TKMcG支持由从加拿大胸科学会的助学金。 作者的贡献所有参加中意境的手稿。此外，TKMcG发起的项目，收集了实验结果，撰写的稿件和严格审查作出了贡献。 AR收集和分析实验结果，起草的手稿，并严格审查作出了贡献。 LF收集的实验结果，并严格审查的稿件做出了贡献。 TFS和JGM严格审查的手稿。

下述步骤由麦吉尔大学机构动物护理委员会批准根据加拿大动物护理（CCAC）理事会的指引。 1。准备步骤<ol><li>解决方案： <ol><li>乙酰甲胆碱：准备的储备液，在50毫克/毫升，并连续稀释（1:1）的基础上进行测试5的浓度。允许的解决方案，以达到室温后雾化5。 </li><li>麻醉剂：不同的方案已经在不同品系小鼠（ 表1）的文献报道。 注 ：1养生下使用本协议。 </li></ol></li><li>设备：本协议适用于要么两个由flexiWare 7软件支持flexiVent几代。该软件的功能被分为三个模块：学习的定义与规划 ， 实验Session和审查报告 。 <ol><li>打开系统（仅flexiVent FX）和/或启动该软件。 </li><li>在第一个实验阶段或之前的任何时间，打开研究的定义与规划模块预定义的研究结构。 </li><li>点击“ 创建一个新的学习按钮，并按照向导创建一个研究，勾勒出协议，并定义实验组和学科加以研究。 </li><li>实验会话启动开放实验会话模块和启动序列的研究和模板选择。 </li><li>分配的对象的测定部位，并确认它的重量。 </li><li>继续进行，通过以下的步骤中描述的操作软件系统的校准。您将被提示在一个点上，所述套管被使用（步骤1.3.3）附加到Y油管用于校准。 </li><li> 关键的一步。Repea吨步骤1.2.6如果所获得的校准值是可接受的范围之外。 （校准值特定于模块的可接受的范围，请参阅flexiVent FX或flexiWare的 7用户手册）。 </li><li>取消提示启动通风和数据记录，除非准备开始实验。这些都可以在以后的某个开始。 </li></ol></li><li>主题： <ol><li>麻醉的主题，使用适当剂量的麻醉剂（ 表1）。 </li><li>验证这个课题已经达到了手术的麻醉水平。主体应到脚趾捏没有反应，其呼吸要定期和没有劳苦的。 </li><li>进行了气管切开手术，导管插入气管。 <ol><li>动物放置在它的后面，并提供一个热源（ 例如，温度控制的加热毯或灯位于从鼠标约45厘米，以避免过度加热，用60瓦的灯泡）。 </li><li>清洁日ê用酒精和咽喉区通过皮肤切口，暴露气管，轻轻分离颌下腺和肌肉层覆盖。 </li><li>使用一对微型镊子轻轻提起气管，并通过它下面的缝合。 </li><li>最近的喉软骨做一个小切口在气管而不切片切两环之间。 </li><li>将先前校准套管切口，轻轻推进气管内的长度约为5环。 注 ：本实验使用一个1.2厘米长的金属18号套管进行。 </li><li> 关键的一步。套管固定的地方使用缝合。附件应形成气密密封套管周围。 </li></ol></li></ol></li></ol> 2。机械通气<ol><li>把动物靠近呼吸机。 </li><li>通过选择预定义开始机械通气或定制的通风泊坞窗通风轮廓。 </li><li>将动物呼吸机通过Y油管。 </li><li> 关键的一步。对准动物的呼吸机，确保气管插管呼吸机在同一水平，以避免可能的导管阻塞或气管扭曲。 </li><li> 关键的一步。执行深通胀扰动通过双击扰动验证套管插入和附件的名称。在泄漏的情况下，系统应该超过3秒的时间内，能够容纳30 水柱的压力，没有过多的位移量（ 图1）。