膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型
Summary
约3-8亿人在美国患有间质性膀胱炎/膀胱疼痛综合征（IC / BPS），其特征在于，部分盆腔疼痛衰弱状态。为了研究的条件神经系统的贡献，膀胱疼痛生理模型是用在小鼠和大鼠。
Abstract
约3-8万人，在美国从间质性膀胱炎/膀胱疼痛综合征患（IC / BPS），其特点是增加了紧迫感和排尿次数，以及夜尿和一般盆腔疼痛，特别是在膀胱充盈或衰弱状态排尿。尽管多年的研究，IC / BPS的原因仍然是难以捉摸和治疗策略无法为病人提供完整的救济。为了研究的条件神经系统的贡献，许多动物模型已经发展到模仿与IC / BPS带来的痛苦和症状。一个这样的小鼠模型是膀胱胀满（UBD）。在该模型中，一个特定的压力的压缩空气被输送到轻度麻醉动物超过一个设定的时间周期膀胱。在整个过程中，在上斜肌腹部肌肉的电线从肌肉记录电活动。这项活动被称为内脏运动反应（VMR）和我SA可靠和可重复的测量伤害性的。在这里，我们描述了在小鼠包括手术操作，生理记录和数据分析执行此技术的必要步骤。随着使用这种模式，初级感觉神经元，脊髓继发传入，以及参与膀胱疼痛更高中枢神经系统领域之间的协调可以解开。这个基本的科学知识，然后可以转换临床治疗患者从IC / BPS的痛苦。
Introduction
慢性疼痛的官方定义为疼痛持续约3个月，或比正常组织愈合时间较长1。这种类型的疼痛是人们被驱赶到求医2的主要原因之一，并且可以花费$ 635十亿每年3美元。目前慢性疼痛的应对策略被认为是过时的;几十年的医疗进步，的NSAIDs（非甾体抗炎药）和阿片类药物后，仍然由医师处方的主要治疗方法。然而，这些治疗方法针对所有不同类型的疼痛以同样的方式，提供镇痛作用超过一切，而不是特别注重，准确的痛苦事件的起因身体。为了更好地帮助那些患有慢性疼痛，研究应注意转向与慢性疼痛最常见的原因相关的病因及可能的疼痛特异性治疗方法。这manuscri的目标角是描述用于更好地理解被称为间质性膀胱炎/膀胱疼痛综合征（IC / BPS）的状态的模型。
IC / BPS是一种使人衰弱的疾病，影响千百万人，主要是妇女的40 4岁以上的IC / BPS的确切原因尚不清楚，但发病率已经与遗传5，特定的饮食和高应力水平6 。 IC / BPS的症状包括但不限于：增加了紧迫性排尿，排尿次数增多，疼痛，穿刺，或膀胱充盈及排尿时灼热疼痛，以及夜尿7。患者出现这些情况有较高的压力水平，而且更着急8。这种增加的压力会导致疼痛加剧，这增强了原有的应力水平。有研究表明，抑郁和焦虑水平降低以下治疗方法，减轻尿疼，从而有效地打破这种积极的反馈循环<sup> 9。其中一个在发现新的治疗IC / BPS的挑战一直是膀胱疼痛基础科学的了解很少。
为了研究的条件神经系统的贡献，许多动物模型已经发展到模仿与IC / BPS带来的痛苦和症状。传统上，膀胱炎已经被重新创建在动物中通过引入各种化学品，例如芥末油，丙酮，脂多糖，盐酸，和环磷酰胺进入膀胱。但是没有外国代理人存在于IC / BPS患者的无菌尿，从而质疑这些模型的有效性。泌尿疼痛另一种小鼠模型是膀胱胀满（UBD）10。在该模型中，一个特定的压力的压缩空气被输送到一个清醒或轻度麻醉动物在一段设定时间的膀胱。在整个过程中，在上斜肌腹部肌肉纪录电气ACTIV电线从性肌（与肌电图（EMG））。此活动被称为内脏运动应答（VMR），是痛觉10的可靠的，可重复的测量。类似的中空器官，腹胀（ 如膀胱，直肠）在健康志愿者产生不舒服的感觉，并显著增加报告的疼痛11,12，这经常被用来诊断IC / BPS的特质。因此，在用刺激或抑制的电，药理学和光遗传学方法结合，UBD是对理解膀胱伤害感受和疼痛两者的外周和中枢神经系统组件的有用和有效的模型。
在这里，我们描述的过程欠平衡钻井是目前用在我们的实验室中常用的小鼠品系，C57BL/6J的女性成员内。由于男性的导尿过程中的困难，本程序主要是在雌性小鼠中进行。该协议是改编自由内斯<开发EM>等10及以前出版的从我们的实验室13。以下方案描述该模型在麻醉的动物的四个主要组成部分：（1）膀胱导管与记录电极手术（2）局部麻醉诱导（3）膀胱膨胀和VMR记录，以及（4）数据的原始VMR /肌电痕迹分析。由于选择的有机体，麻醉深度和诱导，和体温的欠平衡钻井-VMR过程中的细微差别将在下面讨论。
Protocol
Representative Results
Discussion
在人类中，间质性膀胱炎/膀胱疼痛综合征（IC / BPS）表示一个显著的临床问题，因为病人已经衰弱的痛苦，往往是反应迟钝到正规的疼痛治疗15。其中一个在理解和最终治疗慢性膀胱疼痛的主要挑战是要了解所参与对伤害性膀胱膨胀，正常的神经过程。为了克服这个困难，膀胱疼痛的动物模型是必要的。这种模式应该是可重复的，稳定的，并且容易测量。幸运的是，尼斯和同事10?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢博士。罗伯特Gereau，亨利赖，和Lara瓦罐在设立这个制度的有益的帮助。我们也想感谢的资金来源为这项工作（BJK – 疼痛早期职业研究资助由扫描研究国际协会|设计基金会由INGER＆晶石BRUUN和杜肯大学Hunkele可怕的疾病基金）。
Materials
| Infrared heating blanket and monitoring system | Kent Scientific | Right-Temp System | This system is setup to monitor two separate temperatures. This should include the animal and the heating blanket. In addition, the system can automatically adjust the temperature to maintain a set temp. However, this automatic function produces electrical interference during EMG recording and must be turned off. Kent can provide a battery pack for the heating pad for use during the riEMG recording part of the experiment. |
| Isoflurane vaporizer | Draeger | Vapor 19.1 | Any isoflurane vaporizer will work, but it is helpful to have one that has mutliple notches between 0 and 2% isoflurane. |
| Isoflurane | Multiple | n/a | |
| 100% Oxygen air tank | Multiple | n/a | For ventilation of animal |
| Air breathing grade | Multiple | n/a | For bladder distention |
| 24GA 0.56inch IV catheter | BD biosciences | 381411 | For bladder catheterization |
| Surgilube (sterile) | Savage Laboratories | 0281-0205-02 | Any surgical grade lubricate would work fine. |
| Mineral oil (sterile) | Multiple | n/a | |
| Saline (sterile) | Multiple | n/a | |
| AG8W Silver Wire, 2 m, 0.20 mm (.008") D, L, No Insulation | Warner Instruments | W4 64-1318 | Any silver wire with these specifications will work. Wire does not need to be "chlorinated." |
