这项工作是由德意志研究联合会（B＆K 196"Signaltransduktion北adaptativen UND maladaptiven kardialen改建-Prozessen"，FR 1638 / 1-2）以及由DZHK（德国中心心血管研究，健康研究的德国中心的部分支持，这是一个BMBF（德国教育和研究部的）行动）。

注:分离过程的基本步骤示于图1中的所有步骤都在下面详细描述的协议。乳头肌隔离，安装在器官浴室，采集和分析是在一个连续的和强制性的时间表进行。 所有的动物实验均按照有关保护动物的有德国法律进行，并批准了海德堡大学的伦理审查委员会。 1.准备仪表<ol><li>对于收缩测量使用多节器官浴设置。 离体收缩器官浴中设置的组件示于图2。 注意:为尽量减少的时间制备的乳头肌和实验方案的开始之间的时间量，建立设备和事先准备缓冲器收集实验tissue.This provid上课的时间，该组织保持生存能力进行实验的最大量。 </li><li>启动计算机和数据采集设备。打开器官浴热（预设为32℃），并允许单位预先设定的温度。 </li><li>准备采集设备。启动中提到的软件手册记录数据。 </li><li>检查，并进行校准（如有必要）在每个通道上，零所有通道从各自的力换能器（与该信道相关联）提供的电信号标准化到由经认证的重量供给2克力。 </li><li>开启气体供给（95％O 2/5％CO 2）到器官浴室中。连续气在整个协议的所有测量室。 </li></ol> 2.准备缓冲区和生理解决方案<ol><li>准备一个的Krebs-Henseleit缓冲溶液以达到最终浓度为119毫摩尔NaCl; 25毫米的NaHCO 3; 4.6毫米氯化钾; 11MM葡萄糖; 1毫米的Na-Pyruvate; 1.5毫米氯化钙2; 1.64毫米硫酸镁 ; 1.18毫KH 2 PO 4和调节pH值至7.4（见表1的生理溶液）。 </li><li>制备单独的Krebs-Henseleit缓冲溶液如步骤2.1中所述用另外30毫2,3-丁二酮单肟（BDM）和50的IE肝素/毫升隔离期间使用。凉溶液至4℃（见表1制备的溶液）。缺血模拟，如表1中所述制备缺血溶液。 </li><li>对于钙离子的协议-依赖性收缩， 钙离子加入到的Krebs-Henseleit缓冲溶液到请求的最终浓度。 注意:它直接使用前准备这些解决方案，并与卡波盖斯以避免Calciumcarbonate沉淀。使用前Prewarm解决方案，以32℃。 </li></ol> 3.准备的乳头切除术过程中使用的冲气管道和解剖菜<ol><li>连接软气唱管（外径2-4毫米），气体连接（100％的氧气，卡波备选地）与专家工作。 </li><li>创建冲气管解剖盘内配合的封闭环。穿刺放气管数次，一个连续鼓泡是有保证的。此放气管可多次使用。 </li><li>制备夹层菜与硅弹性体在底部（0.5厘米厚），以使销可以卡到其中。此菜可多次使用。 </li></ol> 4.分离的小鼠左前乳头肌注:在启动乳头肌的隔离前，检查煤气管道是畅通无阻的。 <ol><li>准备器官浴设置为启动乳头肌的准备之前提到的协议部分1-3。制备所有溶液在实验当天。 </li><li>颈椎脱位accordi牺牲鼠标（约8-12周龄）NG专家的工作和机构准则。 </li><li>通过固定前爪固定在背侧位置的动物和后爪上的夹层板，通过与锋利的骨剪刀肋切割打开两侧的胸腔侧，然后切断与一个横向切口隔膜。取出心包。现在的心脏和主动脉的访问。 </li><li>修复钝钳的心脏上的血管共干靠近心脏，迅速分开心脏与肺部剪刀和周围的组织。 </li><li>跳动的心脏转移至培养皿装有充以氧（步骤2.2）中的冷却溶液制备。让心脏跳动，并通过触摸心尖钳刺激心脏收缩轻声。 </li><li>一旦该心脏完全exsanguinous，其传输到解剖盘充满冷却制备溶液（步骤2.2）和通入O 2。从这个步骤上使用立体显微镜。 </li><li>修复老天室温通过右心室使心脏从背部可见（右心室位于右侧，从运营商的观点）一个小针。 </li><li>采用显微剪刀分开两个心房的房室水平，切从心室连接组织并丢弃。通过从AV-阀级到心脏，避免任何机械压力的顶点室壁执行剪切。 </li><li>打开心室和修复左路游离壁用钳子，因此具有一起来看看这两个左心室乳头肌。 </li><li>略切去上左前乳头肌的两侧保持在乳头肌制备瓣膜帆的一部分的心室组织的横向和避免接触或拉伸乳头肌尽可能。解剖从乳头肌的其余室壁组织。 </li><li>在乳头肌准备两侧安装丝线（7/0，指标0.5）aration，一个在瓣膜帆，一个在肌肉部分。修复准备在器官浴室充满器官浴的解决方案，并通入卡波，温度32℃的建议。 </li></ol> 5.平衡乳头肌和刺激<ol><li>在器官浴中的固定后立即刺激乳头肌制备的矩形脉冲（2毫秒持续时间和100mA的电流）在1赫兹的刺激频率。 </li><li>确保器官浴溶液的频繁变化，或者通过连续变化或根据所使用的器官浴类型的设置频繁地手动改变（每5分钟）。 </li><li>逐步提高预紧直到最大抽搐力达。后面没有进一步增强在抽搐力预紧的增加时，最大抽搐力达到。 </li><li>平衡后的45-60分钟，开始实验方案。 </li></ol>注意:下面列出的实验方案包括标准的机动生理和病理条件下心肌收缩的特点。在代表节中，我们还详细示出代表性的结果说明这些协议（也见表2）。 <ol><li>记录和试验方案分析<ol><li>对于记录，使用合适的软件，有足够的时间分辨率（采集率≥1kHz时）。 </li><li>分析参数，包括抽搐力的振幅（高度），达峰时间张力（TTP）和半数最大松弛时间（R 50 / TFALL，分别）中提到的软件程序（如实施例图表的ADInstruments 5.5， 参见图4）。 </li></ol></li></ol>

