We would like to thank Prof David Walker for his technical advice and provision of equipment in the development of this technique. This work is supported by the Victorian Government&#8217;s Operational Infrastructure Support Program. This work was partly supported by the Victorian Government&#8217;s Operational Infrastructure Support Program.

注:以下实验步骤是由动物伦理委员会在莫纳什大学批准，并按照惯例动物的护理和使用用于科学目的（2006年），澳大利亚守则进行。用于产生代表结果成年雌性C57BL / 6小鼠购自莫纳什动物服务获得。小鼠饲养在无特定病原体，温度和湿度控制的房间有12小时明暗周期。这些老鼠都可以免费获得食物和水。 1，初始设置<ol><li>连接笔记本/台式机到数据采集设备通过USB电缆录制。 </li><li>通过BNC电缆从"输出1"的连接桥放大器"输入1"的数据采集机。 </li><li>将压力转换成"通道1"的八桥放大器的。把数据采集的机器上，并打开分析软件。本应自动软件matically检测设备安装（见材料和​​设备表）。 </li><li>在软件的设置工具栏中找到明渠设置。改变信道的数目被记录到1。 </li><li>成立晴雨表测量室压力和水柱设备校准桥式放大器。水柱装置包括通过塑料管连接两个5毫升血清注射器吸液管。 </li><li>填充柱，用水和确保水水平是用尺子均衡。连接一块塑料管向每个移液管的顶部， 图2示出了水柱成立。 </li></ol> 2，桥式放大器校准注意:要校准桥式放大器注射空气进入水体，需要建立1厘米的H 2 O的偏转。这将发生在一组条件，并取决于用户的设备时。为了澄清这些步骤demonstrate该实验室将如何进行校准。 <ol><li>撤销1毫升注射器300微升;连接注射器的活塞在上水柱的右手侧的管子的端部。注意:请确保活塞是开放的注射器和水柱，并关闭室内空气。如果水位在这一点上不均衡，旋转活栓，以便它是开放的室内空气和水柱，这将平衡的水。在水柱的左手侧的管道应该被连接到所述压力换能器来测量引起的切入注射器中的压力变化。 </li><li>附上从水柱管在左手侧上的压力换能器（换能器的顶圈）的连接器。 </li><li>选择发现旁边通道1在主屏幕上的软件的右侧，然后选择"桥接放大器"（见材料和​​设备表）的下拉菜单。 </li><li>输入设置为5 mV时，20 Hz的低通，选中"反相"复选框，然后单击"零"。点击"零"设置跟踪在〜0毫伏。减少窗口大小为4:1，方便观看。 </li><li>一切设置，按下1ml注射器的，离开它，持续3秒。这将显示在该软件的突然飙升，因为压力已经改变了。当加入300μl被压下的压力将1厘米移动的水的水柱。这个已知值将有助于校正桥式放大器。 注意:在该腔室由于300μl的抑郁压力的增加对应于用于以后计算为P K值。 </li><li>选择"输入设备"上桥放大器窗口的左下角找到。 </li><li>突出"后台跟踪"也被称为"零区域"秒杀之前。 <ol><li>点击旁边的箭头"1点"，这将产生BAC的-0.002毫伏0.002毫伏的范围内kground信号（该值永远不会恰好在0毫伏）。 </li><li>在窗口中输入"0"相邻的背景信号窗口。 </li></ol></li><li>从当注射器按下突出"的曲线上升压力区域"。点击旁边的箭头指向2和值应在0.9-1.2的范围内毫伏。 <ol><li>在旁边的"压力增大"窗口中的窗口中输入"1"。有关步骤2.7和2.8的可视澄清请参见图规定可能表明损坏八桥放大器的范围外发现3的值。 </li></ol></li><li>转到"定义单位的窗口右上角找到并选择" 水柱 "。如果此选项不可用，也可以手动输入。单击确定。 </li><li>返回到"桥放大器"菜单（参见2.1）。选择1 mV和设置放大器为"零";这将完成的校准和水柱可以安全地取出。 </li></ol> 3，记录肺功能<ol><li>称取鼠标（G）。注:一个星期前的生理评估引入鼠标的体积描记室环境。这将有助于驯化和执行时，这个程序在日后减少压力。为一个整体的概略展示UWBP设置，请参考图4。 </li><li>测量体温与直肠体温计。润滑与插入之前凡士林的温度计。记录的温度读数，用80％（V / V）乙醇清洗润滑剂关闭。如果用非常小的动物，如新生乳鼠中，平均体温值可以用一个红外温度计，而不是确定的。 </li><li>将温度/相对湿度探头上的体积描记术室的一孔端。记录温度，湿度和barometrIC压力之前，将鼠标内的体积描记箱内。 </li><li>鼠标放置在该体积描记室，微微覆盖的开口端。这让鼠标适应环境。关闭室。 </li><li>用在体积描记腔与孔的侧面插入的温度/湿度传感器，现在插入的换能器和注射器在另一侧的两个孔。 </li><li>按软件程序和记录约15-45秒"开始"。录制5-10秒的数据，其中的动物是不动的。