SML 由加拿大卫生研究院的奖学金支持, MG 由 FRQS (全宗 de 研究所) 的呼吸道健康网络的助学金支持, YB 是 Santé的研究学者。
作者的贡献 所有作者贡献了原稿的构想和/或者录影。SML 和 LD 收集了这些数据。SML、LD、YB、DM、DB 和 AR 为数据分析、数字生成和手稿撰写做出了贡献。YB、AR、KL 和 MG 都参与了编写视频脚本的过程。这出戏是由 YB、KL 和 MG 表演的。

根据加拿大动物保育理事会 (管理委员会) 的指导方针, 魁北克心脏和肺研究所动物护理委员会批准了下列程序。
1. 准备
<ol><li>研究
	<ol>
<li>(关键)在进行任何实验之前, 获得适当的批准 (如IACUC) 和培训 (如动物处理)。</li>
		<li>熟悉设备和操作软件。阅读用户手册, 如果需要, 创建一个配置文件来定义站点数量、输入信号、分析器和参数。 注:请确保选择高采样率 (2 赫)。</li>
		<li>为感兴趣的参数配置分析器设置。<ol>
<li>从工具栏和分析器中选择 "调整"。</li>
			<li>将流量阈值调整为正确分隔呼吸的数值 (鼠标: 0.5 毫升/秒), 并选择Ti + Te计算呼吸速率。</li>
			<li>设置气压 (760 毫米汞) 的值, 并指定吸气/呼气容积的最大偏差, 以使呼吸被认为有效 (鼠标: 20%)。</li>
			<li>单击 "计算参数" 字段中的设置以调整接受限制。 注:以下设置用于描述的鼠标实验: 灵感时间, 50 到170毫秒;到期时间, 40 到180毫秒;呼吸频率, 30 到 450 bpm;特定气道阻力, 0 至 15 cmH2O·s;midexpiratory 流的精度至少为3个小数。</li>
			<li>完成后, 选择 "应用并关闭" 以退出对话框窗口。</li>
		</ol>
</li>
		<li>从 "工具栏" 菜单中, 再次转到 "调整", 然后存储以设置所需的数据存储速率。单击 "应用" 和 "关闭" 退出窗口。 注:平均每十年代通常使用。</li>
		<li>在操作软件中创建一个协议, 以定义命令序列和每一个所需的时间。如图 1所示。</li>
		<li>如果实验涉及通过气溶胶对某一物质进行管理, 则根据要测试的浓度准备适当的溶液和稀释。</li>
	</ol></li>
	<li>动物
	<ol>
<li>在一个安静的地方工作, 从住房室偏远。允许动物适应环境的变化。</li>
		<li>权衡动物, 并选择适当的限制大小。</li>
		<li>(关键)在实验开始之前, 将动物适应抑制剂和程序。根据实验设计, 可能需要增加持续时间 (例如, 5-30 分钟) 的多种驯化训练。 注:不适应的动物应该从研究中移除。<ol>
<li>在每个驯化阶段, 在抑制剂内插入动物, 从后开口开始;垂直放置设备可能会有帮助。</li>
			<li>一旦该动物就位, 插入后柱塞, 并轻轻地锁定它到位, 而不施加过度的力量。</li>
			<li>(关键)目视检查动物是否正常呼吸。如果需要, 通过移动锁定机制调整其位置。确保动物的鼻孔在鼻锥外突出, 它的鼻子靠在抑制剂的内壁上。</li>
			<li>将胸腔的后面板分离, 通过胸腔内的橡胶开口插入含有该动物的抑制剂, 并关闭腔室。</li>
			<li>连接头部, 并提供偏置流。对鼠标使用0.5 升/分流。</li>
			<li>允许动物放松5分钟。</li>
			<li>一旦动物平静下来, 就开始记录鼻腔和胸腔的流动信号。在计算机屏幕上验证跟踪是否平滑, 并显示正常的呼吸模式;请参见图 2中的示例。 注:如果该议定书涉及某种物质的气溶胶管理, 则可以将盐水挑战纳入驯化程序。</li>
			<li>在每节课结束时, 将动物从胸腔和抑制剂中取出, 并将其返回到其外壳和房间。</li>
		</ol>
</li>
	</ol></li>
	<li>设备
	<ol>
<li>在实验当天, 启动一个实验会话并加载相应的配置文件。 注:验证它是否包含实验所需的协议。</li>
		<li>转到 "工具栏" 菜单中运行。输入实验和动物信息。完成后, 单击窗口底部的 "运行" 按钮。</li>
		<li>继续校准系统。分别校准每个站点和输入信号。<ol>
<li>打开偏置流发生器, 通过一条油管将其连接到机头, 并调整流量。</li>
			<li>用帽子关闭头室的顶部开口。</li>
			<li>分离胸腔后面板, 牢固地将校准工具插入头部和车身室之间的橡胶开口内, 以创建密封密封。然后关闭并重新连接胸室的后面板。</li>
			<li>验证胸腔的侧端端口是否已盖上。</li>
			<li>从 "软件工具栏" 菜单中, 转到 "调整", 然后进行校准。</li>
