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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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+measuring+airway+resistance+in+man+using+a+body+plethysmograph:+Values+in+normal+subject+and+in+patients+with+respiratory+disease.">A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subject and in patients with respiratory disease.</a> J. Clin. Invest. 35 (3), 327-335 (1956).</li><li>Enhorning, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+plethysmography,+does+it+measure+tidal+volume+of+small+animals.">Whole-body plethysmography, does it measure tidal volume of small animals.</a> Can. J. Physiol. Pharmacol. 76 (10-11), 945-951 (1998).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+unrestrained+single-chamber+plethysmography+provide+a+valid+assessment+of+airway+responsiveness+in+allergic+BALB/c+mice.">Does unrestrained single-chamber plethysmography provide a valid assessment of airway responsiveness in allergic BALB/c mice.</a> Respir. Res. 10 (61), (2009).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:263px;">LabChart 7 software (for Macintosh)</td>
			<td style="width:139px;">ADINSTRUMENTS</td>
			<td style="width:155px;">MLU60/7</td>
			<td style="width:475px;">used in protocol step 4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerLab 8/30 (model ML870)</td>
			<td>ADINSTRUMENTS</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Octal Bridge Amp (model ML228)</td>
			<td>ADINSTRUMENTS</td>
			<td>FE228</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Black BNC to BNC cable (1m)</td>
			<td>ADINSTRUMENTS</td>
			<td style="width:155px;">MLAC01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Macintosh OS&#160;</td>
			<td>Apple Inc.&#160;</td>
			<td>-</td>
			<td style="width:475px;">Mac OS X 10.4 or later</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgipack Digital Rectal Thermometer&#160;</td>
			<td>Vega Technologies</td>
			<td>MT-918</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Grass volumeteric pressure transducer PT5A</td>
			<td>Grass Instruments Co.</td>
			<td style="width:155px;">Model number PT5A; serial No. L302P4.</td>
			<td style="width:475px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1ml Syringe</td>
			<td>Becton Dickinson (BD)</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:263px;">5ml serological syringe pipettes</td>
			<td>Greiner Bio One</td>
			<td>606160</td>
			<td style="width:475px;">Connected via plastic tubing</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Balance/Scales</td>
			<td>VWR International, Pty Ltd</td>
			<td>SHIMAUW220D</td>
			<td style="width:475px;">Any weighing balance with of 0.1 gram resolution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HM40 Humidity &#38; temperature meter&#160;</td>
			<td>Vaisala&#160;</td>
			<td>HM40A1AB</td>
			<td style="width:475px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Barometer</td>
			<td>Barometer World</td>
			<td>1586</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Laboratory tubing</td>
			<td>Dow Corning&#160;</td>
			<td>508-101</td>
			<td style="width:475px;">Used to connect water column to the syringe and pressure transducer</td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;width:263px;">Cylindrical Perspex Chamber</td>
			<td>Dynalab Corp.</td>
			<td></td>
			<td style="width:475px;">Custom built cylindrical chamber with internal dimensions as follows: 50mm(w) x 1500mm(l). There are two lids for each side, with dimensions 80mm(l) x 80mm(w). Each lid has a 60mm wide circular hole cut on the face of the lid 50mm 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 5mm in diameter. For attachment of humidity probe, the openings are 25mm in diameter.&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">80% Ethanol (4L)</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?...

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

Research

JoVE Journal

Biology

30.0K 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

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

使用卷压力记录，一条尾巴袖套法小鼠的血压测量

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

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 ...

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

全身纳米气溶胶吸入暴露

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

小鼠膝盖外展肌肉功能的体内测量

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...