移动会改变动物的基本呼吸生理学，并提供效果差。呼吸应以振荡在节目的线性路径。这些都是有用的数据。注意:排尿或排便可以导致增加的温度和湿度的体积描记腔室内部。这将在分析过程中掩盖的结果。在排尿或排便的情况，应立即停止记录和清洁PLethysmography室用80％（V / V）乙醇。请参考图6为次优的结果，其中数据应被拒绝的视觉表示。 </li><li>记录为45秒，按"停止"的软件后（见材料和​​设备表）程序。从体积描记室取出小鼠，并立即记录该室的温度和湿度。不要连续超过45秒的记录，因为这可能会强调动物。 </li><li>返回鼠标笼子，喷雾和用80％（V / V）乙醇擦拭该室。 </li><li>允许该腔室至干，在进行到下一个鼠标之前返回基线的温度和湿度。重复步骤3.1至3.9为后续的动物。注意:如果多个动物正在研究中，确保室内的温度和湿度返回至接近基线值的每个新的动物之前被放置在腔室中。 </li></ol> 4，体积描记分析无TE:计算呼吸参数，例如潮气量（V T）和分钟量以下变量需要被测定:呼吸率（次/ min），总呼吸周期时间（秒），吸气/呼气时间（钛/碲，秒）和压力的变化，由于各潮气量（P T）， 图1示出了可以从一个跟踪被测量的变量。下面的步骤使用的软件（见材料和​​设备表）来测量这些变量。在分析，避免含嗅或运动轨迹的地区。为可再现的结果，至少5秒钟的良好呼吸迹是必需的。对于不同呼吸痕迹，例如参见图5和图6。 <ol><li>打开屏幕以全屏，设置视图以1:1和选择5秒可利用的数据。这方面的一个有代表性的快照显示在图5。 </li><li>在打开的亲顶部发现的迷你数据焊盘窗口克在DataPad标签。选择通道1，选择"循环测量"的左栏和"循环平均身高"的右栏。 <ol><li>选择"选项"，然后设置规模最小峰值检测到1（毫秒）。这将允许检出每一个峰值，并使用产生小幅度摆动的小动物时，就显得极为重要。 </li><li>点击"确定"。这将显示"压力变形，由于每个潮气量&#39;（P T）测量。 </li></ol></li><li>在迷你资料片，选择"循环测试"之后"事件计数"，然后点击"确定"。这将显示"频率"（f）的测量。 <ol><li>频率需要被转换为次/ min。这是通过由60秒相乘的值并除以回答由记录（分钟）的总时间内完成。 </li></ol></li><li>在迷你资料片，选择"CY骑士测量&#39;后面&#39;时期"，然后单击"确定"。这将显示"共呼吸循环时间"（T TOT，秒）测量。 </li><li>接下来的步骤是用来创建一个宏指令产生峰值吸气和呼气的时间值。确保光标是直接在高峰/低谷的最大和添加注释9连续的波峰和波谷。与振荡的峰值开始， 如图5。 </li><li>随后，选择窗口:数据盘和1列在出现点击左栏"持续时间"在右栏"选择信息"，然后点击"确定"窗口。 </li><li>选择宏在程序的顶部发现了，然后开始录制。现在选择命令:"查找"，"去"，"开始的文件"，然后点击"查找"。 </li><li>选择命令:"查找"和"查找评论"。键入相同的短语输入中提供的"含"框中的评论框。选择"选择要上点"选项卡，"查找"。 </li><li>选择命令:"添加到资料片"。接下来，选择宏:宏命令，并开始重复。出现的重复计数窗应设置为9。 </li><li>选择命令:"查找下一个"。选择命令:"添加到资料片"。最后选择宏命令和结束再说。 <ol><li>现在选择的宏观和停止录制。保存并命名的动物数量后宏。注:设置为每个动物的宏允许用于纵向研究宏观和节省时间。 </li></ol></li><li>宏现在可以运行，以获得每个评论的启示（T I）和到期（T E）的时间。这些数据将出现在datapad的通道1。呼气和吸气时连续的数据将​​出现在这个顺序。 <ol><li>这些数据需要手动分成吸气和呼气值。平均每个参数的四个数据值来获得的平均T I和Tê。 </li></ol></li><li>一旦初级数值已经获得的潮气量（V T，毫升）可以计算出来。为了获得潮气量Drorbaugh和芬恩8的公式用于: V T（毫升）=（P T / pk信息）×（V K）×（（T CORE（P B - P C））/（T CORE（P B - P C） - T C（P B - P CORE））） 在哪里 V T:潮气量 pk信息 :压力偏转由于每次注射1毫升（参见步骤2.5） 牛逼的核心 :每个动物的核心温度 P C:在茶室内温度过高X相对湿度水汽压MBER T C:温度在动物室 P 芯 :压力在体温下（在体温下的水蒸气压力×1.0） P T:压力偏转，由于每个潮气量 V k为容量注射剂用于校准 P B:气压 </li><li>一旦潮气量已经计算下列参数也可以被确定: <ul><li>分钟通气量（毫升/分钟）= V T XF </li><li>分钟通气量（毫升/分钟/公斤）=（V T XF）/体重（千克） </li><li> V T（毫升/千克）= V T（毫升）/体重（千克） </li><li>吸气占空比（％）= T I / T TOT </li><li>吸气流量（ml /秒）= V T / T I </li><li>吸气时间比到期时间为T I / TË </li><li>总周期时间​​（sEC）=吸气时间（秒）+到期时间（秒） </li></ul></li></ol>