			<li>转到输入 1 (胸腔)并选择校准以启动胸腔流量信号的校准对话框。</li>
			<li>(关键)验证 "校准" 对话框窗口中列出的参数是否显示适当的设置,即物理应力应用的低值: 0;物理应力值高:-20 毫升/秒;示例: 集成。完成后, 在 "示例" 窗口中单击 "低"。</li>
			<li>验证在显示窗口中生成的信号是否恒定, 然后单击 "关闭"。</li>
			<li>使用一个塑料连接器和一条油管将20毫升注射器通过胸腔的侧端连接起来。</li>
			<li>(关键)在 "样品" 窗口中选择 "高", 并立即将20毫升的空气注入2秒内的腔内, 流速尽可能恒定。</li>
			<li>验证生成的信号是否完全显示在显示窗口中。使用箭头图标验证信号在零线附近是否居中和对称。然后, 单击 "关闭"。单击 "示例" 窗口中的 "移除交流偏移量", 从零处移除任何偏移量 注:如果需要, 可以重新制作高值校准。</li>
			<li>验证所得到的缩放输入范围是否在建议的范围内 (鼠标: ±280到±420 mL)。(关键)如果值超出可接受范围, 则重复校准步骤。</li>
			<li>以类似于胸腔的方式校准头部腔 (步骤 1.3.3.6)。这一次, 选择输入 2 (鼻腔)。 注:(重要)应用高值的物理应力值必须设置为 +20 毫升/秒。这将改变前腔内流的极性与后腔的关系。因此, 当动物呼吸时, 两个流动信号几乎处于相位, 除了用来计算 sRaw 的延迟。</li>
		</ol>
</li>
	</ol></li>
</ol>2. 肺功能测量
<ol><li>称动物体重, 注意体重。</li>
	<li>将动物插入抑制剂内, 将其放置在胸腔 plethysmograph 腔内 (步骤 1.2.3.1 1.2.3.5)。</li>
	<li>允许动物放松至少5分钟。</li>
	<li>通过选择第一步, 然后单击 "执行", 启动命令的协议。</li>
	<li>检查计算机屏幕上的动物的呼吸信号是定期和平滑 (图 2)。该软件自动显示计算参数在呼吸的基础上。验证动物的参数是否稳定。</li>
	<li>在基线条件下记录呼吸模式长达10分钟。</li>
	<li>对于涉及通过气溶胶管理测试物质的协议, 请执行以下操作:<ol>
<li>根据需要调整雾化器的时间和工作周期。 注:在本文所展示的例子中, 雾化器在十年代的5% 工作周期中运行。</li>
		<li>执行车辆挑战 (如盐水) 并记录反应。</li>
		<li>如果需要, 通过在不断升级的步骤中改变雾化浓度 (例如加倍浓度), 使动物接触到增加测试物质的浓度。记录每个管理后的响应。</li>
		<li>在实验课结束时, 如果没有自动完成, 停止录音并将动物送回其外壳和房间。</li>
	</ol>
</li>
	<li>如果需要, 请从 "工具栏" 菜单中选择 "运行" 以运行另一个实验会话。<ol>
<li>在会议之间, 清洁 plethysmograph 室, 并用水冲洗喷雾器。 注:酒精的使用可能对 plethysmograph 造成不可逆转的损害。</li>
	</ol>
</li>
	<li>如果这项研究涉及反复的评估时间, 重复的整个测量序列在每个选择的 timepoint。 注:明智的建议是补充研究与一些精确测量呼吸力学和/或肺体积1,15。</li>
</ol>3. 数据分析
注:该软件自动保存实验文件, 并在实验会话关闭时导出记录的参数。
<ol><li>计算每个动物和实验组感兴趣参数的基线平均值。 注: 表 1列出了一些典型参数, 根据它们提供的信息类型进行分类。</li>
	<li>当相关时, 用特定点 (例如,最大值或最小数值)、平均值或全时过程来评估雾化物质对每个浓度的兴趣参数的影响;还可以考虑对基线的规范化。</li>
	<li>使用表或图形格式中的组方法和错误报告结果。统计分析结果。 注:在本研究中, 采用双向 ANOVAs 和重复措施, 以评估乙酰甲胆碱, 过敏原-家尘螨 (HDM) 的作用, 以及它们在不同的磷酸氢钙读数 (sRaw 和 EF50), 以及不同的读数 (RN, G 和H), 两株小鼠。Sidak 的多种比较试验被用来确定过敏小鼠与控制小鼠不同的乙酰甲胆碱浓度。同样的试验用于评估天数、HDM 及其在基线 (即乙酰甲胆碱前) 对气道梗阻 (sRaw 和 EF50) 读数和通气模式 (BF、电视、MV 和 EIP) 的影响。用皮尔逊与 sRaw 的相关性来评价用该方法获得的磷酸氢钙与 RN的相关性。所有的统计分析和图表都是使用替代标准统计软件 (如GraphPad 棱镜) 进行的。p ≤0.05 被认为足以拒绝零假设。</li>
</ol>