记录的体积和压力的痕迹，也应该是光滑，没有任何迹象显示偏移或变形，这些可能表明导管阻塞或错位。 </li><li>如果需要，生命体征传感器连接的心跳率和体温监测。数据记录可以被启动eitheŗ手动或自动通过脚本。 </li></ol> 3。肺功能测量测量或命令（ 例如，雾化器激活，事件标记），可以自动使用一个高度控制的和可重复的实验过程中（ 图2）的预定义的或定制的脚本。六个家庭的扰动而产生的一些参数可以被用来描述在基线的主题呼吸系统力学，并按照给定的挑战（ 表2）。 <ol><li> 关键的一步 。当您准备开始进行测量，运行深通胀招聘闭肺区和规范肺容积历史。 </li><li> 关键的一步 。验证的情况下通过运行一个测试的测量（ 如肺静脉-P或的PV-V）的自发吸气努力。观察选定的数据集视图的压力信号的痕迹。随着逐步PV曲线，压力高原应当加以明确界定，没有向下偏转。向下的压力摆在表示吸气努力从动物（ 图3）。 </li><li>通过双击启动选定的脚本，它的标题。在本研究中所用的脚本一般包括测量： <ul><li>基线测量序列，一式三份。 </li><li>激活雾化器吸 ​​入乙酰甲胆碱注气道反应性的评估：当系统提示，进入雾化器装入约100微升生理盐水或乙酰甲胆碱的解决方案。雾化会自动启动和停止。 </li><li>紧密间隔的测量（每10-15秒）一段约3分钟后，激活，雾化器的序列。 </li><li>提示执行另一项挑战，重复序列测量。 注：干燥雾化器支架内用棉签在between挑战可以帮助防止水滴或凝结在吸气线建设。 </li></ul></li><li>在实验结束时，停止通风和分离的主题。 </li><li>切换到下一个主体中的操作软件，并确认其重量。 </li><li> 关键的一步。冲洗和干燥雾化器，转接器，Y型管，套管主体之间。 </li><li>重复步骤1.2.6至3.6。 </li><li>在一天结束的时候，关闭实验会话。请记住，以冲洗和干燥雾化器，适配器，Y-油管和套管，并按照制造商的说明，离开实验室前清洁系统的呼气阀。 </li></ol> 4。数据分析该软件会自动计算并显示相关的参数扰动。它还提供了一个判定系数（COD），这反映了数学模型的数据拟合。每个数据集与绝缘被标记为排除由软件fficient COD。实验会议回顾回顾和报告模块的软件，数据进行重新分析和创造出口情况。 <ol><li>打开审查和报告模块，并创建一个出口的情况下，照顾到只包括数据集，具有足够的COD。 </li><li>出口所需的参数，压力或流量-容积曲线，原始数据集信号或受到信息的电子表格应用程序（ 见表3）。 </li><li>每个参数和情节的平均基线测量所有测量作为时间的函数（ 见图4）。然后，您可以选择曲线下的面积来计算，分析的一般轮廓曲线进行统计分析。 </li><li>为了表达的乙酰甲胆碱浓度作为一个功能的气道反应性的结果，确定为每个主题，或者是特定的参数和实验条件点（ 如峰值）或每个乙酰甲胆碱激发后的特定时间。计算组平均值和的报告或情节业绩为每个实验条件下（ 表4，图5）。 </li><li>您也可以考虑计算的浓度产生一个给定的参数基线值（PC 200; 图5C）增加了一倍，申请归一化（ 如 ％为基准），或进行统计分析。 </li></ol>

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AR，LF，TFS受雇于科学SCIREQ呼吸设备公司TFS还拥有股票。 这篇文章是由SCIREQ科学呼吸设备公司免费使用