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The authors declare that they have no competing interests. The authors have no conflicts to disclose.

这里描述的技术是奔放和麻醉的小鼠呼吸参数评估一种非侵入性的方法。该协议的优势包括它的简单性和精确性纵向测量肺功能以最少的人工制品。有，然而，一些局限性和关键步骤要注意有关的过程。首先，最重要的是，鼠标必须留室至少五秒钟内平静。增加的压力会破坏鼠标的呼吸模式，从而提供不同的结果（ 图6a）。这种缺点可能会保持和处于倍于预期。但是，更换鼠标在其家笼，并允许它的时间来重新安置会很容易纠正。至关重要的是，在动物感觉的室内环境舒适，以获得适当的/有用的数据五秒钟。进一步考虑，需要对室内环境。环境，Unrelated呼吸力学，可以显著影响结果。作为评估的增加，该腔室增加的湿度和温度，以及降低的有效氧的持续时间，显著上通风影响。换气的时间增加了腔室的湿度和温度，以及减少的有效氧21。小量泄漏，在室内可以减少热源生产22,23产生热漂移帮助。讨论的协议是专门针对列出的设备和仪器。在第2桥式放大器的校准将取决于读者的设备。如果等因素油管长度是不同的300微升注入空气会不会导致1 cm.H 2 O偏转。 也有根据分析的时间的生理差异。啮齿动物自然昼伏夜出的动物，昼夜循环，最终产生变化呼吸，应定时实验24时予以考虑。因此，这是必要的时间和计划，使得实验数据可以精确队列之间进行比较的实验。同样重要的是要注意到该跟踪运动。如果振荡不以线性模式运行时，它通常是由于缩合或湿度的积聚腔室（ 图6b）内，或无效的密封件上的腔室中。最终，这些限制可占的UWBP过程中进行适当地提供精确的呼吸测量。同样重要的是要注意，这种方法将需要修改（更小的腔室的大小），用于测量在新生儿标准实验室小鼠的呼吸的变化（ 例如 ，&lt;2周C57BL / 6），为了检测在该大小的动物的呼吸压力的变化。 虽然UWBP表明相当多的优点，还带有争议。调查人员应熟悉辩论，并作出明智的决定，这个技术是否适用于所研究的问题。最初，Drorbaugh和芬恩（1955）8认为增加腔室压力由吸入空气所引起被加热和加湿至肺的值;发生在到期相反。这允许潮气量的计算。随后的研究认为，压力变化，通过改变气流25的生成过程中的肺泡的压力引起的。该工作人员表示用体积描记法的气道阻力的计算。 Enhorning 等人（1998）26所提供的证据表明，潮气量，呼吸速率和呼吸道阻力的体积描记器腔室之内的所有影响的压力波动。当空气在腔室中被加热和加湿，以身体条件的压力波动是由三分之二降低，通过增加电阻值进行放大&lt;SUP&gt; 21。由于所有这些组件反映压力波动存在争议的一个特殊的呼吸参数测量是否准确。因此，它已得出结论，从体积描记法获得的潮气量是定性而不是定量评估26。两个上和下气道阻力是体积描记系统中测量的支气管收缩27产生不确定性的分量。这是作者的意见，UWBP应相互使用侵入性的分析。这是，事实上，某些期刊的稿件仅基于UWBP数据的政策，将不被接受。这将是另一个考虑读者。 总之，UWBP是测量改变在标准实验室啮齿动物，尤其适合于纵向研究呼吸参数的有用方法。这种技术的主要优点是避免了侵入性操作，化学挑战和麻醉的要求。这使得研究人员收集生理数据最能代表自然发生的事件，并减少实验的变异。

