M.Z.和 K.S.F. 和这项工作得到了匹兹堡大学临床和转化科学研究所在皮特创新挑战 （PInCh） 下授予的赠款的支持，NHLBI R01 授予 HL153407，授予 M.Z.

匹兹堡大学机构动物护理和使用委员会在进行这些动物实验之前，批准了本出版物中指定的所有动物协议。
注:此协议详细说明了如何使用双模SPECT/CT扫描仪进行放射性核素成像的体内粘膜清除研究。所展示的技术包括运行系统校准、麻醉小鼠、小鼠气管插管、向肺部灌输同位素、双模成像、这些图像的共同注册以及分析。
1. 光谱/CT 系统设置
<ol><li>设计一个合适的工作流程，并在使用活体动物进行实验之前设置。<ol>
<li>使用由 60 个投影组成的 SPECT 采集，在旋转半径为 40 厘米的投影之间步幅为 6o。CT 采集由 220 个投影组成，投影之间角度为 1.6o。</li>
	</ol>
</li>
	<li>确保系统为小鼠提供正确的 MWB 校准器和 SPECT 成像。如果安装了不适当的校准器，请使用对准器向导安装正确的对准器。</li>
	<li>运行必要的系统校准，为系统准备使用。 注:扫描仪的SPECT和CT组件需要校准。使用源调理和暗/光 （D/L） 校准来校准 CT 组件，每 2 周进行一次中心偏移 （COS） 校准，并每月评估 X 射线硬件。SPECT 组件需要每年校准一次。<ol>
<li>要评估 X 射线硬件，请检查 CT 校准期间 的评估 X 射线硬件 框（补充 CT 校准菜单）。</li>
		<li>要执行源调理，请在 CT 校准期间检查 执行源调理 框（补充 CT 校准菜单）。</li>
		<li>要执行 D/L 校准，请检查 CT 校准期间实验中使用的 CT 采集协议旁边的 D/L 框。取消检查所有其他协议（补充CT校准菜单）。</li>
		<li>要执行 COS 校准，请用校准环工具替换床，调整床型设置以匹配运动控制设置，并在 CT 校准期间的实验中使用的 CT 采集协议旁边检查 COS 框。取消检查所有其他协议（补充CT校准菜单，补充校准环）。</li>
	</ol>
</li>
</ol>2. 鼠标灌输和灌输
<ol><li>称重要扫描的鼠标。如果扫描多个鼠标，请注意使用耳击或尾部标记等方法为识别目的标记小鼠。</li>
	<li>使用 1.5% 异氟化物麻醉鼠标，气体流量为 2 L/min O2，在气室中持续约 5 分钟，以产生足够深度的麻醉，直到呼吸速度减慢到每分钟16 次 55-65次呼吸（图 1A）。</li>
	<li>将鼠标从腔室中取出，在 45o斜坡的灌管支架上由前切口悬挂。用鼻锥装备插管支架，以确保小鼠在插管过程中麻醉（图1B）。</li>
	<li>将 50μm 光纤线的一端连接到光源，并用电线将 20 光纤管穿线（图 1C）。</li>
	<li>打开鼠标的嘴，用钝钳把舌头向前拉。照亮导线，并用它来可视化声带（图1D）。</li>
	<li>通过声带的导线，使电线刚刚超越声带，并在上气管休息。沿着钢丝向前滑动1英寸的管子，给老鼠做管子，通过足够深的管子，使其中心与动物的切口（图1E）相抗使。取出留下管子的电线。</li>
	<li>通过用手指短暂地插入手杖并检查呼吸变化来测试插管。停止呼吸或呼吸紧张，同时堵塞和释放后加速呼吸是适当的气管插管的迹象。如果堵塞手杖后呼吸模式没有变化，后者很可能在食道中。</li>