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DB 和 AR 受雇于 SCIREQ 科学呼吸设备公司, 这个商业实体涉及与本文内容相关的主题。DB 还拥有库存。SCIREQ 有限公司是一家 emka 技术公司。

在呼吸系统研究中, 显然有必要测量有意识动物的肺功能。一般来说, 磷酸氢钙是一个有趣的方法来评价呼吸系统的通气功能的自觉和自发呼吸动物26。更具体地说, 该磷酸氢钙或其头外变体往往在所提供信息的质量和所需的入侵3水平之间取得适当的平衡 (表 2)。该技术可以适应各种物种 (例如,老鼠, 鼠, 豚鼠) 或动物的大小, 可用于许多研究应用。它是特别有用的一次评估许多动物在一次平行的研究设计, 被重复的方式监测呼吸功能, 并且捕获反应的动力学在时间以后。此外, 该技术是直截了当的, 可以在比较及时的方式学习。本文以小鼠为例, 采用磷酸氢钙测定法, 对该约束 plethysmography 技术的实际操作进行了描述, 并讨论了关键步骤和相关结果。
在与有意识的动物一起工作时, 必须控制周围环境的条件 (例如,有数量有限的人或活动的安静的房间), 以便产生可重现的结果。由于抑制剂来自不同的维度, 所以从适当的尺寸开始是很重要的, 这样呼吸运动就不会受到干扰。这也是有益的, 往往需要适应动物的实验设置和程序, 因为它是很好的建立在小鼠, 抑制影响呼吸频率12。根据实验设计或条件, 可能需要多个会话的增量工期。最后, 允许动物实验开始时的时间调整到房间的变化和必要的处理是一个简单的考虑, 证明是有效的, 以确保呼吸模式是一贯定期和放宽基线。在动物舒适、适应性好、镇静的条件下工作, 在结果变异性和质量方面也会有好处。它也限制任何压力诱导的儿茶酚胺释放, 这可能增加气道口径和衰减诱导支气管收缩。
重要的是要明白, 有必要尽可能地分离鼻和胸流。根据所研究的系统或种类, 密封机制的形状和功效都不同。在我们所描述的磷酸氢钙中, 海豹是在动物的鼻子和抑制装置之间创建的。在用磷酸氢钙进行呼吸功能评估时, 还必须提供足够且连续的偏置流, 因为对动物可用氧气水平的降低会产生显著的影响。考虑到抑制剂动物的福祉, 限制了鼓动造成的空气泄漏的倾向, 从而使数据质量最大化。Contrastingly, 密封的断裂会导致被拒绝的数据集或某些参数的低估。
除了启用鼻腔流量信号的单独记录外, 头部腔通常用于使动物接触雾化物质。如本文所示, 这可以用来执行 bronchoprovocation 测试以显示不同程度的响应性。在这样的实验中, 根据所研究的动物的种类、应变或性别, 可能需要调整测试浓度的范围。如前所示的8,9,10,27, 目前的结果表明, 乙酰甲胆碱引起的 sRaw 的变化与侵袭性的气道阻力测量有很好的相关性。研究结果还表明, 磷酸氢钙技术并不像其检测呼吸功能障碍的能力那么敏感, 并确定在肺下部 (肺组织和/或小外围气道) 内局部改变的反应。.由于动物的气道完好无损, 上呼吸道的存在, 占到气流28的总呼吸阻力的最大部分, 可能会影响气溶胶的分布和沉积, 除了减弱的贡献下呼吸道的测量。表 3总结了磷酸氢钙法与该技术的其他区别。最后, 虽然理论上可以估计动物的总气道阻力 (包括上呼吸道) 从测量的 sRaw, 一般建议用一种侵入性测量技术来补充氢钙的评价, 如29获得对详细呼吸力学的直接测量。根据研究的目的, 对上呼吸道阻力的测量也可以被认为是30,31,32。
结论  由于其侵袭性有限, 磷酸氢钙是一种能满足呼吸研究的重要需求的技术。它能同时提供一些无可争议的气流阻塞指标, 准确地读数出意识动物的通气模式。获得的信息也真正补充了从更多侵入性的方法。