肺功能障碍是发病率和死亡率在世界上领先的原因之一。该条件的特点是不充分的氧气交换，等同于咳嗽，胸痛和呼吸困难。呼吸系统疾病占死亡率的全球1％〜10％。根据世界卫生组织，死亡率都将上升，由于长期吸烟，环境污染和职业的刺激。 UWBP是一个有益的补充学习肺的生理，有力地恭维传统的生化和组织学分析2。其他程序用于肺癌的评估不提供相同的优点UWBP。侵入体积描记法是一种常用的技术，需要对动物进行anesthetised 3,4，因此，导致呼吸测量不一定是反射的自然状态。此外，机械通气和化学挑战要求排除未来测量3,4。收集的呼吸数据的另一种方法是通过强迫振荡，这是更敏感的微细化改变呼吸参数相比UWBP 5。强迫振荡，然而，一种侵入性的技术，并且需要动物终止数据收集5-7。 UWBP包括将动物的专业室内。在吸气过程中，潮气的空气被加热和肺部增加水蒸气压力范 ​​围内的加湿，并使气体8的热膨胀。这种效应导致的净变化在空气体积产生的体积描记器室8内的压力增加。过期创建从动物呼吸波形期间发生相反。波形分析，然后用从呼吸迹测量:呼吸率（次/ min），总呼吸周期时间（秒），吸气/呼气时间（钛/碲，秒），由于各潮气量的压力变化（P T）。 <stroNG&gt;图1显示了每个测量起源于呼吸的痕迹。这些测量是简单的计算和多种呼吸参数可以从这些测量得到的。这些参数包括:潮气量（空气的体积的正常吸气和呼气之间的移动），每分钟通气量（气体从每分钟肺部吸入的体积），吸气占空比（吸气时间与总呼吸周期的持续时间的百分比），并吸气流量（空气的启发在一个给定的时间量）。 UWBP提供了在动物模型中的呼吸生理学的精确，非侵入性，定量分析，并且可以用于测量呼吸性疾病和肺功能6,9的进展。相反，其他体积描记技术，UWBP避免使用麻醉，限制和产生的文物和实验变异6,9侵入性操作的。麻醉可以抑制呼吸，改变心脏速率，并且可以是具有挑战性的调节10。限制导致增加呼吸因额外的压力，通过皮质酮和肾上腺素释放出11,13。 UWBP的主要特点是反复的生理评估，使之适合于纵向研究。 UWBP强烈建议肺生理的纵向评估，并提供了一​​个宝贵的技能为未来的呼吸药物评估。 博来霉素，卵清蛋白，和缺氧已被用于诱导呼吸道挑战在几个研究和UWBP已经成功地测量准确肺生理评估7,9,13-16。中描述的协议被设计为标准成人实验小鼠。然而，UWBP已适应其他动物，如大鼠，豚鼠，以及非人类灵长类17-20。 UWBP并不限于只以评估肺功能障碍，但也被用于为肺成熟3的评估。UWBP的多功能性，简洁性和可重复性已经建立起了一支优秀的技术评估动物肺功能。各种软件（见材料和​​设备表）将被要求执行此过程。一个有经验的科学家将能够在1小时内执行该协议与鼠标。