	<li>在10μL的体积中准备 9900万个技术硫胶体（99mTc-Sc）的0.2 mCi，并将移液器放入管子中。让老鼠在1-2分钟（图1F）内自发地吸入肺部。在将鼠标转移到扫描仪托盘之前取出手杖。 注:放射性核素是由红衣主教健康公司制备和过滤的。</li>
</ol>3. 光谱/CT 成像
<ol><li>将鼠标转移到带鼻锥的25毫米托盘中，用胶带固定，注意不要将胸部和腹部粘得太紧，以免损害呼吸。请小心删除连接到鼠标的任何金属耳标。</li>
	<li>在 200 μL 中制备由 0.05 mCi 组成的放射性幻影，并将此量放置在 0.2 mL PCR 管中。通过将管子贴在小鼠下腹部下方的托盘上，避免与肺部重叠，从而放置管子。 注:幻影用于共同注册CT和SPECT图像，以及清除的负控制。</li>
	<li>将鼠标插入SPECT/CT系统，选择成像工作流程并运行 "设置"。</li>
	<li>设置鼠标上探测器的定位，并运行成像工作流程。</li>
	<li>为手术后接受放射性的老鼠准备一个笼子，不受限制地获得食物和水，并使用辐射安全贴纸进行清晰的标签。</li>
	<li>工作流程完成后，将鼠标从成像托盘中取出，并允许鼠标在预制的笼子中恢复，扫描（扫描结束 1 至扫描 2 开始）之间持续 6 小时，并允许广告利比图姆获得食物和水。选择 6 小时，因为它对应于根据 cilia 功能进行线性间隙的时间段，而紫藤间隙很少。</li>
	<li>6小时后，重新麻醉鼠标并扫描，连同幻影，使用相同的工作流程来测量从气道中清除的同位素量。 注:让小鼠恢复至关重要，因为连续6小时用异氟兰麻醉会导致显著的胆汁抑制作用，导致近零粘膜间隙。</li>
</ol>4. 分析
<ol><li>成像后，执行后处理以重建完整的 3D 堆栈图像。<ol>
<li>使用 99mTc 的出厂标准设置对 SPECT 图像进行直方图，然后使用 MAP3D 算法和点点分布功能 （PSF） 重建进行重建。 注:重建使用8次迭代和6个子集进行。有效的重建需要将子集与 1:10 的预测进行比对，或均匀地划分为预测数，因此使用 60 个投影进行收购使用了 6 个子集。</li>
		<li>使用费尔德坎普算法和谢普-洛根过滤器重建CT图像。 注:重建是使用4次迭代进行的。</li>
	</ol>
</li>
	<li>使用残片工具处理 FIJI ImageJ21 中的 CT 和 SPECT 图像，从默认轴向图像生成日冕视图图像。然后在 SPECT 图像上执行 z 堆栈总和投影，以添加每个切片的计数数据，并生成单个图像以便于分析。</li>
	<li>调整大小并共同注册CT和SPECT图像使用幻影埃彭多夫管作为参考（图2A，B）。跟踪并使用所有样品的一致调整尺寸测量。</li>
	<li>使用自动阈值对 CT 图像进行二进制，然后倒置堆栈，并执行 z 堆栈总和投影，生成肺部轮廓以供分析（图 2C）。</li>
	<li>旋转 CT 和 SPECT 图像，并使用通道工具合并图像。通过在右肺周围绘制投资回报率并测量（图 2D）来计算 MCC。 注:此测量将计算右肺 0 和 6 小时时间点的总计数，使用公式更正 6 小时图像以进行放射性衰变:N（t）= N0e+t.99米Tc-Sc 的衰变常数为每秒 3.21e=5，半衰约 6 小时。然后，这些值可用于计算百分之百的间隙。 注:右肺被选为投资回报率图纸和测量计数，因为粘膜间隙将把放射性同位素从肺部输送到咽部，从那里吞咽，最后在胃中。很多时候，计数可以看到在胃，可能与左肺重叠，从而产生错误的计数。这种混淆可以通过测量右肺的计数来避免。</li>
</ol>

