感（10）。大多数研究者研究C57BL / 6小鼠品系在CS曝光模式尽可能多的基因打靶小鼠可在这株。 6个月后的CS曝光，肺气肿和小气道纤维化的发展中的野生型（WT）C57BL / 6小鼠，并且两个病变的严重程度（5,10）相对温和。研究人员使用两种类型的CS曝光:鼻式和全身暴露。鼻式暴露技术的主要缺点是:1）它是一种劳动密集型方法;和2）小鼠在小室能够诱导在动物（11）的应力响应和热疗受到抑制。的全身暴露量（本文中所述）的主要缺点是，动物可以摄取（以及吸气）的尼古丁和焦油的产品时，他们清洁他们的皮毛。暴露于全身的CS小鼠也具有较低的碳氧水平，当与动物暴露于鼻式的CS（12）相比，减少的体重减轻。 肺功能检查（肺功能）:肺顺应性和顺应性的措施通常是在C57BL / 6野生型相似（WT）由于相对温和肺气肿，开发时，这种暴露在空气中或CS 6个月小鼠应变被暴露到CS（10）。然而，当气肿性破坏更加严重，增加肺顺应性和左移位，在压力容积（PV）流可以检测环路。后者可以被观察到，例如，在小鼠菌株更容易受到CS的效果（10），在具有比C57BL / 6野生型小鼠更严重的肺气肿类型的CS-暴露的C57BL / 6株基因靶向的小鼠（13），或者在经受使得它们更容易受到CS的效果（14）的环境变化的CS-暴露的小鼠。该协议使用小动物呼吸机来测量减少肺的弹性回缩力（增加准静态肺顺应性[Cst中]和减少组织顺应性[H]），光伏流动回路，并改变在麻醉小鼠（15,16）的气道和组织电阻。 肺气肿的措施:肺气肿发展的CS暴露C56BL / 6小鼠品系分析是具有挑战性的，因为它的分布空间异质性。几种不同的方法进行量化的小鼠空域放大图。所用的第一种方法是将平均线性截距（L M）（17）。然而，L M法是一种缓慢的，手动的过程，可能无法捕捉疾病（除非肺的所有部分是随机取样的）和它的用途，因此可能会引入偏见观察者到分析的异质性。破坏性指数[DI，（18）]也量化使用的透明片，用50均等地分布点放置在一个苏木精和伊红染色的肺部的印刷数字化图像空域放大。 PI法得分周围的每一点交流区盘带到何种程度上在此区域内的肺泡管和肺泡壁破坏。在DI法的主要缺点是，它是费时和不大于其他的方法（19,20）更加准确。 该协议的措施平均肺泡弦长和肺泡面积上沾满吉尔氏染色石蜡包埋的肺切片​​。形态软件转换的肺切片，以二值图像的图像（在该组织是白色的，空域是黑色），然后叠加水平和垂直线（和弦）的均匀网格，该软件然后量化内确定的区域的每个弦的长度软件领空。使用该方法，可以测量在标准化和相对自动化的方式（21）在肺的所有部分肺泡的大小。 小气道重塑（SAR）:ECM蛋白的沉积增多（尤其是interstitia升胶原蛋白）周围小气道发生在CS暴露的动物，并有助于气流阻塞。研究人员不学习特区COPD的动物模型那样频繁肺气肿发展（22）。量化特区中的CS-暴露的小鼠，这种协议使用图像分析软件来测量ECM蛋白的该沉积周围小气道（气道具有300到899微米之间的平均直径）在石蜡包埋肺切片的层的厚度沾上Masson三色染色。

<table><tbody><tr><td>Whole-body smoke exposure device</td><td>Teague Enterprise</td><td>TE-10z</td><td>Chronic Smoke exposures to induce chronic lung disease in mice</td></tr><tr><td>Research Cigarette</td><td>University of Kentucky</td><td>3R4F reference cigarettes</td><td></td></tr><tr><td>Pallflex&amp;reg; Air Monitoring Filters, Emfab Filters TX40HI20WW, 25 mm</td><td>Pall Corporation</td><td>7219</td><td>For measurement of TPMs</td></tr><tr><td>25 mm filter holder</td><td>Pall Corporation</td><td></td><td></td></tr><tr><td>Filter sampler</td><td>Intermatic</td><td>Metal T100</td><td></td></tr><tr><td>Gas meter</td><td>AEM</td><td>Gas meters G1.6; G2.5; G4</td><td></td></tr><tr><td>Tracheal Cannula for mouse 18 gauge</td><td>Labinvention</td><td></td><td>Analysis of pulmonary function</td></tr><tr><td>Mechanical ventilator</td><td>Scireq</td><td>FlexiVent</td><td></td></tr><tr><td>Gill&#039;s hematoxylin solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>GSH316</td><td>For Gill staining, work under a fume hood</td></tr><tr><td>Hematoxylin solution, Harris modified</td><td>Sigma-Aldrich</td><td>HHS16</td><td></td></tr><tr><td>Cytoseal-60</td><td>Thermo Scientific</td><td>8310-16</td><td></td></tr><tr><td>Micro-Slide-Field-Finder</td><td>Andwin Scientific INC</td><td>50-949-582</td><td>For analysis of emphysema</td></tr><tr><td>Scion Image Program</td><td>Scion Corporation</td><td></td><td></td></tr><tr><td>Mason&#039;s trichrome stain</td><td>Sigma-Aldrich</td><td>HT15</td><td>For analysis of small airway fibrosis</td></tr><tr><td>MetaMorp Offline version 7.0</td><td>Molecullar Devices LLC</td><td>31032</td><td></td></tr></tbody></table>

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.

该协议开始于小鼠CS的全身暴露。适当的监督和维护该装置和监测的TPM的计数确保一致烟雾暴露（ 图1）。重要的是，研究人员使用的充气装置实行的肺充气技术该协议开始于小鼠CS的全身暴露。适当的监督和维护该装置和监测的TPM的计数确保一致烟雾暴露（ 图1）。重要的是，研究人员实践使用充气装置（ 图2），并仔细的肺膨胀技术移?...

Watch this Scientific Journal Video about Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice at JoVE.com

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

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 ...

automated-measurement-pulmonary-emphysema-small-airway-remodeling

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.2K 次观看.  Brigham and Women's Hospital - Harvard Medical School. 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. 

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

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

科研

医学

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

免疫与感染

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

在小鼠肺压力容积曲线的测量

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

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

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

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 ...

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 ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

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 ...

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...