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）。

下面的协议已被批准的机构动物护理和使用委员会在杜肯大学，并与来自美国国立卫生研究院的指引一致。 电极丝和膀胱导尿管1。利用无菌操作技术植入手术（总时间5-10分钟） <ol><li>下列项目前，应开始手术准备: <ol><li>主动监视和使用温度监控系统与电池供电的加热垫和动物体温探头调节体温到37.5±0.5℃（材料清单）连接的自动系统来为体温的附加控制的低瓦数加热灯。 </li><li>所需要的异氟醚麻醉设备包括压缩空气（ 例如 100％O 2），异氟烷蒸发器，异氟醚，异氟醚感应腔（用于初始击倒），鼻锥和排气系统。 </li><li>第组装Ë适当的手术器械，静脉导管和电极（材料清单） </li></ol></li><li>在2-3％异氟醚诱导感应腔麻醉状态。从腔取出鼠标时翻正反射消失（0-1分钟）。 </li><li>鼠标放置在鼻锥与2-2.5％异氟醚。捏脚趾，检查全麻醉。应该没有从动物的反应。启动定时器。 </li><li>将1滴对小鼠每只眼睛眼药膏。 </li><li>转动鼠标移到所以背面朝下。 </li><li>通过尿道进入膀胱插入导管。 </li><li>浸导管手术润滑油来润滑导管。 <ol><li>轻轻握住尿道开口在垂直于动物的身体尖头镊子。持导管与第二组镊子，轻轻插入导管的一端插入尿道口在垂直于动物的身体的角度。 </li><li>当导管已进入尿道〜1-2毫米，轻轻地调节导管，使得它现在平行于本体旨在向头部。这一运动是必要的，以避免耻骨。 </li><li>轻轻推导管向老鼠的身体。导管应滑入体腔平稳，无阻力。不要强迫导管。如果导管不能顺利进入，返回到垂直导管位置，并再次启动。 </li><li>用导尿管在膀胱完全插入，轻轻向下按鼠标的腹部驱逐膀胱内容。推一小块纸巾入导管开口从导管中取出所有的尿液。当纸巾浸泡尿液，更换。在整个过程中膀胱膨胀实验剩余的协议，包括连续取出尿（见协议3下文。） </li></ol></li><li>插入两个银线分为左腹部肌肉和一个银线到胸部（如地面）。 <ol><li>用酒精或碘酒消毒区域外侧导管的右侧（身体的动物的左侧。） </li><li>暴露左腹部肌肉。 <ol><li>按住左附近腹肌用钳子皮肤。做一个小1-2厘米的切口中等手术剪。 </li><li>将剪刀尖插入切口，轻轻地扩大切口至2厘米。 </li><li>使用镊子移动覆筋膜暴露上斜肌腹部肌肉。注意不要剪/撷取任何都位于这一地区的主要血管。 </li></ol></li><li>弯三银线的一半。使用锋利的21号针头取一小口的肌肉。然后，通过肌肉推针。在肌肉中插入的时候要小心，不要刺穿任何内脏器官或诱导肌肉造成不必要的损伤。 </li><li>坚持一银线的自由端插入21号针头的开幕式。轻轻一拉退针穿过肌肉，通过肌肉咬绘制银线。当针不受肌肉，轻轻通过肌肉拉银线直到在导线环路是与冲水肌肉咬。 </li><li>重复步骤1.7.3-1.7.4的第二肌丝。这根电线应放在从第一肌肉线约0.5厘米它的两个导线不要触摸插入之后是很重要的。 </li><li>插入第三个银线（地线）在胸前逊色于心脏。 <ol><li>使用锋利的21号针头取一小口的皮肤（这是没有必要做一个切口或刮皮肤），并通过皮肤推针。 </li><li>将上述银线，转危为安的皮肤。 </li></ol></li></ol></li><li>将少量的摄氏37.5度的无菌矿物油覆盖在外露的肌肉，以保持湿润，拉皮肤上尽可能多暴露的肌肉尽可能。 </li><li>注射0.5毫升皮下注射生理盐水，以帮助在随后的步骤中保持液面高度。 </li><li>轻轻摇动动物到一边，这样体温可以很容易地保持。 </li></ol> 2，局部麻醉，降压过程（托塔升时间〜75分钟） 下面麻醉协议已被批准的机构动物护理和使用委员会在杜肯大学，并与来自美国国立卫生研究院的指引一致。 <ol><li>立刻科后，我（上图）是完整的，异氟醚降低至1.5％。保持在这个水平上15分钟。 </li><li>较低的异氟烷，使得该动物被部分地麻醉。 <ol><li>由0.125％降低异氟醚，每15分钟，直到动物表现出屈曲响应脚趾捏，但不发声，或以其他方式移动。 </li><li>通常，0.8〜1.0％的异氟醚的水平是最优的，但是水平可以有所不同，因为在麻醉设置差异。发声和/或下床活动是极其罕见的事件时，麻醉是在适当的水平。 </li></ol></li></ol> 3，肌电图（EMG）记录在膀胱膨胀与压力梯度（15-75毫米汞柱）（总时间 - 变量取决于实验） <ol&gt; <li>安装肌电图记录和膀胱充盈以下项目: <ol><li>设置一个放大器，数字转换器，以及录音软件。