越来越多的人使用小动物来建模人类呼吸系统疾病, 敦促发展技术来定量评估这些动物呼吸系统的功能。目前, 强迫振荡技术被认为是评估小动物呼吸力学的最精确方法1,2。然而, 正如分型不确定原理所指出的, 在测量精度方面得到的是与 noninvasiveness3的损失进行交易的。事实上, 在受高度控制的实验条件下, 需要进行麻醉、气管切开或口服插管, 以及机械通气, 这些测量都是获得的。一个远离现实生活的场景。
在实验要求禁止使用麻醉药或要求很少或不偏离动物的自然生理状态的情况下, 可以考虑双腔 plethysmography (DCP)。顾名思义, 磷酸氢钙的设置由两个连接的刚性腔组成, 它们建立在前腔内, 从其胸腔, 在后室中尽可能地隔离动物的头部 (或鼻子)。在安装过程中, 动物是有意识的, 在被抑制时会自发地呼吸。由于周围空气的压缩/减压, 分庭的墙壁不能扩张或收回, 在动物的空气进出的运动产生一个对应的, 但相反的波形在后方腔内。由于前腔内鼻腔流动而引起的波形与后腔内的胸部运动有关, 因此可以同时分离和捕获。根据 DCP 设置的设计, 这些波形可以通过一组压力传感器或 pneumotachographs 来获得, 以分别记录腔室压力的变化或腔内的气流, 作为时间的函数。后一种方法现在比较常见。
当动物的呼吸频率可以通过任何一种 plethysmography 技术精确地确定时, 在潮汐体积的测定和相关的通风参数 (例如, 分钟通风,呼气量等)。与全身 plethysmograph (人造板) 技术不同的是, 该动物的潮汐体积估计从箱信号4,5, DCP 技术提供了准确的潮汐量评估。这与动物在后室的胸部运动直接获得有关, 这与呼吸期间肺容积的变化成正比。
除了这些准确的通气参数 (如潮汐容积, 呼吸频率, 和分钟通风), 一些干扰的形状的呼吸循环也可以用来调查神经元方面, 控制呼吸驱动或呼吸反射。该应用的一个具体例子是评估吸入物质对上呼吸道感觉神经元的刺激电位6。在这里, 在失效开始时暂停的持续时间是使用称为端吸气停顿 (EIP) 的参数确定的, 也称为制动6的持续时间。这种暂停的刺激物质的延长是与动物的声门关闭有关, 导致一个可测量的制动周期的第一部分的失效6,7。
磷酸氢钙的另一个重要优点是, 它提供了两个验证和无争议的参数, 对气流阻塞敏感。一个被称为流在中潮汐呼气容积和缩写 EF508,9,10。它是气流在每个潮汐呼吸的中途容量在失效期间。EF50 从胸腔的流动轨迹中提取出来, 因此可以在没有前腔的情况下进行测量 (即在头部外的配置中)。另一种称为特定气道阻力和缩写 sRaw11,12,13。sRaw 的确定需要同时记录动物的鼻腔和胸腔的流动, 因为它是从这些单独的呼吸痕迹之间的时间延迟计算, 在零流点的灵感结束。阐述了这一拖延与 sRaw 有关的基础的理由, 以前11。简而言之, 肺容积的变化在空气运动之前, 因为压力梯度需要开发为了驾驶气流。在健康的动物安静地呼吸, 这种延迟通常是非常小的。然而, 适应给定流量所需的压力梯度 (例如, 足以提供足够通风的流量) 受气道阻力的影响。例如, 在支气管收缩过程中, 适应给定流量所需的压力梯度更大, 这意味着动物必须更加努力地呼吸。更大的压力梯度在动物的胸腔也意味着更大一部分的流动进出的后方室是由于减压/压缩空气内的胸腔, 这是部分的总胸部扩张/退缩, 这是与鼻腔流出相脱离。由于支气管收缩而增加的阻力会增加后方和前室之间的延迟, 从而增加 sRaw。驱动气流进出肺的压力梯度也受初始胸气容积 (TGV) 的影响。例如, 在更大的 TGV 上, 为了产生给定压力梯度所需的胸腔的扩张/缩进更大 (仅仅是因为产生给定压力梯度所需的体积位移更大), 这也意味着动物必须更努力地呼吸。再次, 这些额外的胸部置换是需要减压/压缩的空气在胸腔, 从而与鼻流的阶段。因此, 增加的 TGV 也会增加分庭之间的延迟, 从而增加 sRaw。正如可以看到的, 支气管收缩和增加的 TGV 会导致更重要的努力, 以吸引空气进出肺。这实质上是 sRaw 的生理意义。它代表呼吸5,14所需的工作。
因此, 重要的是要明白, 两个不同的因素影响 sRaw: 气道阻力和 TGV。事实上, sRaw 可以表示为气道阻力和 TGV11的产物。有意识的动物可以随时修改他们的 TGV, 以便使他们的通风适应特定的环境。在这种情况下, 动物的自然生理状态是不变的, 因此无法辨别 sRaw 的变化是否源于气道阻力的改变, 从 TGV 的变化, 或两者的混合。因此, 建议用更多侵入性测量呼吸力学和/或肺容积, 如1、15所提供的方法来补充 DCP 评价。
迄今为止, 该磷酸氢钙已用于各种研究应用。该技术可用于或没有头部室, 以定量和准确地评估各种物质, 如药物, 过敏原, 刺激剂或其他调解者的作用, 对意识小动物的呼吸功能16,17,18。前室还可用作曝气室, 以雾化物质或变化的气体浓度 (缺氧、高碳酸血症等)19。方便地, 它允许一个人伴随地测量这些暴露的急性影响。事实上, 磷酸氢钙的一个常见用途是评估在不同型号的呼吸道疾病中雾化乙酰甲胆碱的反应程度,20,21,22,23,24,25。
虽然 DCP 技术看似简单明了, 但一些实际的挑战可能会阻止没有经验的用户, 或损害结果的准确性和重现性。本文详细介绍了在有意识、克制、自发性呼吸小鼠中成功记录呼吸功能的推荐程序。说明是特定于所述设备 (请参阅材料表)。在基线和反应雾化乙酰甲胆碱的两株小鼠肺过敏性炎症的常见模型中也显示了磷酸氢钙的效用和价值。