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与此工作无关。

湿润的呼吸道在疾病和发展中的作用继续发展，并得到更好的赞赏。气管支气管树内衬细胞的apical表面的多个摩尔西利亚的同步、元时间跳动产生头皮流，产生粘膜间隙或 MCC。MCC在像PCD22这样的心肌病中受到损害，像COPD18这样的疾病，其重要性在CHD中得到了承认，传统上并不被认为是心肌病。最近的数据显示，在患有异位轴23和无异质轴7的CHD中，呼吸系统肝功能障碍。这种移动性胆汁功能障碍被证明转化为更大的呼吸症状9以及更大的术后发病率8。大多数，如果不是全部，这些疾病，有小鼠模型可用，我们的协议，测量MCC在小鼠是一个有价值的工具，可以用来测试潜在的治疗方法。
动物模型为了解疾病和治疗的发展提供了效用。体内动物成像提供了进一步的效用，能够从同一动物获得多个数据点，而无需牺牲动物，使调查人员能够跟踪疾病的纵向过程以及治疗效果的研究持续时间。MCC的老鼠模型是由多个研究者在几十年的时间里开发的，最初是使用平面扫描技术（二维核成像技术24）在小猎犬身上进行的。十年后，这项技术被改用在小鼠身上，随后在25、26年之后的10年适应了SPECT成像。这种技术在小鼠模型中的发展是该技术相关性的一个重大发展，因为有多种人类疾病小鼠模型，如PCD，其中硅功能发生显著变化。MCC已被评估在小鼠模型的肺衰减和免疫抑制，并有可能与其他模型19，26一起使用。在人类气道疾病（如CF、哮喘、PCD和与CHD相关的心肌病）患者中进行了MCC测量研究，并取得了该技术可同时帮助肺生理学和治疗疗效的研究。
此协议的一个重要部分是设置具有正确成像参数的获取，以获取用于量化的准确图像。在设计 SPECT 收购设置时，有许多因素是关键，包括使用哪些校准器、每次革命获得的预测数和旋转步数大小。合金选择是收购的灵敏度和分辨率的一个主要因素，收购设置可能需要根据正在使用的27的对准器进行定制。或者，当使用更大的动物，如老鼠，碰撞器将需要调整。例如，多个针孔校准器更敏感，但在选择步骤大小时应小心，以避免重叠的投影和造成不受欢迎的多路复用，这可以进一步提高收购的灵敏度，牺牲一些图像模糊性，可能会导致重建文物25。重建设置也是生成可量化图像的关键。MAP3D 是常用的迭再重建算法，PSF 是常见的重建模型。两者都可用于重建图像，但在设置迭代和子集数量时应小心谨慎。更多的迭代将增加重建所需的计算时间，并随着回报的进一步增加而提高重建的质量。
为了量化 ImageJ 中的图像，理想的测量工具是 RawIntDen，它在选择中输出像素的总值。当在不同大小的肺 ROI 上量化 SPECT 数据时，RawIntDen 的使用提供了绝对的计数量度，并避免将测量结果调整到 ROI 的区域，就像平均测量结果为 21一样。
此技术具有许多相关的错误来源，调查人员在应用此技术时应了解这些错误源。一个值得注意的混淆是使用麻醉剂。伊索夫卢兰是一种快速作用，吸入麻醉剂，小鼠在完成采集后迅速恢复。然而，应小心为小鼠提供充足的时间在笼子里恢复，并且不要超过必要的时间进行麻醉。根据我们个人的经验（未公布的数据），在0至6小时时间点之间连续使用吸入的异氟兰进行麻醉的小鼠的间隙可以忽略不计。同样，控制剂量的麻醉剂也是必要的，以确保快速恢复。当将动物固定到托盘进行成像时，用于共同登记的幻影管应保持在腹部低位，以避免文物与肺部重叠。同样，为了确保高质量的 CT 图像，请注意从鼠标中删除任何金属标签，以避免从 X 射线散射中分离出工件。
目前的MCC协议可以应用于无数的动物模型。这种技术对被扫描动物的健康影响微乎其微，老鼠可以很好地耐受，因此它可以与疾病模型一起使用，而不会危及已经很脆弱的小鼠的健康。这种方法的强度来自于它是一种体内技术，它允许获得一致和可重复的气道功能测量，而无需牺牲动物来切除气管进行视频显微镜检查，前体内模型需要26。这种技术在对同一动物的多次扫描中产生可重复测量的一致性，允许用不同的剂剂或潜在的治疗方法对同一动物进行治疗，并在同一动物之间进行统计比较，以减少任何动物模型固有的生物变异性，从而减少显示统计显著差异所需的样本量。
使用MCC技术对气道功能的评估可以调整到各种动物模型，并应用于许多不同的气道健康模型，以及测试新的疗法。可使用此技术以及 COPD 模型评估 PCD 小鼠模型的气道。我们的方法也可以用来研究各种麻醉剂对MCC的差异效应，这是常见的临床用途。最后，也可以使用此模型评估治疗剂对气道的影响。如前所述，但值得重复，因为它是一种体内测量，它允许在疾病过程中重复MCC评估，以及随着时间的推移治疗干预的测试益处。此外，小鼠是用于模仿/研究人类疾病的最常见实验室动物，在某些情况下，有多种转基因小鼠模型可供选择。