持续气道功能障碍，因为它涉及到哮喘和其他肺部疾病的研究仍然是最重要的了解相关疾病和治疗方案的发展机制。利用小鼠模型气道疾病已经逐渐认识到这些疾病的机制至关重要。当考虑为小鼠标在某一学科评价气道功能障碍，具有可靠和准确的工具来衡量肺功能关键。此外，有工具，能够提供见解的位置上气道功能障碍或治疗效果是非常宝贵的。 FOT技术结合所有这些属性，并且提供了强大的，综合和翻译的方法来评估生理变化。 为了取得成功，这种类型的测量小鼠，应特别注意几个步骤，即校准系统，气管CA的阻力nnula，雾化器（以及它的工作设置）不同的动物和标准化的肺容积历史的定位。此外，它是必要的，以获得有效的数据集，被检者的呼吸系统，在测量过程中保持被动。肌肉瘫痪剂的管理，可以实现深的麻醉平面或过度换气的主题，引起呼吸暂停（ 见表1）。研究者可以开始掌握了系统和操作软件，如果需要的话，试验载荷，同时获得必要的技能，在小鼠测量。然后，它将逻辑之前在天真的动物疾病模型或治疗的小鼠产生重复性的结果。由于疾病模型的一个重要比例在呼吸道的研究涉及动物暴露剂，如过敏原，毒素，污染物，香烟烟雾或气体，得到的结果与测量 - 变异吨在这篇文章中描述的技术，因此可使用曝光程序的影响。标准化的关键实验过程（ 例如，使用电脑控制曝光和测量系统6，13，14），可能有一个显着减少变异的影响。 在这篇文章中提出的例子代表了天真和氯气暴露小鼠实验的典型结果，而选择突出的强项，以及该技术的局限性。例如，在图6中可以看出，该技术是能够产生可重复的肺功能测量。虽然类似的基准电阻 ​​值之间的小鼠品系，不同的顺应性然而观察15。实质性的变化也是可以预期的婴儿和成年小鼠16之间。至于其他的在体内的生理评估，精度高的结果，如第OSE所产生的FOT，让步为主体的自然状态。这条原则，这被称为表型的不确定性原理1，适用于本协议，在这个意义上，测量需要进行麻醉，tracheotomised（或口头气管插管）和机械通风科目。该技术的另一个限制是观察图5D-5F如果没有数据可在最高浓度氯气暴露组，因为恒定相位模型数据的拟合较差中等以上水平的支气管收缩。然而，可以评估严重bronchoconstricted动物，分析Zrs的直接15，或者使用第三方分析软件，以适应更复杂的数学模型， 例如考虑到机械功能的异质性17。也可以观察到，如果动物的气道不能充分p排除的数据集assive或如果所述套管的电阻过高。作为一个经验法则，在套管的电阻不应超过动物的电阻在基准。插管内径较大和/或长度较短工作将有助于减少套管电阻。最后，本示范FOT测量小鼠可能会​​被看作是一个耗时的，因此效率较低的方法或不太适用于纵向相比，微创技术的研究。但是后者具有很大的不确定性，他们的成果的基础上，被视为由许多硬伤1。口服插管的动物有可能在反复创测量，虽然技术上更具挑战性的17。 从提供的例子，结果表明两代的flexiVent系统的等价性呼吸力学测量，以及呼吸道hyperreac的吸入乙酰甲胆碱的灵敏度和过敏氯曝光后的作用。当用于表征或理解的生理变化或疾病模型，有关的技术的详细的测量方面可以向延长的当前状态的知识。