<table><tbody><tr><td>Acetyl-&amp;beta;-methylcholine chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A-2251</td><td>Methacholine</td></tr><tr><td>Phosphate buffered saline</td><td>Multicell</td><td>311-506-CL</td><td>PBS 10X</td></tr><tr><td>House dust mite extract</td><td>GREER</td><td>290902</td><td>HDM&amp;nbsp;</td></tr><tr><td>DCP complete system</td><td>&amp;nbsp;SCIREQ Inc. /emka TECHNOLOGIES</td><td></td><td></td></tr><tr><td>iox software</td><td>&amp;nbsp;SCIREQ Inc. /emka TECHNOLOGIES</td><td></td><td></td></tr><tr><td>Aerogen Aeroneb nebulizer</td><td>&amp;nbsp;SCIREQ Inc. /emka TECHNOLOGIES</td><td></td><td></td></tr><tr><td>flexiVent FX complete system</td><td>&amp;nbsp;SCIREQ Inc. /emka TECHNOLOGIES</td><td></td><td></td></tr></tbody></table>

assessment of respiratory function in conscious mice by double-chamber plethysmography

由有意识的主体在机体内自发呼吸而产生的风量变化是在 plethysmography 的基础上进行的, 这种技术用于非侵入性评估人类和实验动物呼吸功能的某些特征。本文主要研究双室 plethysmography 在小动物中的应用。它提供了有关方法的背景信息, 以及一个详细的分步程序, 以无侵入的方式成功地评估有意识的、自发呼吸的动物的呼吸功能。该磷酸氢钙可用于监测多个动物的呼吸功能, 并确定在选择的时间段和重复方式的雾化物质诱导的变化。本文采用控制和过敏小鼠的实验方法, 论证了该技术的实用性, 解释了相关的结果参数, 并讨论了相关的优缺点。总的来说, 磷酸氢钙提供了有效的和理论上的声音读数, 可以信赖的评估的意识小动物的呼吸功能, 在基线和挑战后, 雾化物质。