Cilia 是基于微管的细胞器，保存于从藻类到人类的进化历史中。它们来自细胞表面，具有多种功能1，从识别局部环境感官信号到运动性，其功能可以追溯到人类早期的单细胞真核生物2、3。Cilia 可以是非移动和单体，作为细胞处理环境信号的专用天线:或移动和多，在同步的，元时波跳动，以产生流体流动，如在输卵管的衬里和上下气道，除了终端支气管导致肺泡1，2。
呼吸道广泛的上皮表面不断受到各种潜在危险吸入污染物和病原体形式的污染，因此需要防御。一个关键的防御机制是气管支气管树的粘膜装置，通过敲击气管支气管上皮细胞的apical表面的多块粘液，机械地将连续的分泌粘液流出气道。这些功能诱捕吸入的污染物，并通过其连续，同步殴打，运输他们头孢4，5。
Cilia已被证明在胚胎发育中具有关键作用，如在胚胎发育中左右模式，其中胚胎节点的摩尔西利亚断裂对称性6。由于心脏的不对称结构，与西莉亚相关基因的突变与先天性心脏病（CHD）等疾病有关。最近的研究报告，有染病患者呼吸道功能障碍的高发率，以及术后呼吸道并发症和慢性呼吸道症状在上、下气道7、8、9、10的患病率上升。有或无异质性心肺功能障碍的病人，在术后5、8、10日，已证明有呼吸系统并发症及负面呼吸道结果的风险增加。除了在信号和开发中的作用之外，气道西莉亚的重要性也由心肌病所证明，其中一个典型的例子是主要的胆囊性发育不良（PCD）。PCD是一种先天性疾病，由一些影响湿润的呼吸道的突变引起，导致肺部反复感染，支气管切除术，并有可能需要肺移植11。此外，即使西莉亚在囊性纤维化（CF）中是正常的，这是高加索人群中最常见的先天性疾病，但MCC由于CFTR基因12的突变引起的粘稠粘液而受损。PCD 和 CF 的多个鼠标模型，以及不断增加的 CHD 模型。最终，西莉亚是多功能结构，具有许多关键作用，评估活体中移动呼吸系统西莉亚功能的方法对于临床前研究、评估突变以及药物对粘结清除 （MCC）13的影响是有价值的。这种方法对于评估这些小鼠模型中新药、基因疗法或干预措施对MCC的影响也很有价值。
有许多不同的模型已经用于评估MCC。一种值得注意的方法是使用已灌输到支气管中的甲基蓝色染料，通过纤维光学测量染料运动的间隙14。这种方法受制于观察染料运动的能力，这种染料在人类中比在临床前小鼠模型中更为常规。另一个值得注意的方法是同步加速器相对比X射线成像（PCXI），它可以用来跟踪气道中的单个粒子。这种方法是比较新的，并不广泛访问15。有许多外活体方法来评估气道通过切除气管的视频显微镜，但这些模型提供很少的效用，在人类患者16。Cilia成像的高分辨率技术，如光学相干断层扫描，以同样的方式受到限制。
本文介绍了一种可重复的方法，用于测量无数动物模型中的肺间隙，以及研究慢性阻塞性肺病的MCC，并评估免疫抑制药物18、19的影响。这种方法跟踪放射性制药9900万技术硫胶体（99mTc-Sc），一种不溶性颗粒放射性跟踪器，在注入肺部后清除。然后，放射性核素可以使用单光子发射计算断层扫描（SPECT）18，20进行跟踪。我们进一步完善了这种技术，通过使用双模频谱和计算断层扫描 （CT） 成像与放射性同位素计数的共同本地化到肺部，并测量这些计数在 6 小时内的减少。双模成像，CT 和 SPECT 图像的共同注册，可准确定位辐射计数到我们感兴趣的区域，肺部。虽然我们详细描述了小鼠的MCC测量方法，但可以调整协议以研究大鼠的MCC。校准器需要调整以及辐射剂量。我们认为，由于动物体积小，小鼠MCC扫描在技术上更具挑战性，但由于人类疾病的既定小鼠模型数量众多，因此比大鼠更有用。此外，由于动物群落的成本和维护成本较低，较大的样本量在小鼠中更为可行。