<table><tbody><tr><td>LabChart 7 software (for Macintosh)</td><td>ADINSTRUMENTS</td><td>MLU60/7</td><td>used in protocol step 4</td></tr><tr><td>PowerLab 8/30 (model ML870)</td><td>ADINSTRUMENTS</td><td>PL3508</td><td></td></tr><tr><td>Octal Bridge Amp (model ML228)</td><td>ADINSTRUMENTS</td><td>FE228</td><td></td></tr><tr><td>Black BNC to BNC cable (1 m)</td><td>ADINSTRUMENTS</td><td>MLAC01</td><td></td></tr><tr><td>Macintosh OS</td><td>Apple Inc.</td><td></td><td>Mac OS X 10.4 or later</td></tr><tr><td>Surgipack Digital Rectal Thermometer</td><td>Vega Technologies</td><td>MT-918</td><td></td></tr><tr><td>Grass volumeteric pressure transducer PT5A</td><td>Grass Instruments Co.</td><td>Model number PT5A; serial No. L302P4.</td><td></td></tr><tr><td>1 ml Syringe</td><td>Becton Dickinson (BD)</td><td>309628</td><td></td></tr><tr><td>5 ml Serological syringe pipettes</td><td>Greiner Bio One</td><td>606160</td><td>Connected via plastic tubing</td></tr><tr><td>Balance/Scales</td><td>VWR International, Pty Ltd</td><td>SHIMAUW220D</td><td>Any weighing balance with of 0.1 gram resolution</td></tr><tr><td>HM40 Humidity &amp;amp; temperature meter</td><td>Vaisala</td><td>HM40A1AB</td><td></td></tr><tr><td>Barometer</td><td>Barometer World</td><td>1586</td><td></td></tr><tr><td>Laboratory tubing</td><td>Dow Corning</td><td>508-101</td><td>Used to connect water column to the syringe and pressure transducer</td></tr><tr><td>Cylindrical Perspex Chamber</td><td>Dynalab Corp.</td><td></td><td>Custom built cylindrical chamber with internal dimensions as follows: 50 mm(w) x 1,500 mm(l). There are two lids for each side, with dimensions 80 mm(l) x 80 mm(w). Each lid has a 60 mm wide circular hole cut on the face of the lid 50 mm deep. This allows the chamber to fit into the lid. A rubber ring is fitted around each hole of the lid where the chamber will fit. For attachment of syringe and pressure transducer, the openings are 5 mm in diameter. For attachment of humidity probe, the openings are 25 mm in diameter.</td></tr><tr><td>80% Ethanol (4 L)</td><td>VWR International, Pty Ltd</td><td>BDH1162-4LP</td><td></td></tr></tbody></table>

measuring respiratory function in mice using unrestrained whole-body plethysmography

呼吸功能障碍的发病率和死亡率在世界上领先的原因和死亡的发生率持续上升的。肺功能的啮齿动物模型定量评估是未来疗法的发展的一个重要工具。常用的技术评估呼吸功能，包括侵入式体积描记法及强迫振动。尽管这些技术提供了有价值的信息，数据集合可以是充满了伪像和实验变异性，因为需要对麻醉和/或动物的侵入性仪器。与此相反，无节制的全身体积描记法（UWBP）提供了一个精确的，非侵入性，定量由此来分析呼吸参数的方式。该技术避免了使用麻醉和限制的，这是常见的传统的体积描记技术。这部影片将展示UWBP程序包括设备安装，校准和肺功能记录。它将解释如何分析收集到的数据，以及确定实验离群和文物而导致的动物的运动。使用这种技术获得的呼吸参数包括潮气量，每分钟通气量，吸气占空比，吸气流量和吸气时间到期时间的比值。 UWBP不依赖于专门的技能和价廉来执行。 UWBP的一个关键特征，并且最吸引潜在的用户，是在同一个动物进行肺功能的重复测量的能力。