在控制和过敏性 BALB/c 小鼠中, 在基线条件下连续三天 (如图 1所示的协议12天、13和 14) 对呼吸功能进行反复评价的结果如图3所示. .选择用于评估呼吸模式的参数包括呼吸频率 (图 3A)、潮汐容积 (图 3B)、分钟通风 (图 3C) 和端吸气停顿 (图 3D)。用于评估气道梗阻的参数为 EF50 (图 3E) 和 sRaw (图 3F)。两组连续三天, 各选择参数的结果均稳定, 过敏性炎症无明显影响。
在控制和过敏性 BALB/c 小鼠的连续天数 (图 1所示的协议12天、13和 14) 中对乙酰甲胆碱的反应程度进行了评估。结果如图 4所示, 显示了对气道阻塞敏感的两个参数的变化, 即 sRaw (图 4A、 B和C) 和 EF50 (图 4D、 E和F)。正如预期的那样, 乙酰甲胆碱的增量浓度逐渐增加 sRaw, 并逐渐减少 EF50。这些反应是增强过敏性炎症, 特别是在最后浓度测试, 这证明了存在的高反应性。结果还表明, 夸大的反应程度仅限于第一天 (12 天), 因为在随后的两次评估 (即13 和14天) 期间没有观察到这种情况。
在实验性议定书的最后一天 (15 天) 进行的呼吸力学评估结果;图 1)在控制和过敏 BALB/c 小鼠, 如图 5所示。这些实验包括在研究中, 以补充磷酸氢钙的评价。这是公认的更精确的方法来评估呼吸功能2。它的优点之一是, 它提供了地形上的洞察力, 在哪些部位的肺 (传导气道与周围呼吸道和肺部组织) 受到测试干预 (如过敏原和乙酰甲胆碱) 的影响。前文描述了评估呼吸力学的建议方法,1。本文用三参数描述了过敏性炎症和乙酰甲胆碱引起的呼吸力学变化。这些参数包括: 1-牛顿电阻 (RN;图 5A), 其值变化主要反映大型导电气道电阻变化的参数;2-组织阻尼 (G;图 5B), 其值的变化主要反映组织阻力变化的参数;和 3-组织 elastance (H;图 5C), 其值的变化主要反映组织刚度变化的参数2。正如预期的那样, 每个参数的增加都是针对乙酰甲胆碱的增量浓度。与从前一天 (14 天) 获得的 sRaw 和 EF50 结果一致;图 1), 乙酰甲胆碱诱导的 RN的变化 (图 5A) 在控制和过敏小鼠之间是可比较的。事实上, sRaw 在14天的值与 RN在15天的值相关 (图 5D)。乙酰甲胆碱诱导的 H 的上升也类似于控制和过敏小鼠 (图 5B)。然而, 乙酰甲胆碱诱导的 G 的增加在过敏小鼠中显著增大 (图 5C)。这一结果表明, 在15天的过敏小鼠中存在一个持续的 hyperresponsive 表型, 这是在前两天进行的磷酸氢钙评价未发现的。
整个研究重复了 C57BL/6 小鼠。如图 15所示, 12、13和14号议定书 (图 1) 的 sRaw 的连续磷酸氢钙评价的结果, 以及 RN的估计值, 见6。在特定的小鼠菌株中, 在三天 (图 6A、 B和C) 中, 在过敏小鼠中观察到的夸张的乙酰甲胆碱反应保持不变。这种 hyperresponsive 表型也被描述为15天, 由乙酰甲胆碱诱发的 RN的上升, 在过敏小鼠更明显 (图 6E)。这些与 BALB 小鼠的结果形成鲜明对比, 高反应性从12天到 14 (图 4) 的逐渐消退, 观察到乙酰甲胆碱引起的 RN的升高缺乏差异。15天 (图 5A)。这些结果表明, 过敏原对乙酰甲胆碱诱导的两株小鼠的反应有时变的影响。重要的是, 这两种菌株的差异都是由磷酸氢钙和其所描述的。和谐, 在14天的 sRaw 测量的值与 RN的值 (图 6F) 的测量结果相关, 这与 BALB/c 小鼠 (图 5D) 观察到的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57778/57778fig1.jpg"> 图 1.用于诱发肺部过敏性炎症并评估乙酰甲胆碱反应程度的协议.这项研究是对女性 BALB/c 和 C57BL/6 小鼠7至9周的年龄进行的。在整个研究中进行的干预顺序显示在小组 (A) 中。半数小鼠暴露于50µg 的家尘螨 (HDM) 提取物 intranasally 连续14天, 诱发肺部过敏性炎症。另一半则暴露在盐水中, 用作控制。呼吸功能由双室 plethysmography (DCP) 在三个不同的场合 (12 天, 13 和 14, 黑色圆圈) 评估后的驯化疗程 (天 11; 灰色圆圈), 其中包括一个挑战, 雾化盐水。在每届会议期间, 使用小组 (B) 所示的自动协议评估基线呼吸功能和对乙酰甲胆碱的反应。15天, 用强迫振荡技术对呼吸力学进行了侵入性评估, 如前所述1。<a href="/files/ftp_upload/57778/57778fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57778/57778fig2v2.jpg"> 图 2.来自健康 BALB/c 鼠标的典型流量信号.这些面板显示了在基线条件下, 控制鼠标中的双腔 plethysmography 获得的典型记录痕迹。胸流显示在上板, 鼻流显示在下部板。负值是在灵感和积极的价值在到期期间。<a href="/files/ftp_upload/57778/57778fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57778/57778fig3.jpg"> 图 3.反复评价意识 BALB 小鼠的呼吸功能.基线呼吸功能评估的双室 plethysmography (DCP) 的控制 (开放符号) 和过敏 (固体符号) 小鼠在 12, 13 和14的协议, 说明在图 1。用于评估呼吸功能的磷酸氢钙参数包括呼吸频率 (A), 潮汐容积 (电视), (B), 分钟通风 (MV) (C), 结束吸气停顿 (EIP) (D), 在中潮汐呼气容积的流动 (EF50) 在 (E) 和特定气道阻力 (sRaw) (F)。每只老鼠的呼吸频率、电视、MV、sRaw 和 EIP 的值都是记录在1.5 分钟以上的平均值。EF50 的值是在这个记录期间获得的最小值。结果以组为标准差 (n=5/group) 表示。<a href="/files/ftp_upload/57778/57778fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57778/57778fig4.jpg"> 图 4.乙酰甲胆碱 bronchoprovocation 试验在有意识的 BALB/c 小鼠.乙酰甲胆碱反应性评估的双室 plethysmography (DCP) 的控制 (开放符号) 和过敏 (固体符号) 小鼠在 12, 13 和14的协议, 说明在图 1。用于评估反应的磷酸氢钙参数包括 (A) 通过 (C) 的特定气道阻力 (sRaw) 和在 (D) 通过 (F) 的中潮汐呼气量 (EF50) 中的流动。该 bronchoprovocation 是由 aerosolizing 乙酰甲胆碱在磷酸氢钙总商会十年代的增量浓度。