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in vivo evaluation of mucociliary clearance in mice

呼吸摩尔胆汁，细胞的专门细胞器，线的上皮细胞的上皮表面衬里呼吸道。通过以元时间、同步的方式跳动，这些多重、多动、基于行为素的细胞器产生一个头孢流体流，清除吸入的污染物和病原体的呼吸道。随着环境污染的加剧、新型病毒病原体和新兴的耐药细菌的出现，胆汁产生的粘液清除（MCC）对于维持肺部健康至关重要。MCC也患有多种先天性疾病，如原发性囊性发育不良、囊性纤维化以及慢性阻塞性肺病等后天性疾病。所有这些疾病已经建立了，在某些情况下，多个鼠标模型。在本出版物中，我们详细介绍了一种使用少量放射性和双模SPECT/CT成像技术来准确和可重复地测量体内小鼠的MCC的方法。该方法允许在成像后恢复小鼠，使连续测量成为可能，并随着时间的推移对潜在的治疗方法进行纵向测试。野生型小鼠的数据表明，只要充分注意细节，并严格遵守协议，MCC测量的可重复性。

使用此协议，我们在异氟室（图1A）中麻醉小鼠。在达到足够的麻醉水平后，小鼠被放置在垂直支架上（图1B），声带使用照明导线（图1C-1D）进行可视化。这些小鼠通过管子和允许自发吸入肺部的小鼠（图1E-1F）被灌输并灌输0.2mCi 99mTc-Sc，体积为10μL。在图像采集和处理后，CT 和 SPECT 图像以幻影管为地标（图 2B）进行本地化 （图 2A）。肺部的口罩来自CT图像（图2C），用于在右肺周围绘制ROI，以0（图2D）和6小时（图2E-2F）进行分析。为了测试协议的可重复性，在实验条件相同的不同天共扫描了 8 只小鼠两次，使用配对的 t 测试进行分析，显示重复扫描（p 值=0.9904）（图 3A）之间没有显著差异。另有2只小鼠在实验条件相同的不同天被扫描了三次，使用单向ANOVA进行分析后显示重复扫描（p值为0.0041）（图3B）之间有显著匹配。共扫描了8只老鼠，并展示了两张有代表性的图像（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61929/61929fig01.jpg"> 图1:鼠标灌输和同位素灌输。将同位素灌输到气道所需的步骤的图像。 A） 鼠标在室内麻醉。 B） 麻醉鼠标被放置在垂直支架上，由前切口悬挂。 C） 通过20G的管线运行，准备了照明的0.5毫米光纤线作为导线。 D） 鼠标嘴使用钳子打开，并使用照明导线照明以可视化声带。 E） 管子被推过声带，导线被移除。 F） 可溶性同位素被灌输到管子使用移液器和老鼠允许自发吸入同位素到肺部。 <a href="https://www.jove.com/files/ftp_upload/61929/61929fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61929/61929fig02v2.jpg"> 图2:MCC扫描的SPECT/CT图像。A） 已与 CT 图像共同本地化的 SPECT 图像。 B） 带有可视幻管的CT图像，用于共同定位。 C） 通过将 CT 图像比纳化并执行 z 堆栈投影而衍生的气道面罩。 D） CT 面膜与 SPECT 图像共同本地化。在右肺周围绘制了用于分析的 ROI。 E） 6 小时后气道的口罩。 F） 在 6 小时内与投资回报率共同定位气道的 CT 和 SPECT 图像，以供分析。 <a href="https://www.jove.com/files/ftp_upload/61929/61929fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61929/61929fig03.jpg"> 图3:通过多次扫描对同一鼠标进行清除测量。A） 为8只实验条件没有变化的小鼠测量了两个单独的重复间隙。配对 t 测试表明，p 值为 0.9904 的重复扫描之间没有显著差异。 B） 为两只实验条件没有变化的小鼠测量了三个单独的重复间隙。单向 ANOVA 显示重复扫描与 p 值 0.0041 之间有显著匹配。 <a href="https://www.jove.com/files/ftp_upload/61929/61929fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61929/61929fig04.jpg"> 图4:2只小鼠0小时和6小时气道的共同本地化SPECT/CT图像，0和6小时绘制的ROI概述右肺。<a href="https://www.jove.com/files/ftp_upload/61929/61929fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