足够小动物的肺中的力学性能表征已成为必不可少的，因为在呼吸道科学蓬勃发展的小鼠模型。当进行强迫振荡技术（FOT），这种技术也可用于人类受试者，这些测量提供了一个强大的，综合性和平移的方法来研究有意义的生理变化。 FOT测量通常是通过分析得到的压力和体积反应一个预定义的，小振幅振荡气流波形（也称为作为扰动或输入信号）施加在被检者的气道开放1获取的信号。在其最简单的形式中，一个FOT扰动在一个明确定义的频率将是一个单一的正弦波波形。更复杂的扰动通常包括一个涵盖广泛的选择特定的（互质）频率波形叠加。分解的多频他们的组成部分，使用傅立叶变换的输入和输出信号转换成允许呼吸系统的输入阻抗（Zrs的）的计算， 即输入和输出信号之间的传递函数，在每一频率中包含的扰动2。因此，在一个范围内的频率在一个单一的机动2 FOT允许同时评估呼吸力学。进入呼吸道（中枢和外周）和肺实质组织相关的参数1，3，安装先进的数学模型（ 如恒定阶段模型3）的阻抗数据，然后允许分割响应。由于有很多因素影响的生理反应（ 例如，呼吸频率，潮气量，肺容积，上呼吸道，自主呼吸努力，定时测量）的控制和标准的测量系统和实验程序，帽1的技术是能够产生精确和可重复的测量，它是正确执行。这篇文章的目的是为了执行测量小鼠所需的程序提供了一个详细的，按时间顺序描述。该协议包括四个部分：准备步骤（试剂，设备和学科），机械通气，肺功能测量和数据分析。呼吸系统力学的例子具有代表性的结果产生，使用一台电脑控制的活塞的呼吸（flexiVent，公司SCIREQ，蒙特利尔，QC，加拿大）提供。在这些得到从天真的小鼠，以及从气道损伤，其特征在于气道炎症，上皮细胞的损伤及气道反应性增加雾化乙酰甲胆碱吸入4的氧化应激的驱动模型。虽然该协议通常被用来评估吸入乙酰甲胆碱的气道反应性，它延伸到其他的结果和各种Ş的病症，包括哮喘，慢性阻塞性肺疾病（COPD），肺气肿，肺纤维化，肺损伤，以及类似于人类疾病的病理学的转基因小鼠模型。研究发现，使用此工具可以促进一个更好的特性和生理变化或疾病模型的理解，以及对新的研究领域进军。

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evaluation of respiratory system mechanics in mice using the forced oscillation technique

强迫振荡技术（FOT）是一个强大的，综合和翻译工具，允许实验小鼠在一个全面，细致，精确和可重复的方式评估肺功能。它提供了呼吸系统力学的测量，通过分析压力与容积采集的信号反应预定义的，小幅度的振荡气流波形，这是典型的适用于主体的气道开放。本协议所需的步骤，以充分执行在使用计算机控制的活塞呼吸（flexiVent公司SCIREQ，蒙特利尔，QC，加拿大）的小鼠强迫振动测量。的说明，分为四个部分：预备步骤，机械通气，肺功能测量，和数据分析。它还包括如何评估麻醉小鼠吸入乙酰甲胆碱气道反应，常见的应用本techniquE，这也延伸到其他的成果和各种肺部病症。天真的小鼠获得的测量以及气道损伤的氧化应激的驱动模型都说明这个工具可以有助于更好的研究生理变化或疾病模型的表征和理解以及应用新的研究领域。

