Name: bladder smooth muscle strip contractility as a method to evaluate lower urinary tract pharmacology
Uploaded: 2014-08-18T14:00:00.000+00:00
Duration: 10 min 26 s
Description: Watch this Scientific Journal Video about Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology at JoVE.com

这项研究是由美国国立卫生研究院R37 DK54824和R01 DK57284补助磅。支持

这里描述的所有程序是由美国匹兹堡大学IACUC委员会批准。 1，解决方案<ol><li>根据配方制备的Krebs溶液。组成MM:氯化钠118，氯化钾4.7， 氯化钙 1.9， 硫酸镁 1.2， 碳酸氢钠 24.9，KH 2 PO 4 1.2，葡萄糖11.7。 </li><li>充气的Krebs用95％O 2，5％CO 2，并将其放置在37℃水浴中在整个实验过程中使用。放置一旁〜要用于组织解剖将200ml在室温下充气的Krebs溶液。 </li><li>测量充气的Krebs的pH值（〜7.4）和渗透压摩尔浓度（约300毫渗透摩尔浓度）。 </li></ol> 2，实验装置（原理图1A） <ol><li>填充气（95％O 2，5％CO 2）用10毫升的Krebs腔室。 </li><li>启动循环水泵，加热腔室至37℃;打开必要的设备:放大器（S），刺激（S）和录音软件。 </li><li>校准传感器以1克重的。 </li></ol> 3，组织（图1B） 从一个天真的成年人雌性SD大鼠取出膀胱（200-250克;〜10-12周龄）执行以下步骤: <ol><li>准备清扫面积和必要的工具:电动剃须刀，镊子与牙齿，手术刀，解剖剪刀，microscissors两个解剖钳（作者喜欢杜蒙钳＃3），组织夹（或丝线缝合），A的Sylgard涂层剥离菜和克雷布斯组织解剖针。 </li><li>麻醉用异氟烷吸入（在O 2 4％）在感应腔室中的动物。使用对眼睛的兽医药膏，以防止干燥时在麻醉下。连续地观察呼吸速率，响应于外部刺激，和后肢撤回反射丧失监测麻醉的水平。 </li><li>当动物被麻醉，剃毛的下腹部Ä意图。通过中线腹部切口暴露盆腔器官。确定膀胱和尿道。通过切割在膀胱颈附近的近端尿道取出膀胱。在的Sylgard涂层菜充满了加气Krebs溶液立即将组织。 </li><li>如果需要的话，除去额外的组织，此时:尿道，胃肠道（GI）和/或前列腺件等 </li><li>祭祀用IACUC批准的方法（ 例如 ，麻醉过量或CO 2窒息后的第二法）的动物。 </li><li>将组织解剖针穿过膀胱顶部，颈部，输尿管，稳定的组织作进一步解剖。不要伸展组织。去除脂肪，结缔组织，近端尿道，输尿管和如果存在的话。 </li><li>从基部打开膀胱圆顶创建的平片，浆膜侧向下/腔侧上（ 图1B）。放置夹层引脚上的组织的各个角落。删除膀胱DOMe和颈部组织。 </li><li>如果该实验的目的是确定在粘膜（上皮和粘膜固有层-参见图图1C）的贡献，平滑肌收缩，有和没有附着在粘膜比较逼尿肌的属性。对于这一点，前切削成条的组织，使用光圈弹簧在解剖显微镜下剪刀和细镊子小心去除粘膜层。在实验结束时，固定片与H＆E染色，以确认完整切除粘膜。注意，此过程是容易在小鼠膀胱比在大鼠的膀胱。 </li><li>从基部纵向切割组织到圆顶成〜2×8毫米（ 图1B）的条。领带或附上组织夹到每个条带的两端。 注意:一鼠膀胱通常可切成4片，但带条的数目可以增加或减少取决于动物/膀胱大小。 </li><li>转移带的EXPERimental室。将每个条带的力传感器，其测量组织收缩的一端，另一对固定的玻璃/金属杆。 注:组织室大小不等（0.2毫升〜20毫升或更大）。对啮齿动物的囊典型腔是5-20毫升，这提供足够的高度的条带，以在溶液被完全浸没。有些室配备了内置的刺激电极，别人没有。应注意，以确保电极的所有连接都在良好的状态，否则电场刺激是不可靠的。 </li><li>轻轻拉伸的组织，直到基本张力达100克（约10 MN）施涂的力量，每个条。最初的组织趋向于放松被记录为基线张力的降低。洗净组织大约用温水加气克雷布斯和每15分钟调整基线张力各1g后洗净。让组织中平衡​​约1-2小时，或直至基本张力稳定（即没有进一步的组织松弛）。 </li><li>加入氯化钾（80毫米）直接连接到该浴〜5分钟，或直至高原反应试验组织活力被达到。也可在实验过程中，或在实验结束时重复和用于归一化的反应的其它药物或间条带（参见下的数据分析部分正常化）响应于高浓度的KCl。 </li><li>与温暖充气的Krebs洗涤组织多次（3-5x），以允许所述组织返回到前处理的条件。 </li></ol> 4，刺激方案<ol><li>调查病理学上自发肌活动或平滑肌张力的影响，使用来自不同的动物模型，如脊髓损伤，BOO或新生儿平滑肌条， 图2示出了使用该方法的研究开发过程中的变化在膀胱自发活动和脊髓损伤后。此外，药物制剂可被用来调制藻ontaneous活性。 图3示出的KCNQ通道调节剂，氟吡汀和XE991，对自发活性和平滑肌紧张的效果。 </li><li>通过添加来自浓缩的储备溶液的化合物直接向浴中在定义的时间间隔的药理平滑肌刺激构建体的浓度响应曲线。使用平行条带药和车辆占车辆和时间的影响。 <ol><li>使母液所需的测试化合物在1000倍的最终工作浓度。对于卡巴（CCH），毒蕈碱受体激动剂，请准备以下个股:10 -5米，3×10 -5男，10 -4米，3×10 -4男，10 -3米，3×10 -3 M ，10 -2 M。最终浓度在浴为10 -8 M〜10 -5 M（ 图4C，D）。对于神经介素B（NMB）中，蛙皮素受体 ​​亚型1激动剂，请准备以下个股:10-8男，10 -7男，10 -6男，10 -5 M，10 -4男，10 -3 M和终浓度在洗澡都是10 -11 m到10 -6 M。 CCh引起双方和仲裁委员会预计将增加组织的收缩。 </li><li>为10ml的组织浴中，立即加入10微升每CCh引起原液作为响应达到平台（ 图4C，D）。在平行条增加车辆（水）的等量。同样，加入10微升的每个神经介素B货解决方案的各个〜5分钟。 注意:观察图4 NMB和CCH对平滑肌张力在来自不同物种的条带的兴奋作用。 </li><li>与兴奋性剂，通常CCh引起或氯化钾调查平滑肌预收缩组织的松弛性能。 </li><li>要阻止激动剂的反应，预处理组织10-20分钟的拮抗剂，使组织穿透性，以激动剂刺激之前。 </li></ol></li><li>对于神经STIM平滑肌的ulation，也称为电场刺激（EFS）按照步骤1至3.13，继续如下所述。 EFS是为了有选择地激活的神经与平滑肌。为刺激参数应慎重选择，以避免直接平滑肌的刺激。 <ol><li>建立的刺激参数:刺激（单脉冲或火车），持续时间（脉冲持续时间和列车的持续时间），频率和强度，如在下面的步骤中所描述和在图5A，B所示的类型。 <ol><li>对于单脉冲刺激，置位脉冲的持续时间，刺激间间隔和期望的刺激的数量。通常的刺激持续时间的参数是单个的脉冲以期望的间隔（ 图5A）递送0.05-0.3毫秒的持续时间。遵循刺激强度的步骤4.3.1.4。 </li><li>对于火车的刺激，将列车时间和列车间的间隔。膀胱组织的典型值3-10秒交付至少1m在分开（ 图5B）。如果发生组织疲劳（在控制周期即 EFS的收缩降低），提高了列车间的间隔。 </li><li>建立在火车列车刺激脉冲的频率（数 - 图5B）。运行频率响应曲线，从0.5〜50赫兹。典型频率为膀胱是10-20赫兹，这给了ATP和乙酰胆碱介导的可重复性和稳定的收缩。观察到的EFS刺激的频率依赖性反应在小鼠膀胱条带在图5演示了如何此方法可用于评估胆碱和嘌呤机制的神经传递相关的贡献。 </li><li>建立激励强度:系统增加刺激的强度（电压），直到收缩的幅度达到了高原（如果使用的列车保持频率不变）。 </li><li>将刺激取决于强度瞄准实验。如果目的是增加neurally诱发的收缩，然后用次最大强度，使得收缩的幅度〜最大收缩的50％。如果目的是要减少neurally诱发的收缩，然后将强度到〜80％的最大振幅的，以避免组织的疲劳。 </li></ol></li><li>一旦刺激参数（时间，频率和强度）建立，允许〜20-30分钟的EFS-诱发宫缩前的药物测试稳定。 注意:要验证EFS的选择性神经传输，神经区块传输与钠离子通道阻断剂河豚毒素（TTX; 0.5-1微米）。执行此步骤在实验开始时，作为TTX洗掉相对容易。此外，在实验结束时执行此（见步骤4.3.5。下文）。 </li><li>准备原液在1000倍的最终工作浓度为:α，β-亚甲基三磷酸腺苷（ABMA;有嘌呤受体激活剂和脱敏剂）10 -2男，阿托品（毒蕈碱受体拮抗剂）10 -3米（ 图5C）。观察其它实施例在图6中的5HT4受体激动剂，西沙必利（3×10 -6 M，10 -6 M，3×10 -5 M，10 -5 M，3×10 -4 M，10 -4 M， 3×10 -3 M，10 -3 M），增加组织收缩和SB-203186（3×10 -3 M）中，5HT4受体拮抗剂，反向的西沙必利的影响。 </li><li>为了测试ABMA对EFS（ 图5C）的影响，以及阿托品，执行两个控制频率响应曲线。加入10微升10 -2 M ABMA到该浴中的10μM的终浓度。这将收缩的组织由于嘌呤能受体的平滑肌的直接刺激。响应返回到基线后，重复频率响应曲线。加入10微升10 -3 M阿托品作最后concentrat离子为1μM。后〜10分钟（以方框毒蕈碱受体所需要的阿托品），重复频率响应曲线。在平行条带加10微升的车辆，水，在每一个步骤。 注意:对于图6中的其它实施例中，加10微升每西沙比利原液在定义的时间间隔（〜每隔15分钟;见讨论）中，随后加入10μl的SB-203186原液直接向浴和监测其效果EFS引起的收缩。在平行条带加10微升的车辆，二甲基亚砜。观察西沙必利的效果，5HT4受体激动剂对人膀胱和回肠组织中的图6的EFS诱发的收缩，另外，观察DMSO的作用，该车辆为西沙必利，对EFS诱发的人膀胱和回肠组织中的收缩。 </li><li>在EFS协议的端部通过阻断神经传输用的Na +通道阻断剂，河豚毒素（TTX验证EFS的选择性; 0.5-1及＃181，M）。如果TTX抗性的收缩仍然存在，建议调整刺激在随后的实验的持续时间和强度。 </li></ol></li><li>为了确定预或突触后部位药物的影响（ 图7A）按照步骤1至4.3.2。建立可重复的反应，卡巴和EFS，再加入药物十</li><li>在实验结束时，中松开或解开的条带，轻轻吸干它们放在一张薄纸，消除多余的流体，并用天平测量每个条的重量。使用卡尺以确定横截面面积还测量组织的长度。此信息用于数据标准化（参见5.4节）。 </li></ol> 5，数据分析分析使用适当的软件数据（ 例如 ，Windaq，LabChart）。 <ol><li>对于自发活动，之前和药物引起的反应和我的高峰期至少选择30秒的窗口ASURE振幅和肌活动的频率（ 图3）。 <ol><li>使用快速傅立叶变换分析，以确定频率有助于收缩反应和是否频谱有气囊的不同部分之间的差异（ 例如 ，圆顶与颈部）或具有发展，病理学和药物8。 </li></ol></li><li>对平滑肌张力作用之前和在药物诱导的响应的峰值选择至少10-30秒的窗口，并测量收缩的幅度。 </li><li>对neurally诱发的收缩效应测量收缩之前和在药物诱导的响应的峰值的曲线（至少3个）下的幅度，持续时间和面积。 注:这是必要的EFS引起的收缩曲线下测量幅度和面积，因为嘌呤和胆碱成分具有不同的动力学。的嘌呤成分是速度快，瞬态（ATP激活purinergiÇ离子型渠道，如P2X1，使钙的快速涌入，然后他们慢慢适应），从而曲线下促成更多的峰值响应和更少的区域。胆碱能元件是较慢的和持续的胆碱（ACh激活代谢型毒蕈碱受体，它需要更多的时间来激活胞内途径，最终激活离子通道去极化的平滑肌诱导的收缩）。因此，毒蕈碱成分被捕获由曲线下测量面积更好。 </li><li>归一化数据，以便能够比较不同条带和药物治疗的结果。选择用于归一化的参数不应当由试验化合物的影响，病理状况的研究或实验设计。在这些参数中，使用带材的重量，剖面积，氯化钾的反应（ 图4B），％至另一个收缩剂的最大反应的最大反应（ 图7B）或％的（ 例如 ，CCH3）或放松剂（ 如罂粟碱）。 </li></ol>