补充图1:由光纤线照亮的声带视频，其呼吸效果可视化。<a href="https://cloudflare.jove.com/files/ftp_upload/61929/61929supfig01.jpg" target="_blank">请点击这里下载此数字。</a>
补充文件。<a href="https://cloudflare.jove.com/files/ftp_upload/61929/Supplemental_Files.zip" target="_blank">请点击这里下载这些文件。</a>

Watch this Scientific Journal Video about In vivo Evaluation of Mucociliary Clearance in Mice at JoVE.com

In vivo Evaluation of Mucociliary Clearance in Mice

在本出版物中，我们描述了利用双模放射性核素成像评估小鼠体内气道粘膜间隙 （MCC） 的协议。此协议是专为单光子发射计算断层扫描 （SPECT） 和计算断层扫描 （CT） 采集协议而设计的，使用双 SPECT/CT 系统中的鼠标全身 （MWB） 校准器。

在体内评估小鼠的粘液清除

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

in-vivo-evaluation-of-mucociliary-clearance-in-mice

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JoVE Journal

Biology

4.2K 次观看.  University of Pittsburgh School of Medicine. 在本出版物中，我们描述了利用双模放射性核素成像评估小鼠体内气道粘膜间隙 （MCC） 的协议。此协议是专为单光子发射计算断层扫描 （SPECT） 和计算断层扫描 （CT） 采集协议而设计的，使用双 SPECT/CT 系统中的鼠标全身 （MWB） 校准器。

视频: In vivo Evaluation of Mucociliary Clearance in Mice

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

科研

生物学

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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 ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

伸趾长肌的肌肉功能评价<em&gt;体外</em和胫骨前肌<em在原位</em在小鼠

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

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 ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

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

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

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

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

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