呼吸系统力学测量 。 表4显示了从天真的A / J小鼠在基线和乙酰甲胆碱诱导的支气管（12.5毫克/毫升）使用两个由flexiWare 7软件支持flexiVent几代的典型结果。力学呼吸道系统进行评估，，即胸外条件下，在一个紧密排列的方式（快照-150，快速首相-3），分别交替扰动单和宽带频率强迫振动家庭。由于通风在测量过程中，快速首相-3，其中涵盖了类似的频率范围为总理8暂停，但持续时间较短（3比8秒），为了缩短呼吸暂停期间选择的影响降到最低血气和扰动的响应提供更好的分辨率。扰动与每个相关联的参数，计算出的自动化LLY由操作系统软件。结果表明，两代的flexiVent系统产生相同的呼吸力学测量。 网站肺癌响应 。辨肺癌响应的部位允许，研究者进一步查明受影响的地区，以及找出潜在的药物干预6点。例如，天真的A / J小鼠显示基线阻力增加时，对测量呼气末正压增加3至9 水柱 （ 图6A，快照150）。另外，在本实施例中，宽带FOT测量（连结素-3）的使用提供了细节澄清产生的电阻变化的基础：在呼气末正压的变化导致减少气道阻力（R N）一致的支气管扩张效果较高的肺容积和较大的通货膨胀出版社URE（ 图6D），并增加组织阻尼（G， 图6E），组织电阻，反映组织的粘弹性和可能小气道7的电阻密切相关的参数。后者是已知会增加肺容积增加。 气道高反应性 。加氯气后曝光，吸入乙酰甲胆碱的气道反应性相比，暴露在空气中，在Balb / c小鼠作为气道损伤4（ 图2）的结果。氯是众所周知的诱导氧化应激，导致呼吸道细胞结构的破坏，特别是上皮细胞，诱导炎性细胞的招聘。正如图5中所示，可以看出，所有参数的变化描述呼吸系统力学响应增加的乙酰甲胆碱的挑战。小鼠暴露在空气中暴露的小鼠相比，氯化在所有的FOT参数（ 图5A，5B，5D-5F），以及一个统计上显着的浓度-反应曲线左移举出引起的电阻增加一倍所需的乙酰甲胆碱浓度降低，NE气体显示的更大的最大响应和弹性（PC 200; 图5C）。这些结果表明，气道高反应和乙酰胆碱吸入氯气暴露后过敏。 其他测量 。除FOT，的flexiVent系统也可以被用于收集肺功能8-10或心血管11测量的其他类型的图7显示了有代表性的逐步，压力驱动的压力-体积曲线在基准条件下的幼稚的A / J小鼠。通缩肢体的上部的曲线适合Salazar的诺尔斯式（12） </<html>燮&gt;参数自动计算软件。 <img src="/files/ftp_upload/50172/50172table1.jpg" alt="表1" fo:src="/files/ftp_upload/50172/50172table1highres.jpg" fo:content-width="6in" /> 表1中。小鼠中使用的麻醉方案的例子。 <a href="https://www.jove.com/files/ftp_upload/50172/50172table1large.jpg" target="_blank">点击这里查看更大的表</a> 。 <img src="/files/ftp_upload/50172/50172table2.jpg" alt="表2" fo:src="/files/ftp_upload/50172/50172table2highres.jpg" fo:content-width="5in" /> 表2中。用于肺功能测量在小鼠中的摄动。*扩展所需的系统。的问题还需要在测量过程中，在一个封闭的体积描记器室。平直“&gt;点击这里查看更大的表。 <img src="/files/ftp_upload/50172/50172table3.jpg" alt="表3" fo:src="/files/ftp_upload/50172/50172table3highres.jpg" fo:content-width="6in" /> 表3中。例如出口参数单和宽带频率强迫振动扰动家庭。 <a href="https://www.jove.com/files/ftp_upload/50172/50172table3large.jpg" target="_blank">点击此处查看大表</a> 。 <img src="/files/ftp_upload/50172/50172table4.jpg" alt="表4" fo:src="/files/ftp_upload/50172/50172table4highres.jpg" fo:content-width="6in" /> 表4中。肺力学参数采集系统的比较。比较两代操作flexiVent系统由flexiWare 7软件。结果中产生幼稚的A / J小鼠（N = 5 /<html>组）在基线和下面的乙酰甲胆碱诱导的支气管收缩（位3 12.5毫克/毫升）。 *组间比较采用一个双向重复测量的方差分析和日志10个人响应方差齐性（GraphPad棱镜，5.03版GraphPad软件，圣地亚哥，美国）。 <img src="/files/ftp_upload/50172/50172fig1.jpg" alt="图1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50172/50172fig1highres.jpg"/> 图1。面板上显示的深肺通胀。截图呼吸机（红色线）和本科目（灰色轨迹）的交付量由活塞的体积。下面板显示缸的压力升高到设定压力为30 水柱超过3秒的一段时间，并在同一时期内保持恒定。 172/50172fig2.jpg“ALT =”图2“/&gt; 图2。