作者宣称，他们没有竞争的经济利益。

在本文中，我们在这可以用来解决一些涉及到膀胱的生理和病理，以及协助新药的发现治疗膀胱功能障碍的重要科学问题， 体外平滑肌收缩的方法描述了一个简单。我们已经示出了使用这种方法来评估的膀胱平滑肌收缩的发育，病理和药理学特性（ 图2-4），神经传递的调制（ 图5-7A），种间差异（ 图4），器官的差异（ 图6）和特定...

膀胱平滑肌松弛，使储尿和合同引出尿液排除。松弛是由固有平滑肌性质，并通过从交感神经补药释放的去甲肾上腺素（NE），其中激活在逼尿肌β肾上腺素受体​​（β3 AR人类）介导的。排尿是通过抑制交感神经的输入和激活会释放乙酰胆碱/ ATP的收缩膀胱平滑肌1副交感神经实现。许多病理状况，包括脑和/或脊髓损伤，神经变性疾病，糖尿病，膀胱出口梗阻或间质性膀胱炎，可以极大地改变膀胱功能，对生命2的患者的质量造成严重影响。的平滑肌中，传入或传出的神经和/或所述:这些条件通过影响一种或膀胱的多种组分改变平滑肌的收缩性粘膜。 几种体内和体外方法来研究膀胱功能已被开发出来。 在体内 ，膀胱是膀胱功能的主要测量。虽然这是一个完整的准备，允许收集在接近生理条件的信息，有若干在其中使用的平滑肌条是优选的情况。这些包括当外科手术和/或药理学的操作会影响生存和体内制剂的稳定性，或情况下，当研究需要使用的人体组织或昂贵的化学品。该方法还有助于药物，年龄和病理膀胱的各成分的作用的检查， 即平滑肌，粘膜，传入和传出神经。 膀胱条已受聘多年来受到许多团体来回答一些科学问题。他们用EVA在肌自发性活动luate变化引起的病变。这项活动被认为是导致膀胱过度活动症（OAB）的紧迫性和频率的症状，因此对OAB 3-9正在开发的药物的目标。膀胱条带也被用于研究该调节平滑肌张力与发现的离子通道和/或受体和/或可能被靶向诱导或者松弛或平滑肌的收缩，细胞内途径的目的肌和神经因子3,10- 13。其它的研究集中在神经传递的性质，包括诱导病理14,15发射机和参与受体和变化。此外，该方法已被用于组织之间的比较，从不同物种16-18，器官19-21，和药物的构效关系22-24评价之间。这种方法的一个扩展已被用来测量电电子药物对递质释放的传出神经25的效果。此外，各种组织（膀胱，尿道，胃肠道，胃肠道），从动物或人体（从批准用于研究的手术或器官供体组织）收集并从多种动物模型，包括脊髓损伤（SCI），膀胱出口梗阻（BOO），或间质性膀胱炎（IC），可以使用该技术进行研究。 在本文中，我们演示了如何使用这种方法，连同必要的实验方案，来解决上面提到的几个科学问题。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Tissue Bath System with Reservoir</td><td>Radnoti, LLC</td><td>159920</td><td>isolated tissue baths</td></tr><tr><td>Warm water recirculator pump</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>TPZ-749</td><td>to keep tissue baths to 37 &amp;#176;C</td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr><tr><td>Data Acquisiton System</td><td>DataQ Instruments</td><td>DI-710-UH</td><td>To view, record and analyze data</td></tr><tr><td>Transbridge Transducer Amplifier</td><td>World Precision Instruments</td><td>SYS-TBM4M</td><td>Transducer amplifier</td></tr><tr><td>Grass stimulator</td><td>Grass Technologies</td><td>Model S88</td><td>Stimulator</td></tr><tr><td>Anesthesia System</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>ACV-1205S</td><td>To anesthetesize the animal</td></tr><tr><td>Anesthetizing Box</td><td>Harvard Apparatus</td><td>500116</td><td>To anesthetesize the animal</td></tr><tr><td>Anesthesia Masks</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>AC-09508</td><td>To anesthetesize the animal</td></tr><tr><td colspan="4">&lt;strong&gt;Materials and Surgical Instruments&lt;/strong&gt;</td></tr><tr><td>Sylgard</td><td>Dow Corning Corp</td><td>184 SIL ELAST KIT</td><td>To pin, dissect, &amp;amp; cut tissue</td></tr><tr><td>Petri Dish</td><td>Corning</td><td>3160-152</td><td>To dissect/cut