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 border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Name of Material/ Equipment</td>
			<td style="width:123px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:180px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Equipment</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Tissue Bath System with Reservoir</td>
			<td>Radnoti, LLC</td>
			<td style="width:119px;">159920</td>
			<td style="width:180px;">isolated tissue baths</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Warm water recirculator pump</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">TPZ-749</td>
			<td style="width:180px;">to keep tissue baths to 37 C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Computer</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Data Acquisiton System</td>
			<td style="width:123px;">DataQ Instruments</td>
			<td style="width:119px;">DI-710-UH</td>
			<td style="width:180px;">To view, record and analyze data</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Transbridge Transducer Amplifier</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-TBM4M</td>
			<td style="width:180px;">Transducer amplifier</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Grass stimulator</td>
			<td style="width:123px;">Grass Technologies</td>
			<td>Model S88</td>
			<td style="width:180px;">Stimulator</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Anesthesia System</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td>ACV-1205S</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:237px;"><a name="RANGE!A10">Anesthetizing Box</a></td>
			<td style="width:123px;">Harvard Apparatus</td>
			<td style="width:119px;">500116</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Anesthesia Masks</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">AC-09508</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Materials and surgical instruments</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">sylgard</td>
			<td>Dow Corning Corp</td>
			<td>184 SIL ELAST KIT</td>
			<td style="width:180px;">To pin, dissect &#38; cut tissue</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Petri Dish</td>
			<td>Corning</td>
			<td>3160-152</td>
			<td style="width:180px;">To dissect/cut tissue</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:237px;">Insect Pins</td>
			<td style="width:123px;">ENTOMORAVIA Austerlitz Insect Pins</td>
			<td style="width:119px;">Size 5</td>
			<td style="width:180px;">To pin tissue</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Bench Pad</td>
			<td style="width:123px;">VWR International</td>
			<td style="width:119px;">56617-014</td>
			<td style="width:180px;">Absorbent bench underpads</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Rat surgical Kit</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INSRATKIT</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">2 Dumont #3 Forceps</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500064</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Tissue Forceps</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500092</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Scalpel</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500236</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Scalpel blade</td>
			<td style="width:123px;">Kent Scientific Corporation&#160;</td>
			<td style="width:119px;">INS500239</td>
			<td style="width:180px;">To remove and dissect tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Professional Clipper&#160;</td>
			<td style="width:123px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">CLP-223&#160;45</td>
			<td style="width:180px;">To remove fur</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Suture Thread</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:119px;">18020-50</td>
			<td style="width:180px;">Tie tissue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Tissue Clips</td>
			<td>Radnoti, LLC</td>
			<td style="width:119px;">158802</td>
			<td style="width:180px;">Attach tissue to rod/transducer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">1g weight</td>
			<td style="width:123px;">&#160;Mettler Toledo</td>
			<td>11119525</td>
			<td style="width:180px;">For transducer calibration</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Chemicals</td>
			<td style="width:123px;"></td>
			<td></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="170">
			<td height="170" style="height:170px;width:237px;">Krebs Solution:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Sodium Chloride 
			Potassium Chloride 
			Monobasic Potassium Phosphate 
			Magnesium Sulfate 
			Dextrose 
			Sodium Bicarbonate 
			Calcium Chloride 
			Magnesium Chloride</td>
			<td style="width:123px;">Sigma&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
			Fisher 
			Fisher 
			Fisher 
			Fisher 
			Sigma 
			EMD 
			Baker</td>
			<td style="width:119px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; S7653 
			P217-500 
			P285-3 
			M65-500 
			D16-500 
			S5761 
			CX0130-2 
			2444</td>
			<td style="width:180px;">To prepare Krebs solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:237px;">Isoflurane</td>
			<td style="width:123px;">Henry Schein</td>
			<td style="width:119px;">029405</td>
			<td style="width:180px;">To anesthetesize the animal</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">&#160;Oxygen tank</td>
			<td style="width:123px;">Matheson Tri Gas</td>
			<td style="width:119px;">ox251</td>
			<td style="width:180px;">To use with anesthesia system</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:237px;">Carbogen Tank (95% Oxygen; 5% Carbon Dioxide)&#160;</td>
			<td style="width:123px;">Matheson Tri Gas</td>
			<td style="width:119px;">Moxn00hn36D</td>
			<td style="width:180px;">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

Research

JoVE Journal

Medicine

26.1K 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

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

测量方法轴向和近端肌肉音

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

科研

医学

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

一种改进沉淀法来隔离尿外来体

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

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 ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

微电极镀膜法记录从可吸入性中脑动脉的膜电位

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

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 ...

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity.&#160;While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

美味的西式自助餐厅饮食作为模拟啮齿动物饮食诱发肥胖症的可靠方法

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

Speed

Subtitles

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学