例如一个典型的脚本在基线用来评估呼吸系统力学。 <img src="/files/ftp_upload/50172/50172fig3.jpg" alt="图3" /> 图3。自燃吸气努力的执行过程中的一个逐步的压力-体积曲线。 <img src="/files/ftp_upload/50172/50172fig4.jpg" alt="图4" fo:content-width="2.6in" fo:src="/files/ftp_upload/50172/50172fig4highres.jpg"/> 图4。时间当然响应以下增加吸入乙酰胆碱挑战。结果表示为一组5天真的自发性高反应性的A / J小鼠平均（±标准偏差）。 <a href="https://www.jove.com/files/ftp_upload/50172/50172fig4large.jpg" target="_blank">点击这里查看大图</a> 。 &lt;P类=的“jove_content”FO：保持together.within“=”总是“&gt; <img src="/files/ftp_upload/50172/50172fig5.jpg" alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/50172/50172fig5highres.jpg"/> 图5。呼吸系统力学变化挑战氯乙酰胆碱增加和暴露在空气中的BALB / C小鼠。峰值被确定为每个参数在每一个主题和实验条件。集团的平均值，然后计算（平均值±标准差，N = 4-6）。群体之间的差异进行了评估，经方差分析，方差齐性使用日志10个人回应。产生倍增的基线（PC 200）的乙酰甲胆碱的浓度，得到一个二阶多项式拟合个别的剂量-反应曲线和拟合曲线的插补。缺少数据点在D，E和 F中的氯暴露小鼠在两个最高的乙酰胆碱浓度由于不够高系数反映的数学模型与数据的拟合较差的决心。 <a href="https://www.jove.com/files/ftp_upload/50172/50172fig5large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50172/50172fig6.jpg" alt="图6" fo:content-width="6in" fo:src="/files/ftp_upload/50172/50172fig6highres.jpg"/> 图6。分区的呼吸进入气道和肺组织力学响应。说明单（2.5赫兹）和宽带（1-20.5Hz）频率的A / J小鼠实验跟踪从一个天真的强迫振动测量呼吸力学，一式三份，在两个不同的呼气末压力（2 O 3和第9 CMH） <a href="https://www.jove.com/files/ftp_upload/50172/50172fig6large.jpg" target="_blank">点击此处查看大图</a> </A&gt;。 <img src="/files/ftp_upload/50172/50172fig7.jpg" alt="图7" fo:content-width="4in" fo:src="/files/ftp_upload/50172/50172fig7highres.jpg"/> 图7。压力-容积曲线在基准条件下的天真A / J小鼠。压力-容积曲线采用逐步的压力驱动扰动（PVS-P），以确保每只小鼠肺膨胀到相同压力下，独立于自己的病情产生。 Salazar的诺尔斯提取个别的压力 - 体积曲线的方程的参数也被平均，并以表的格式报告。结果表示为平均值±标准偏差组（n = 6）。

Watch this Scientific Journal Video about Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique at JoVE.com

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

本协议提供了一个详细的一步一步的程序需要执行呼吸系统力学测量以及使用强迫振荡技术在小鼠吸入乙酰甲胆碱气道反应性的评估（flexiVent的描述; SCIREQ公司，蒙特利尔，加拿大）。

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 ...

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JoVE Journal

Biology

56.0K 次观看.  McGill University. 本协议提供了一个详细的一步一步的程序需要执行呼吸系统力学测量以及使用强迫振荡技术在小鼠吸入乙酰甲胆碱气道反应性的评估（flexiVent的描述; SCIREQ公司，蒙特利尔，加拿大）。

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

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

科研

生物学

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

用奔放的整体体积描记测量呼吸功能的影响

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...