在此过程中一直遵循正确的，一致的摆动轨迹上的数据分析软件创建的。该程序提供了一个几分钟内呼吸一丝设置了简单的计算计算后确定上市的呼吸参数。 图5表示适当的呼吸跟踪从控制（健康的）鼠标。适当的摆动数据时产生的动物没有积极地移动。 UWBP是控制和肺纤维化的队列之间肺功能的一个非常有用的和可靠的评估。 图7展示了一个鼠标用博来霉素诱导的肺纤维化，在第14天的肺的功能相比较，以控制图， 图7示出了视觉差与博来霉素管理7保持一致。如先前所讨论的，该过程可以重复使我们能够观察变化呼吸parameteRS在这两个群体之间的时间。 得到的结果将被表示为平均值±SEM。建议复制粘贴收集到一个简单的Excel电子表格中的数据。这将成为用于执行在步骤4.13和4.14中讨论的计算是有用的。呼吸功能可以比目测之间的两个基团作为表现出在图8中。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig1highres.jpg" width="500" /> 呼吸循环的图1不同的组件使用激衅。此图说明了一个）中的压力由于吸气（ΔPi的变化），b）由于上述各潮气量（PT）中的压力的变化，c）该图示在压力因过期（ΔPE）中，d）总呼吸周期时间（TTOT）中，e）吸气时间（TI）和f）的到期时间变化（特） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig1highres.jpg" target="_blank">点击此处查看大图。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig2highres.jpg" width="500" /> 图2中的气压计和水柱设置的可视化表示。该图的目的是帮助在建立晴雨表和水柱的校准过程中的阅读器。请注意，水是由统治者资助的两列内的水平。的两列是通过塑料管15厘米连接。在右边（65 cm）处，管连接在1ml注射器和向左（75厘米）连接到数据采集设备的压力传感器。注:管材的长度决定了移动1厘米需水的体积（300微升） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig2highres.jpg" target="_blank">点击这里查看</a>大图。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig3highres.jpg" width="500" /> 图3:执行步骤2.4和2.5安培桥校准 。该图中示出了用于该设备的校准步骤2.7和2.8。关键是要纠正桥放大器来获得精确的结果。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig3highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig4highres.jpg" width="500" /> 图4。UWBP设置的总体示意图 。在左侧是连接到含有动物的体积描记术腔的一侧上的湿度/温度传感器。右边是校准注射器和压力传感器的领先从体积描记室的数据采集系统生产计算机上呼吸道痕迹。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig4highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig5highres.jpg" width="500" /> 图5。呼吸呼吸跟踪使用UWBP时获得C57BL / 6小鼠的控制的一个例子，这一丝喘息从控制动物展示适当的，一致的数据。连续九年评论添加在高峰和呼吸的波动来获得上市呼吸参数的低谷按下面的步骤4.1-4.13。时间沿x轴（SEC）和沿y轴的压力变化代表（cm.H 2 O）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig5highres.jpg" target="_blank">点击这里为vi</a>EW放大图像。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig6highres.jpg" width="500" /> 图中使用UWBP当从C57BL / 6小鼠获得的不同次优痕迹6实施例。次优结果可以混淆成适当的数据，并且是差分析的最常见来源。这个数字说明，不应该被用于分析最常见的次优痕迹。这些呼吸的痕迹证明，而动物嗅探和移动改变动物的基本呼吸生理学）的呼吸跟踪记录。 二）记录产生振荡逐渐增加随着时间的推移一丝通常是通过冷凝和湿度建立所致。然而，跟踪可能被擦拭体积描记室用乙醇或重复校准执行步骤c更正。）一丝丝体积描记室的运动过程中，当动物或研究人员正在从事与设备记录下来。时间沿x轴（SEC）和沿y轴的压力变化（cm.H 2 O）表示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig6highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig7highres.jpg" width="500" /> 图7使用UWBP当从C57BL / 6小鼠诱导的肺纤维化获得了一丝喘息的例子，这一丝喘息的使用本文中介绍的UWBP过程时获得性肺动脉动物展示适当的，一致的数据。连续九年评论添加在高峰和呼吸的波动来获得上市呼吸参数的低谷按下面的步骤4.1-4.13。时间是represented沿x轴（SEC）和沿y轴的压力变化（cm.H 2 O） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig7highres.jpg" target="_blank">点击此处查看大图。</a> <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51755/51755fig8highres.jpg" width="500" /> 控制和博莱之间比较图8中呼吸功能挑战C57BL / 6小鼠。使用UWBP将允许用户以类似的结果是什么这里表示后执行体积描记分析。该图演示了博莱霉素质疑动物（灰色虚线）和对照组动物（黑色实线）之间的生理差异。这些图表显示的）的到期时间（秒比较），B），吸气时间（秒），C）励志占空比（％），D）吸气流速（毫升/秒）E）呼吸频率（次/分），F）分钟通气量（毫升/分钟/公斤），G），潮气量（毫升/千克）和h）总周期时间（秒）。肺功能数据收集纵向在动物在第0天，第7相同的队列中，并且14以下博莱挑战。代表性的数据是改编自Murphy 等人（2012）16。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51755/51755fig8highres.jpg" target="_blank">点击这里查看大图。</a>