tissue</td></tr><tr><td>Insect Pins</td><td>ENTOMORAVIA Austerlitz Insect Pins</td><td>Size 5</td><td>To pin tissue</td></tr><tr><td>Bench Pad</td><td>VWR International</td><td>56617-014</td><td>Absorbent bench underpads</td></tr><tr><td>Rat surgical Kit</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INSRATKIT</td><td>To remove and dissect tissue</td></tr><tr><td>2 Dumont #3 Forceps</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500064</td><td>To remove and dissect tissue</td></tr><tr><td>Tissue Forceps</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500092</td><td>To remove and dissect tissue</td></tr><tr><td>Scalpel</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500236</td><td>To remove and dissect tissue</td></tr><tr><td>Scalpel blade</td><td>Kent Scientific Corporation&amp;nbsp;</td><td>INS500239</td><td>To remove and dissect tissue</td></tr><tr><td>Professional Clipper&amp;nbsp;</td><td>Braintree Scientific, Inc.</td><td>CLP-223&amp;nbsp;45</td><td>To remove fur</td></tr><tr><td>Suture Thread</td><td>Fine Science Tools</td><td>18020-50</td><td>Tie tissue</td></tr><tr><td>Tissue Clips</td><td>Radnoti, LLC</td><td>158802</td><td>Attach tissue to rod/transducer</td></tr><tr><td>1 g weight</td><td>&amp;nbsp;Mettler Toledo</td><td>11119525</td><td>For transducer calibration</td></tr><tr><td colspan="4">&lt;strong&gt;Chemicals&lt;/strong&gt;</td></tr><tr><td>Krebs Solution:&lt;br /&gt;Sodium chloride&lt;br/&gt; Potassium chloride&lt;br/&gt; Monobasic potassium phosphate&lt;br/&gt; Magnesium sulfate&lt;br/&gt; Dextrose&lt;br/&gt; Sodium bicarbonate&lt;br/&gt; Calcium chloride&lt;br/&gt; Magnesium chloride</td><td>&amp;nbsp;&lt;br /&gt;Sigma&lt;br/&gt; Fisher&lt;br/&gt; Fisher&lt;br/&gt; Fisher&lt;br/&gt; Fisher&lt;br/&gt; Sigma&lt;br/&gt; EMD&lt;br/&gt; Baker</td><td>&amp;nbsp; &lt;br /&gt;S7653&lt;br/&gt; P217-500&lt;br/&gt; P285-3&lt;br/&gt; M65-500&lt;br/&gt; D16-500&lt;br/&gt; S5761&lt;br/&gt; CX0130-2&lt;br/&gt; 2444</td><td>To prepare Krebs solution</td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>029405</td><td>To anesthetesize the animal</td></tr><tr><td>&amp;nbsp;Oxygen tank</td><td>Matheson Tri Gas</td><td>ox251</td><td>To use with anesthesia system</td></tr><tr><td>Carbogen Tank (95% Oxygen; 5% Carbon Dioxide)&amp;nbsp;</td><td>Matheson Tri Gas</td><td>Moxn00hn36D</td><td>To aerate Krebs solutions</td></tr></tbody></table>

bladder smooth muscle strip contractility as a method to evaluate lower urinary tract pharmacology

我们描述了一种体外方法来测量膀胱平滑肌收缩，而将其用于调查平滑肌的生理和药理性质以及诱导病理变化。这个方法提供了对于理解膀胱功能，同时克服体内实验中遇到的主要方法困难，如手术和药理操纵影响稳定性的制剂和生存，利用人体组织，和/或使用昂贵的化学品的重要信息。它也提供了一种方法来调查在健康和病理条件下的每个膀胱组分（ 即平滑肌，粘膜神经）的属性。 膀胱是从麻醉动物，放在Krebs溶液和切成条除去。带被置于填充有温暖的Krebs溶液的腔室。一端被连接到一个等角tensioÑ​​传感器来测量收缩力，其另一端被连接到一个固定的杆。组织被直接添加化合物的浴或由电场激励电极的激活神经，类似于触发膀胱收缩的体内刺激。我们证明了使用该方法的过程中发展和实验性脊髓损伤后，评估自发平滑肌收缩，神经传递（发送器和受体参与），参与平滑肌的活性调节因子的性质，个体膀胱部件的作用，和物种，并且响应于药剂器官的差异。此外，它可以被用于研究参与的收缩和/或平滑肌的松弛，药物的构效关系和评价递质释放的胞内途径。 在体外平滑肌收缩力的方法已被广泛用于FOr动用超过50年，提供了数据显著促成了我们的膀胱功能的理解以及对目前临床上用于膀胱管理化合物的药物开发。

自发性肌活动 肌自发性的活动是经过与出生后发育6-9和病理（ 如脊髓损伤，BOO）3-5变革的重要平滑肌的特性。因为此活性被认为是造成膀胱过度活动症（OAB）2的症状，受体，细胞内途径和药理学试剂的评价能调节它，是开发有效的治疗OAB和其它平滑肌功能障碍高的兴趣。这里介绍的方法，可以很容易地调查这些问题， 图2</...

Watch this Scientific Journal Video about Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology at JoVE.com

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

此稿件提出了一个简单但功能强大， 体外方法应对药物制剂或神经刺激评估平滑肌收缩。主要应用于药物筛选和理解组织生理学，药理学，病理检查。

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

bladder-smooth-muscle-strip-contractility-as-method-to-evaluate-lower

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

26.2K 次观看.  University of Pittsburgh School of Medicine. 此稿件提出了一个简单但功能强大， 体外方法应对药物制剂或神经刺激评估平滑肌收缩。主要应用于药物筛选和理解组织生理学，药理学，病理检查。 

视频: Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

鼠标膀胱壁注射液

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

科研

医学

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients 

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

危重病人中的应用手册肌力测试：下肢肌力测量方法

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

生物工程

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

的的膀胱测压小老鼠：A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

人子宫平滑肌的收缩力测量对药物开发的帮助

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

用于尿功能测试的成人男性尿道超声检查

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

分散（Ex Vivo）的毛骨球模型，去除了除位肌肉，用于在膀胱填充期间直接进入苏布罗氦

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

生物学

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

体内 啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 &#176;C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

制备和利用新鲜分离的人类分离器平滑肌细胞，用于9-苯酚敏感阳离子电流的表征

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学

摘要

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危重病人中的应用手册肌力测试：下肢肌力测量方法

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

的的膀胱测压小老鼠：A膀胱Chemosensation研究的使用

人子宫平滑肌的收缩力测量对药物开发的帮助

用于尿功能测试的成人男性尿道超声检查

分散（Ex Vivo）的毛骨球模型，去除了除位肌肉，用于在膀胱填充期间直接进入苏布罗氦

平滑肌功能中分离出来的组织浴，应用药理学研究测量

体内啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量

制备和利用新鲜分离的人类分离器平滑肌细胞，用于9-苯酚敏感阳离子电流的表征

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学

摘要

探索更多视频

此视频中的章节

鼠标膀胱壁注射液

危重病人中的应用手册肌力测试：下肢肌力测量方法

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

的的膀胱测压小老鼠：A膀胱Chemosensation研究的使用

人子宫平滑肌的收缩力测量对药物开发的帮助

用于尿功能测试的成人男性尿道超声检查

分散（Ex Vivo）的毛骨球模型，去除了除位肌肉，用于在膀胱填充期间直接进入苏布罗氦

平滑肌功能中分离出来的组织浴，应用药理学研究测量

体内 啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量

制备和利用新鲜分离的人类分离器平滑肌细胞，用于9-苯酚敏感阳离子电流的表征

体内啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量