| opthalamic ointment | Multiple | n/a | |
| Small surgical scissors | Multiple | n/a | |
| Sharp forceps | Multiple | n/a | |
| 21 Ga needle | Multiple | n/a | |
| Grass amplifier P511 with 3-lead input cable | Grass Instruments | P511 (F-P5IC3/REV1) | This is the "amplifier" used in the protocol. Amplifier with the following settings: Calibrator = 1 mV; Lo freq = 300 Hz; Amplification = 20; Hi freq = 10 kHz; Line filter = in. |
| Cambridge Electronic Design (CED) 1401 Plus (or equivalent) | Cambridge Electronic Design | 1401 Plus | This is the "digitizer" used in the protocol. Other digitizer systems from WINDAQ or other companies would work fine; Need inputs for pressure signal, EMG, and stimulus signal. |
| CED Spike2 software | Cambridge Electronic Design | Spike 2 | This is the the "digitizer software" used in the protocol. Should be from same manufacturer as digitizer. Program should be setup with 3 channels for pressure (0-100 mmHg scale), EMG signal (typically -5 to +5 V range), and stimulus marker (0-2 V) range. |
| Flow chart from air tank to bladder catheter | n/a | n/a | Sequence of connections from pressurized air tank to animal bladder: Air tank to 1/4 inch tubing to Gilmont flowmeter to Y connector. Branch 1 of y connector to to sphygmomanometer. Branch 2 to a single input on the 4-way gang valve to 4-way valve output to the timed pressure regulator to 3/32 tubing from timed pressure regulator to 2nd Y connector (branch 1 to sphygmomanometer) with branch 2 to 3/32 tubing to a 3rd Y connector. Branch 1 of Y connector to a 3rd sphygmomanometer and branch 2 to animal bladder catheter. |
| Gilmont Flowmeter | Gilmont | GF8321-1401 | Multiple brands of flowmeters will work. For bladder distention air, flow meter should be able to handle high pressures (such as this Gilmont meter). For breathing air, flow rate should be adjustable down to 100 cc/min (typical mouse rate is 500-1000 cc/min). |
| 4-way gang valve | Elite | This is a specific piece of equipment. The Elite gang valve is designed for fish tanks at low air pressures. In the bladder distention setup, this valve acts as a safety valves in case the pressure spikes. Too high pressure during initial turning on of the tank will ruin the pressure transducer in the Timed pressure regulator and/or the sphygnometers. In addition, by providing a small amount of leak in the system, this valve makes it easier to adjust the pressure between 10-80 mmHg. | |
| 1/4 inch tubing | Multiple | n/a | |
| 3/32 inch tubing | Multiple | n/a | |
| "Y" connectors (1/4 and 3/32 inch) | Multiple | n/a | |
| Sphygmomanometer | CVS | Any analog sphygmomanometer from a drug store will work for this application. | |
| Timed Pressure Regulator | Custom | This is a custom built machine (Washington University in St. Louis Machine shop) that allows for automated pressure delivery including digitization of air pressure, control of trial length, inter-trial interval automation, control of trial number, and stimulus onset digital signal. However, the basic components of the system (pressure on and off for a given trial period) could be controlled with a simple on/off in-line switch. Such analog control of a trial would necessitate additional analysis parameters (see below section 4). In addition, one would have to manually assign the pressure based on the analog sphygmomanometer during data analysis. | |
| IGOR Pro | Wavemetrics | n/a | For analysis of EMG signal. Many different types of software can be used for data analysis in these experiments. |
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