Watch this Scientific Journal Video about Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography at JoVE.com

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

呼吸生理的评估方法时，需要克制的动物或镇静历来依赖。无节制的全身体积描记法，然而，提供了在动物模型中的呼吸生理学的精确，非侵入性，定量分析。此外，该技术允许重复小鼠允许纵向研究的呼吸系统评估。

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 ...

measuring-respiratory-function-mice-using-unrestrained-whole-body

Universidad San Sebastian

Research

JoVE Journal

Biology

30.2K 次观看.  Monash Institute of Medical Research. 呼吸生理的评估方法时，需要克制的动物或镇静历来依赖。无节制的全身体积描记法，然而，提供了在动物模型中的呼吸生理学的精确，非侵入性，定量分析。此外，该技术允许重复小鼠允许纵向研究的呼吸系统评估。 

视频: Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

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

Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and minute ventilation are routinely reported. Moreover, information can be collected regarding the neural output of breathing, including the existence of central apneas and augmented breaths. An important consideration for UBP is obtaining a breathing segment with a minimal impact of anxious or active behaviors, to elucidate the response to breathing challenges. Here, we present a protocol that allows for short, quiet baselines to be obtained in aged mice, comparable to waiting for longer bouts of quiet breathing. The use of shorter time segments is valuable, as some strains of mice may be increasingly excitable or anxious, and longer periods of quiet breathing may not be achieved within a reasonable timeframe. We placed 22 month-old mice in a UBP chamber and compared four 15 s quiet breathing segments between minutes 60&#8211;120 to a longer 10 min quiet breathing period that took&#160;2&#8211;3 h to acquire. We also obtained counts of central apneas and augmented breaths prior to the quiet breathing segments, following a 30 min familiarization period. We show that 10 min of quiet breathing is comparable to using a much shorter 15 s duration. Additionally, the time leading up to these 15 s quiet breathing segments can be used to gather data regarding apneas of central origin. This protocol allows investigators to collect pattern-of-breathing data in a set amount of time and makes quiet baseline measures feasible for mice that may exhibit increased amounts of excitable behavior. The UBP methodology itself provides a useful and noninvasive way to collect pattern-of-breathing data and allows for mice to be tested over several time points.

Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

通过无节制的乳腺造影获得衰老小鼠呼吸模式的方法

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and ...

行为学

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 ...

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

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

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

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

医学

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 ...

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ensure that both animal welfare and scientific insights advance with the availability of new technologies. This article demonstrates the use of Simplified Whole Body Plethysmography (sWBP) to non-invasively study respiratory failure in a model of lethal respiratory melioidosis. sWBP has the sensitivity to detect breathing in mice through the entirety of the course of the disease, allowing for the moribund-associated symptoms (bradypnea and hypopnea) to be measured and potentially used to develop humane endpoint criteria.
Some of the benefits of sWBP in the context of respiratory disease are that host breath monitoring comes the closest of any physiologic measurement at assessing dysfunction of the primary infected tissue, namely, the lung. In addition to biological significance, the use of sWBP is rapid and non-invasive, minimizing stress in research animals. This work demonstrates the use of in-house sWBP apparatus to monitor disease throughout the course of respiratory failure in the murine model of respiratory melioidosis.

This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively.

简化全身体积描记法以表征呼吸类鼻疽期间的肺功能

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ...

无创实时全身体积脉搏波检测小鼠咳嗽敏感性

Methods for Detecting Cough and Airway Inflammation in Mice

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

作者聚焦:调查慢性咳嗽的病理生理学

Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

检测小鼠咳嗽和气道炎症的方法

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge ...