膀胱平滑肌收缩带作为一种方法来评估下尿路药理学
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 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…
Representative Results
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</…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Equipment | |||
| Tissue Bath System with Reservoir | Radnoti, LLC | 159920 | isolated tissue baths |
| Warm water recirculator pump | Kent Scientific Corporation | TPZ-749 | to keep tissue baths to 37 C |
| Computer | |||
| Data Acquisiton System | DataQ Instruments | DI-710-UH | To view, record and analyze data |
| Transbridge Transducer Amplifier | World Precision Instruments | SYS-TBM4M | Transducer amplifier |
| Grass stimulator | Grass Technologies | Model S88 | Stimulator |
| Anesthesia System | Kent Scientific Corporation | ACV-1205S | To anesthetesize the animal |
| Anesthetizing Box | Harvard Apparatus | 500116 | To anesthetesize the animal |
| Anesthesia Masks | Kent Scientific Corporation | AC-09508 | To anesthetesize the animal |
| Materials and surgical instruments | |||
| sylgard | Dow Corning Corp | 184 SIL ELAST KIT | To pin, dissect & cut tissue |
| Petri Dish | Corning | 3160-152 | To dissect/cut tissue |
| Insect Pins | ENTOMORAVIA Austerlitz Insect Pins | Size 5 | To pin tissue |
| Bench Pad | VWR International | 56617-014 | Absorbent bench underpads |
| Rat surgical Kit | Kent Scientific Corporation | INSRATKIT | To remove and dissect tissue |
| 2 Dumont #3 Forceps | Kent Scientific Corporation | INS500064 | To remove and dissect tissue |
| Tissue Forceps | Kent Scientific Corporation | INS500092 | To remove and dissect tissue |
| Scalpel | Kent Scientific Corporation | INS500236 | To remove and dissect tissue |
| Scalpel blade | Kent Scientific Corporation | INS500239 | To remove and dissect tissue |
| Professional Clipper | Braintree Scientific, Inc. | CLP-223 45 | To remove fur |
| Suture Thread | Fine Science Tools | 18020-50 | Tie tissue |
| Tissue Clips | Radnoti, LLC | 158802 | Attach tissue to rod/transducer |
| 1g weight | Mettler Toledo | 11119525 | For transducer calibration |
| Chemicals | |||
| Krebs Solution: Sodium Chloride Potassium Chloride Monobasic Potassium Phosphate Magnesium Sulfate Dextrose Sodium Bicarbonate Calcium Chloride Magnesium Chloride |
Sigma Fisher Fisher Fisher Fisher Sigma EMD Baker |
S7653 P217-500 P285-3 M65-500 D16-500 S5761 CX0130-2 2444 |
To prepare Krebs solution |
| Isoflurane | Henry Schein | 029405 | To anesthetesize the animal |
| Oxygen tank | Matheson Tri Gas | ox251 | To use with anesthesia system |
| Carbogen Tank (95% Oxygen; 5% Carbon Dioxide) | Matheson Tri Gas | Moxn00hn36D | To aerate Krebs solutions |
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Cite This Article
Kullmann, F. A., Daugherty, S. L., de Groat, W. C., Birder, L. A. Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology. J. Vis. Exp. (90), e51807, doi:10.3791/51807 (2014).