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作者宣称，他们有没有竞争的财务权益。

在这个手稿，我们描述的方法来研究的鼠的乳头肌收缩力在体外可被用来回答有关心脏生理和病理小鼠几个科学问题以及支持的转基因品系的分析和新药物的方法的发现治疗心脏功能障碍。我们示出了使用这种方法来评估的心肌收缩性生理，病理和药理性质（参照图3）。这里未示出的其它应用包括使用不同的药理学试剂的细胞内途径的评价（参见12）。 心脏疾病，如缺血性心脏疾病和心脏衰竭显示杰出的临床问题，并保持对死亡率和发病率全世界22的首要原因。为了表达在心脏许多蛋白质​​，其对心肌收缩作用是STIL升知之甚少或没有研究到现在。对离体乳头肌收缩力的测量表明，研究体外心脏收缩在小鼠模型中有意义的和有效的方法。 尽管该方法在技术上是可行并显示了良好的再现性，也有必要的几个关键步骤，以确保它的成功:首先，分离的心脏后，确保在制备过程期间血液中的游离溶液，以便能够正常工作（可视化）。另一个关键步骤是组织准备工作认真执行，以确保可行性，避免任何接触或拉伸乳头肌。如在方法部分中描述尽量保持瓣膜帆连接到乳头肌，以确保无故障附着丝线。制备在器官浴室的固定后，在第一分钟改变器官浴溶液数次冲洗出BDM。 Increa瑟制剂的预紧略微逐步直到没有更多的增加抽搐力发生。有一些排除标准的单一协议的评估，如心律失常节拍和预紧作为缺血性条件的标志自发的增加。几个实验方案如postrest增强和力频率的关系可以用相同的制剂样品进行，而一个新的制剂样品应当用于对收缩药物介导的效应的分析。制备协议被优化以分离左前乳头肌。适配协议，也后部肌肉可用于收缩的测量此方法。 隔离的乳头肌的测量该方法提供了关于钙，温度和频率有关的收缩力的各种信息，以及有关应用药理学试剂或SIM后收缩反应ulation的病理状况。然而，这些结果的解释和外插需要与科学小心处理。所描述的方法是在体外模型心肌，从它的正常环境和神经支配断开。因此，实验条件不生理和在这个协议中接收到的结果不能无需深入解释被转移到体内的情况。例如，该方法没有考虑到在血压，激素或外在神经控制帐户改变。的氧和代谢补充剂的制剂在器官浴中的供给受到扩散限制。的氧和代谢补充剂供应不足会导致缺血性病症，随后无效的实验结果。为了避免这种情况，在试验条件应该是最佳的，特别是关于氧合器官浴溶液，钙的浓度和温度。与这里描述的协议的实验条件下，有效的结果可以得到。 在这份手稿模拟类缺血病症的方法。此外缺氧的仿真，所述器官浴中的溶液的所有含能补充被耗尽模仿缺血状态体内尽可能好。在对比缺氧，它表明，氧和能源供应的同时消耗导致的变化的钙瞬变媲美见于体内 23的改变。 对小鼠的乳头肌的隔离中描述的方法可以适用于其它物种，例如大鼠。然而，器官浴设置的最佳条件可以在不同的动物模型之间，具体费用可能会进行调整。可能需要自适应参数是温度和生理溶液中的钙浓度。 在总表Y，这种收缩方法提供了一个非常有效的方法来评估心脏生理学，病理生理学和药理学。如果使用得当，它为研究心肌收缩在一个孤立的，但良好的控制环境的能力。

像离子通道参照它们的作用为心脏收缩蛋白的调查是必不可少的发现不同的病理机制，并建立新的治疗策略用于心脏疾病，如局部缺血和心脏衰竭。 哺乳动物心肌收缩功能已知通过各种离子通道，转运和其他蛋白质被调制。动作电位诱发激活电压依赖性肌膜L型钙通道引出的Ca 2+内流 ，从细胞外空间，并随后到的 Ca 2+ -诱导的 Ca 2+释放（CICR）1，这将触发蜂窝收缩2。钙 -信号起着心脏收缩力和适应方面发挥核心作用的生理或病理压力。儿茶酚胺激活心脏β肾上腺素能受体，从而刺激腺苷酸环化酶（AC）的合成营。被激活，proteiÑ ​​激酶A（PKA）的磷酸化细胞内的不同和膜相关蛋白质如L-型Ca 2+通道 ，磷蛋白和兰尼碱受体导致的钙瞬变和心脏收缩1,3,4修饰。的cAMP通过磷酸二酯酶（PDE）的降解。 G s - 偶联受体比β肾上腺素能受体其它的活化也导致的cAMP蓄积。 心室肌细胞肌条收缩测量的技术已经非常成熟的大型哺乳动物物种5-8。基于基因的可能性靶向小鼠建立的方法来分析鼠心脏生理是重要的。然而，大约在小鼠中分离肌肉制剂的生理特性的现有数据的差异取决于实验条件9-12。 所描述的方法被用来分析左心室乳头肌的预心脏收缩parations 体外 。在没有影响改变心肌收缩力在体内 ，如血压，神经体液刺激和生理或代谢应激进行心肌收缩力的研究。承包肌肉制剂的跳动速率可以严格定义，改变随意。抽搐力能够在特定刺激，如钙浓度，跳动频率或温度的情况下进行分析。此外，这种方法可用于研究不同信号传导途径的组件和通过控制上述实验条件下进行比较的转基因小鼠模型的心脏的性能。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P 5655</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F3917</td>
			<td>Hazard statement H312, solve in DMSO</td>
		</tr>
		<tr>
			<td>3-?Isobutyl-?1-?methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>Hazard statement H 302, solve in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>I5627</td>
			<td>Hazard statement H 315-H319-H335</td>
		</tr>
		<tr>
			<td>Sodium Heparine 250.000 IE/10ml</td>
			<td>ratiopharm</td>
			<td>PZN 3874685</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H7250</td>
			<td>Hazard statement H 315-H 317-H319- H334-H335</td>
		</tr>
	</tbody>
</table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

乳头肌从成年小鼠心脏分离，可用于在不同的生理/病理条件下，研究心脏收缩。独立的外部影响，如血管紧张或神经体液状态的收缩特性可以被评估。它描述了单细胞测量孤立心肌细胞之间， 像超声心动图体内研究科学的方法。因此，乳头肌制剂作为一个很好的模型来研究心脏生理/病理生理学和可用于像由药剂的调制或转基因动物模型的探索调查。这里，我们描述分离鼠左前乳头肌以调查在器官浴设置心脏收缩的方法。在对比一个肌肉条准备从心室壁分离，乳头肌可以全盘而不损伤肌肉Tissu酒店制备Ë严重。器官浴设置包括几个温度控制，毒气和电极配备器官浴室。将分离的乳头肌固定在器官浴室和电刺激。诱发抽搐力是使用压力传感器和参数，如抽搐力的振幅和抽搐动力学分析记录。不同的实验方案，可以执行以调查收缩剂如儿茶酚胺或其他药物的碳酸钙和频率相关的收缩以及剂量 - 反应曲线。此外，像急性缺血病理状况可以模拟。

本手稿的孤立鼠的乳头肌收缩制剂测量值的协议被调谐到最佳条件，以实现在生理条件下可再现的实验结果。为了确定最佳的实验条件下，我们进行试点实验，不同的器官浴温度和细胞外钙离子浓度（参见12）。这里描述的协议用的1.5mM的胞外钙浓度和32℃的温度下进行。 基底收缩特性要抽搐力振幅表征基底收缩性质，参数，抽搐率，达峰时间（定义为达到从5％基底起始最大收缩力所需的时间）和松弛50时间（半最大松弛时间;定义为所需的时间达到一半松弛力）在本协议中使用。 TW的代表痕迹痒和这些参数的分析示出图3A-B中 钙依赖收缩细胞外钙浓度关键确定心脏兴奋收缩耦联以及通过改变该参数，收缩装置的心肌细胞的钙敏感性进行评估。 如图3C中改变钙瞬变的胞外钙浓度导致在改变的抽搐力产生的增加和随后。钙浓度的胞外溶液的增大一步一步（即，在1.5至7毫米）。通过应用该协议，关于收缩装置的钙依赖性收缩的钙稳态可以评估。在该手稿中使用的器官浴设置，通过改变器官浴SOLUT执行的胞外钙浓度的变化离子手动导致起搏的短时中断，并随后向一个人工postrest增强（见非起搏间隔后力增强）。 非起搏间隔后力增强（PRP） 在一个收缩周期钙的端部螯合成sarcoplasmatic网（SR）主要由ATP依赖钙泵（SERCA，肌浆-内质​​网钙ATP酶），从而在舒张期间降低细胞内钙浓度。如果两个收缩之间的时间间隔变大，更多的钙可以抽回SR并且随后更钙能在下一个激励期间被释放。该postrest-增强协议（PRP）描述了先前的刺激定​​义的持续时间的休整期之后，抽搐力的振幅的变化。在人类，大鼠以及小鼠心脏组织，抽搐力的增大显示出10,12-14,16（ <stroNG&gt;图3D）。已知的是，一个正交的其余的增强被减少或甚至在没有人和大鼠13,14,16,17的心脏组织的不利。该协议用 ​​于评估舒张钙积累的功能在细胞内储4,15。 频率相关的收缩（FFR） 力频率的关系描述了跳动率和收缩力抽搐（鲍迪奇效应）18之间的相关性。力-频率关系哺乳动物物种和实验条件19之间显著不同。在小鼠中，显示出打浆速率和收缩强度之间的特定相关性。在32℃，0.1-1赫兹刺激率之间的初始负FFR被示出，而FFR被证明是在1-4赫兹刺激9-12（图3E）的范围内积极。 在没有myocardium搏动频率和收缩描述20,21之间的负相关关系。在该手稿，标准起搏频（1赫兹）降低到0.1赫兹，并从那里开始，则起搏频率被逐步升高到5赫兹（0.2; 0.5; 1; 2; 3; 4; 5赫兹）。 β肾上腺素能刺激像肾上腺素和去甲肾上腺素儿茶酚胺这期间（病理）生理应激条件下通过激活β肾上腺素能受体调节心肌收缩力被释放激素。儿茶酚胺医源性应用起着临床治疗急性心脏衰竭，暂时加强心肌收缩力显着的作用。这种效果也可以看到并研究在体外研究 。作为β肾上腺素受体 ​​的激动剂，异丙肾上腺素增加收缩抽搐力。 在体内，增加跳动频率也会发生（正性肌力作用），其此外调制收缩反应（鲍迪奇效果，另见上文FFR协议中所述）。使用此处描述的方法中，异丙肾上腺素的肌力作用可以在一个固定的打浆速率进行调查没有这种变时作用（图3F）。 β-肾上腺素受体的刺激也可以通过其他激素如组胺实现。在小鼠心室组织，在其中包含的初始正性肌力作用，但与在抽搐力的瞬时下降，如图3G双相反应应用组胺结果。出于这个原因，通过应用组胺测量数据的解释似乎很难;尤其是其受体激活小鼠并没有完全解决。 缺血性条件急性心肌缺血被定义为血液流动，导致与氧心脏组织的供应不足一个严格的限制，并养分导致的损失的收缩，缺血性挛缩和细胞死亡的发展。为了模拟缺血体外，肌肉被暴露30分钟，以葡萄糖和丙酮酸盐-自由胞外溶液鼓泡用95％ 的 N 2/5％CO 2。这一做法的ATP在心肌细胞耗尽，应引起。在开始缺血模拟后几分钟，抽搐力的下降发生在外观描绘成在预置张力（ 图3H）自发增加缺血性挛缩。 <img alt="表1" src="/files/ftp_upload/53076/53076table1.jpg" /> 表1:成分及所使用的缓冲溶液的浓度。 <img alt="表2" src="/files/ftp_upload/53076/53076table2.jpg"<html> /&gt; 表2:建议试验性的协议列表调查孤立乳头肌的准备心肌收缩力。 <img alt="图1" src="/files/ftp_upload/53076/53076fig1.jpg" /> 图乳头肌准备1.关键步骤。这两个心房（A）解剖。 （二）对左室前壁乳头肌查看。 （ 三）夹层从心室壁乳头肌。 （四）在器官浴室固定前两丝线的附件。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53076/53076fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> 6fig2.jpg"/&gt; 图2.器官浴设置。器官浴设置的（ 一）示意图设置。配备有通气玻璃料水套器官浴室提供稳定的温度控制。以产生方波脉冲场电极被附连到组织支撑。的力传感器检测的肌肉，从而放大所产生的信号的收缩。这个信号被滤波并传送回计算机由A / D转换器。该信号然后被数字化并可以保存供以后的分析。 （二）照片的单个乳头肌固定编制从器官浴设置（C）器官浴室。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53076/53076fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html>皮克"/&gt; 图鼠左前乳头肌收缩力的测量代表性的成果。（A）抽搐力的振幅的代表分析，达峰时间紧张（TTP）和单抽搐半最大舒张时间（R 50）。根据基底刺激1赫兹（B）的搏动频率鼠乳头肌制剂刺激抽搐，期间增加细胞外钙离子浓度（C）代表的录音，在规定的休息时间间隔（后剩下的增强，PRP）（D），或不同刺激率（力频率的关系，FFR）（E）和应用后Isoprenaline-（F）或组胺的应用程序（G）的浓度增加。刺激抽搐和预紧期间缺血刺激（H）代表记录。S://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53076/53076fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53076/53076fig4.jpg" /> 收缩参数图4.分析。为了表征收缩功能，参数，包括抽搐力的振幅（高度），达峰时间张力（TTP）和半数最大松弛时间（R 50 / TFALL，分别）在这个协议中使用的软件分析计划表5.5（的ADInstruments）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53076/53076fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

鼠左心室乳头肌可用于研究在体外心脏收缩。本文详细介绍了隔离和实验方案，研究心脏收缩的特点。

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

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Nanyang Technological University

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14.9K 次观看.  Universität Heidelberg. 鼠左心室乳头肌可用于研究在体外心脏收缩。本文详细介绍了隔离和实验方案，研究心脏收缩的特点。 

视频: Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

科研

生物学

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

"体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替"

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

The Mouse Isolated Perfused Kidney Technique

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

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

鼠标离体肾技术

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

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

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. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>小鼠内淋巴囊解剖

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

小鼠内淋巴囊解剖

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...