每次浓度后1.5 分钟内对反应进行监测。每个浓度的每只老鼠的 sRaw 值是记录在1.5 分钟以上的平均值。EF50 的值是在这个记录期间获得的最小值。结果以组为标准差 (n=5/group) 表示。星号符号 * 指定统计学上的显著差异 (p <img src="https://wikimedia.org/api/rest_v1/media/math/render/svg/e1dd3e3e5dbf6fc91ddef46a531a96e5e3f57143" style="border: 0px; vertical-align: -0.671ex; display: inline-block; color: rgb(34, 34, 34); font-family: monospace; font-size: 11.375px; text-align: center; width: 1.808ex; height: 2.676ex;"> 0.05)。<a href="/files/ftp_upload/57778/57778fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57778/57778fig5.jpg"> 图 5.BALB 小鼠呼吸力学的侵袭性评价.根据图 115 天的强迫振荡技术, 在基线和对乙酰甲胆碱的反应中对呼吸力学进行了评估。控制 (开放符号) 和过敏 (固体符号) 小鼠是相同的, 在12天, 13 和14的双室 plethysmography (DCP) 测试。用于评估呼吸力学的参数是牛顿电阻 (RN) (A)、组织 elastance (H) (B) 和组织阻尼 (G) (C)。bronchoprovocation 是由雾化增量浓度的乙酰甲胆碱直接进入气管插管麻醉, 气管切开, 瘫痪, 机械通气小鼠仰卧位。每次浓度后5分钟内对反应进行监测。每个浓度下每个鼠标的每个参数的值是在此记录期间获得的峰值值。结果以组为标准差 (n=5/group) 表示。小组 (D) 显示了在14天和 RN测量的特定气道阻力 (sRaw) 之间的相关性由15天。open 符号表示基线上的值, 而实体符号表示最高浓度的乙酰甲胆碱的最大值, 测试的是控制 (圆圈) 或过敏 (正方形) 小鼠。该插图显示了测定系数 (r2)。星号符号 * 指定统计学上的显著差异 (p ≤ 0.05)。<a href="/files/ftp_upload/57778/57778fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57778/57778fig6.jpg"> 图 6.C57BL/6 小鼠呼吸功能和侵袭性呼吸力学.特别气道阻力 (sRaw) 是由双室 plethysmography (DCP) 评估的基线和响应乙酰甲胆碱控制 (开放标志) 和过敏 (固体符号) 小鼠在天 12 (A), 13 (B) 和 14 (C) 的协议如图 1所示。牛顿电阻 (RN) 在基线和响应乙酰甲胆碱是评估的强迫振荡技术 (日) 在15天 (D)。bronchoprovocations 的执行情况如图 4和图 5所示, 分别为磷酸氢钙和。结果以组为标准差 (n=5/group) 表示。小组 (E) 显示了 sRaw 测定的14天和 RN在15天测量的。open 符号表示基线上的值, 而实体符号表示最高浓度的乙酰甲胆碱的最大值, 测试的是控制 (圆圈) 或过敏 (正方形) 小鼠。该插图显示了测定系数 (r2)。星号符号 * 指定统计学上的显著差异 (p ≤ 0.05)。<a href="/files/ftp_upload/57778/57778fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>参数</td>
			<td>单位</td>
			<td>描述</td>
			<td>信息</td>
		</tr>
<tr>
<td>F</td>
			<td>Bpm</td>
			<td>呼吸频率</td>
			<td rowspan="12">通风方式</td>
		</tr>
<tr>
<td>电视</td>
			<td>毫升</td>
			<td>潮汐容积</td>
		</tr>
<tr>
<td>Mv</td>
			<td>毫升</td>
			<td>分钟通风</td>
		</tr>
<tr>
<td>钛</td>
			<td>女士</td>
			<td>吸气时间</td>
		</tr>
<tr>
<td>Te</td>
			<td>女士</td>
			<td>呼气时间</td>
		</tr>
<tr>
<td>PIF</td>
			<td>毫升/秒</td>
			<td>吸气峰值流量</td>
		</tr>
<tr>
<td>Pef</td>
			<td>毫升/秒</td>
			<td>呼气峰值流量</td>
		</tr>
<tr>
<td>电动汽车</td>
			<td>毫升</td>
			<td>呼气量</td>
		</tr>
<tr>
<td>NTV</td>
			<td>毫升</td>
			<td>鼻潮容积</td>
		</tr>
<tr>
<td>内华达州</td>
			<td>毫升</td>
			<td>呼气容积</td>
		</tr>
<tr>
<td>Eip</td>
			<td>女士</td>
			<td>结束吸气暂停</td>
		</tr>
<tr>
<td>Eep</td>
			<td>女士</td>
			<td>结束呼气暂停</td>
		</tr>
<tr>
<td>Dt</td>
			<td>女士</td>
			<td>延迟时间</td>
			<td rowspan="4">气流阻塞</td>
		</tr>
<tr>
<td>sRaw</td>
			<td>cmH2O·s</td>
			<td>特定气道阻力</td>
		</tr>
<tr>
<td>sGaw</td>
			<td>1/cmH2O·s</td>
			<td>特定气道电导率</td>
		</tr>
<tr>
<td>EF50</td>
			<td>毫升/秒</td>
			<td>中潮汐呼气量流量</td>
		</tr>
<tr>
<td>锶</td>
			<td>%</td>
			<td>成功率</td>
			<td rowspan="2">质量控制</td>
		</tr>
<tr>
<td>N</td>
			<td></td>
			<td>有效呼吸次数</td>
		</tr>
</tbody></table>表1。从双腔 plethysmography 获得的典型参数列表.这些参数根据它们在呼吸功能评估过程中提供的信息的性质进行分组。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>优势</td>