在此设置中，数字转换器具有一个输入放大器和两个输入压力调节器（压力和刺激标记输入，见下文第3.1.2节）。确切的硬件和软件设置为自定义（见材料清单的具体说明）。 </li><li>建立一个系统，提供空气压力的具体阵阵动物。这个系统被称为在下面的协议的"定时压力调节器"。它允许自动压力输送，包括数字化的空气压力，试验长度的控制，试验间隔自动化的试验数控制，以及刺激呈现数字信号（参见Anderson 等人 14的一个定时压力调节器示意图）。 </li></ol></li><li>连接所有导线从鼠标（2腹线+ 1个接地线）到放大器和数字化仪和数字化仪的软件开始记录的肌电信号。注意:在数字化仪软件中选择什么时间"写"。 </li><li>从导尿管取出纸巾。连接导管空气管道（从定时压力调节器。） </li><li>提供一个单一的20秒试验在60毫米汞柱扩张的压力。 <ol><li>将流量调节至60毫米汞柱（检查压力使用模拟血压计。） </li><li>设定的定时压力调节器提供一个20秒的预压力的时间间隔（这将被记录在数字化仪软件的2V刺激信号），后跟一个20秒的压力膨脉冲。 </li><li>启动试验（预压力区间+膨压试验）。当实际压力试验开始，加倍使用3血压计连接到系统检查压力。 </li><li>在压力膨胀试验，强大的肌电信号（&gt; 0.5 V）应在与20秒腹胀可能是后观察。该动物可显示出经腹部NAL运动，但动物不能发声或以其他方式移动。虽然罕见，如果出现异常运动或发声的观察，增加异氟醚的水平。 </li></ol></li><li>如果第一 60毫米汞柱腹胀产生适当的肌电信号，重复步骤3.4两次以1-2分钟的intertrial间隔。 </li><li>当从三个60毫米汞柱试验接收强信号，进行实验distentions。 <ol><li>许多研究使用渐变distensions开始在低压（10-15毫米汞柱），并工作到有害的压力（75〜80毫米汞柱）在10〜15毫米汞柱为单位。请参阅下面的代表性成果，例如痕迹在不同压力。 </li><li>典型的试验包括一个20秒的预压力区间，一个20秒的压力扩张试验，以及1-2分钟intertrial间隔。 </li><li>执行三个distentions在每个压力，然后平均在三个试验中（以获得针对每个压力单个内脏运动响应）。在另一种方法，对erform 5 distensions在每个压力，丢弃高和低的试验，然后平均剩余的三个反应。 </li></ol></li><li>一旦实验distensions是完整的，停止录音，保存实验文件，并使用认可的方法牺牲动物。 </li></ol>的原始痕迹肌电图4。分析<ol><li>内脏运动反应（ 即肌电图记录）的分析是很容易做到使用板载数字转换器软件或通过第三方程序（材料清单）以下说明，可以使用多个程序或使用手动分析可用于分析这些数据的一般说明。 </li><li>要导出肌电图，压力，并从数字化仪软件的刺激信号数据请执行以下操作: <ol><li>打开实验文件。 </li><li>点击"文件""导出为"。 </li><li>更改文件类型为"电子表格文本（。TXT）"，命名文件，然后单击"保存"。 </li><li>中宝对窗口，改变输出采样率到"1000"，更改开始时间的那一刻开始数据采集和变更结束时间为"MAXTIME（）"选择框标记"时移输出，所以第一行是一个0.0秒"，并单击"确定"。 </li><li>的数据现在将导出为可以在各种程序中打开一个文本文件。该文件应包含四列（定时器，VMR（EMG），压力，刺激）。 </li></ol></li><li>每个实验动物进行测试，执行以下的数学运算。 <ol><li>纠正肌电图信号，通过计算各时间点的绝对值设置整个的数据。 </li><li>确定实验的背景技术部分中的平均EMG反应。 </li><li>减去在实验中每一个数据纠正这点平均背景肌电反应，以获得背景校正的数据集。 </li><li>在曲线（AUC）为每20秒前决定下的面积在背景校正的数据设定压力的时间间隔。 </li><li>确定AUC为每20秒压胀试验在背景校正后的数据集。 </li><li>由最低预压力间隔AUC从整个实验将每个压力膨胀试验的AUC（在步骤4.3.5中获得）（在步骤4.3.4得到）。这是归一化和背景校正后的AUC为每个完整的膨压试验。 </li></ol></li><li>对于每个实验的动物，从一个单一的压力平均归一化的多个试验，以获得归一化的VMR中伏特*秒（VSEC）。 </li><li>进行适当的统计数据上的数据取决于实验设计。 </li></ol>

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The authors have nothing to disclose.

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?...

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&gt; 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&lt;开发EM&gt;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。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Infrared heating blanket and monitoring system</td>
			<td>Kent Scientific</td>
			<td>Right-Temp System&#160;</td>
			<td>This system is setup to monitor two separate temperatures.&#160; This should include the animal and the heating blanket.&#160; In addition, the system can automatically adjust the temperature to maintain a set temp.&#160; However, this automatic function produces electrical interference during EMG recording and must be turned off.&#160; Kent can provide a battery pack for the heating pad for use during the riEMG recording part of the experiment.</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Draeger</td>
			<td>Vapor 19.1</td>
			<td>Any isoflurane vaporizer will work, but it is helpful to have one that has mutliple notches between 0 and 2% isoflurane.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Oxygen air tank</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td>For ventilation of animal</td>
		</tr>
		<tr>
			<td>Air breathing grade</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td>For bladder distention</td>
		</tr>
		<tr>
			<td>24GA 0.56inch IV catheter</td>
			<td>BD biosciences&#160;</td>
			<td>381411</td>
			<td>For bladder catheterization</td>
		</tr>
		<tr>
			<td>Surgilube (sterile)</td>
			<td>Savage Laboratories</td>
			<td>0281-0205-02</td>
			<td>Any surgical grade lubricate would work fine.</td>
		</tr>
		<tr>
			<td>Mineral oil (sterile)</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (sterile)</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>AG8W Silver Wire, 2 m, 0.20 mm (.008&#34;) D, L, No Insulation&#160;</td>
			<td>Warner Instruments</td>
			<td>W4 64-1318&#160;</td>
			<td>Any silver wire with these specifications will work.&#160; Wire does not need to be &#34;chlorinated.&#34;</td>
		</tr>
		<tr>
			<td>opthalamic ointment</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>21 Ga needle</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Grass amplifier P511 with 3-lead input cable</td>
			<td>Grass Instruments</td>
			<td>P511 (F-P5IC3/REV1)</td>
			<td>This is the &#34;amplifier&#34; used in the protocol.&#160; Amplifier with the following settings: Calibrator = 1 mV; Lo freq = 300 Hz; Amplification = 20; Hi freq = 10 kHz; Line filter = in.</td>
		</tr>
		<tr>
			<td>Cambridge Electronic Design (CED) 1401 Plus (or equivalent)</td>
			<td>Cambridge Electronic Design</td>
			<td>1401 Plus</td>
			<td>This is the &#34;digitizer&#34; used in the protocol.&#160; Other digitizer systems from WINDAQ or other companies would work fine; Need inputs for pressure signal, EMG, and stimulus signal.</td>
		</tr>
		<tr>
			<td>CED Spike2 software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike 2</td>
			<td>This is the the &#34;digitizer software&#34; used in the protocol.&#160; Should be from same manufacturer as digitizer.&#160; 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.</td>
		</tr>
		<tr>