			<td>限制</td>
		</tr>
<tr>
<td>·有意识的动物</td>
			<td>·控制周围环境的必要性</td>
		</tr>
<tr>
<td>·准确的通风参数</td>
			<td>·动物的事先驯化</td>
		</tr>
<tr>
<td>·无争议的气流阻塞指标 (sRaw, EF50)</td>
			<td>·鼻与胸流的分离要求</td>
		</tr>
<tr>
<td>·适应不同种类和动物的大小</td>
			<td>·某些结果参数的绝对值可变性</td>
		</tr>
<tr>
<td>·用于许多研究应用中</td>
			<td>·sRaw 不是一个真正的阻力测量</td>
		</tr>
<tr>
<td>·简单的技术</td>
			<td>·上部航空公司的存在</td>
		</tr>
<tr>
<td>·对更改敏感</td>
			<td>·用侵入性评估补充测量结果</td>
		</tr>
</tbody></table>表2。与双室 plethysmography 相关的优点和限制的列表。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>双室 Plethysmography</td>
			<td>强迫振荡技术</td>
		</tr>
<tr>
<td>动物的意识状态</td>
			<td>改变</td>
			<td>麻醉 (通常瘫痪)</td>
		</tr>
<tr>
<td>动物的位置</td>
			<td>直立</td>
			<td>仰卧</td>
		</tr>
<tr>
<td>动物的可及性</td>
			<td>密闭室内</td>
			<td>访问</td>
		</tr>
<tr>
<td>动物与测量装置的集成</td>
			<td>鼻或颈部密封</td>
			<td>气管切开或口服插管</td>
		</tr>
<tr>
<td>动物气道树</td>
			<td>完整</td>
			<td>部分-上呼吸道段排除 (即鼻腔传导, 咽和喉)</td>
		</tr>
<tr>
<td>获得结果参数的肺容积</td>
			<td>动物所采用的可变自发体积</td>
			<td>标准化-使用受控的招聘机动和正端呼气压力。</td>
		</tr>
<tr>
<td>评估结果参数的频率</td>
			<td>动物所采用的可变自发呼吸频率</td>
			<td>控制-使用指定频率的预定义波形</td>
		</tr>
<tr>
<td>上呼吸道段对结果参数的贡献</td>
			<td>预期</td>
			<td>规避</td>
		</tr>
<tr>
<td>气溶胶输送现场</td>
			<td>在顶头房间里</td>
			<td>直接在气管</td>
		</tr>
<tr>
<td>上呼吸道段对吸入剂量/气溶胶沉积模式的影响</td>
			<td>预期</td>
			<td>阻止</td>
		</tr>
<tr>
<td>检测变化的能力-基于本研究的结果</td>
			<td>观察</td>
			<td>观察</td>
		</tr>
<tr>
<td>技术的固有变异性--基于本研究的结果</td>
			<td>sRaw 的变化系数在基线上的波动: 7.5-20.6%</td>
			<td>RN的变化系数在基线上的波动: 3.6-13.4%</td>
		</tr>
</tbody></table>表3。双室 plethysmography 与强迫振荡技术的比较。

Watch this Scientific Journal Video about Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography at JoVE.com

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

本条的目的是提供一个详细的描述的建议程序, 以评估在意识小鼠呼吸功能的双室 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 ...

assessment-respiratory-function-conscious-mice-double-chamber

Research

JoVE Journal

Medicine

42.8K 次观看.  Université Laval. 本条的目的是提供一个详细的描述的建议程序, 以评估在意识小鼠呼吸功能的双室 plethysmography。

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

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

科研

医学

Murine Model of Allergen Induced Asthma

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

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

过敏原诱发的哮喘小鼠模型

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

免疫与感染

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

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

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

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 ...

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

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

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

使用强迫振动技术在小鼠呼吸系统力学评价

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

生物学

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 ...

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 ...