			<td>Flow chart from air tank to bladder catheter</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Sequence of connections from pressurized air tank to animal bladder:&#160; Air tank to 1/4 inch tubing to Gilmont flowmeter to Y connector.&#160; Branch 1 of y connector to to sphygmomanometer.&#160; 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&#160; to a 3rd Y connector.&#160; Branch 1 of Y connector to a 3rd sphygmomanometer and branch 2 to animal bladder catheter.</td>
		</tr>
		<tr>
			<td>Gilmont Flowmeter&#160;</td>
			<td>Gilmont</td>
			<td>GF8321-1401</td>
			<td>Multiple brands of flowmeters will work.&#160; For bladder distention air, flow meter should be able to handle high pressures (such as this Gilmont meter).&#160; For breathing air, flow rate should be adjustable down to 100 cc/min (typical mouse rate is 500-1000 cc/min).&#160;</td>
		</tr>
		<tr>
			<td>4-way gang valve</td>
			<td>Elite</td>
			<td></td>
			<td>This is a specific piece of equipment.&#160; The Elite gang valve is designed for fish tanks at low air pressures.&#160; In the bladder distention setup, this valve acts as a safety valves in case the pressure spikes.&#160; 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.&#160;&#160;</td>
		</tr>
		<tr>
			<td>1/4 inch tubing</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>3/32 inch tubing</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Y&#34; connectors (1/4 and 3/32 inch)</td>
			<td>Multiple</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphygmomanometer</td>
			<td>CVS</td>
			<td></td>
			<td>Any analog sphygmomanometer from a drug store will work for this application.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Timed Pressure Regulator</td>
			<td>Custom</td>
			<td></td>
			<td>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.&#160; 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.&#160;&#160; Such analog control of a trial would necessitate additional analysis parameters (see below section 4).&#160; In addition, one would have to manually assign the pressure based on the analog sphygmomanometer during data analysis.&#160;&#160;</td>
		</tr>
		<tr>
			<td>IGOR Pro</td>
			<td>Wavemetrics</td>
			<td>n/a</td>
			<td>For analysis of EMG signal. Many different types of software can be used for data analysis in these experiments.</td>
		</tr>
	</tbody>
</table>

urinary bladder distention evoked visceromotor responses as a model for bladder pain in mice

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。

整体UBD-VMR设置的图示可以看出，在图1A中 。越来越大的压力的步骤诱导的增加原材料VMR（ 即 EMG）（ 图1B）。原始VMR，压力和刺激标记物的信号，即应使用数字化仪软件在录制过程中可观察到在图2中可以看出，顶部通道见于图2是肌电图跟踪（中五）。肌电图跟踪的背景信号应该是稳定的，低振幅（&lt;0.2 V）。如果背景是高还是包含了零星...

Watch this Scientific Journal Video about Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice at JoVE.com

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

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。

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 ...

urinary-bladder-distention-evoked-visceromotor-responses-as-model-for

Research

JoVE Journal

Medicine

17.7K 次观看.  Duquesne University. 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。 

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

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

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

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 ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

神经病理性疼痛小鼠坐骨神经掴型号

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

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

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

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 ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...