这项研究得到了 NIH-NINDS （R21NS130241）、IND DEPT HLTH （55051、74247、74244） 和美国陆军 （HT94252310700） 的支持。

动物研究方案已获得印第安纳大学医学院机构动物护理和使用委员会的批准。批准代码：21098MD/R/MSS/HZ 批准日期：2021 年 9 月 29 日。
1. 导管的准备
<ol><li>切割一根 30 厘米的聚乙烯 PE-30 管（0.017 英寸 x 0.030 英寸）。使用打火机在管子的一端张开，确保它不会接触到火焰，并在管子形成适当圆形的钟形尖端后撤回打火机。</li>
	<li>小心地将大约 3/4 的 25G 针头插入管的另一端。准备一个 1 mL 注射器，并用无菌的 0.9% NaCl 填充。将注射器连接到 25 G 针头。</li>
	<li>轻轻注入生理盐水，检查针头尖端是否合适，针头末端是否泄漏。确保没有压力并且盐水顺利流过导管。</li>
</ol>2. 电极的制备
<ol><li>准备 2 根 20 厘米长的钢丝。取钢丝，在涂层区域的两端涂上砂光，剥去 5 毫米的钢丝。</li>
	<li>取一根 25G 针头，将其插入电线的一侧。确保小心插入针头，以免损坏电线。像钩子一样弯曲电线的剥皮部分。钩子将有助于将电线连接到 EUS 肌肉。</li>
	<li>使用焊料将引脚连接到带状线的另一端。焊接有助于将引脚固定到电线上并确保牢固连接。确保加热锡铅焊料，直到它熔化并覆盖电线和引脚。</li>
</ol>3. 动物的准备
<ol><li>根据机构动物护理，动物设施中的家养雌性 C57BL/6 小鼠（8 周龄，体重 18-20 克），具有 12 小时的明暗循环，可无限制地获得水和标准食物颗粒。</li>
</ol>4. 手术中的麻醉
<ol><li>将动物置于 2% 异氟醚和纯氧 （1 L/min） 的腔室中。在将动物转移到面罩之前，使用阴性脚趾捏检查确认动物的完全麻醉。确认后，将气体条件更改为面罩。</li>
	<li>确保麻醉面罩固定在无菌手术野上的适当位置。将动物仰卧在无菌窗帘上，鼻子放在吸入小面罩（0.8-1 L/min，2% 异氟醚）中以继续给药。</li>
</ol>5. 手术准备
<ol><li>用胶带固定动物的四肢。使用电动剃须刀剃除下腹部和尿道口周围（生殖器区域）的皮毛。</li>
	<li>涂抹眼药膏以防止任何潜在的眼睛干燥。使用聚维酮碘溶液准备剃须区域，并用 70% 乙醇擦去溶液。在手术区域贴上无菌窗帘。</li>
</ol>6. 外科手术
<ol><li>膀胱导管植入术<ol><li>在手术显微镜下，使用 Metzenbaum 直钝剪刀，在腹盆皮肤中线切开一个 1-2 厘米的切口。继续切开中线的筋膜和肌肉，通过切口伤口露出膀胱。</li>
		<li>一旦通过切口伤口可以看到膀胱，就根据需要继续缩回周围的任何器官或组织，以获得手术区域的清晰视野。注意避免对膀胱进行任何不必要的操作或紧张，因为这可能会导致并发症，例如漏尿或损伤周围结构。</li>
		<li>抓住膀胱圆顶并用锥形尖端针使用 5-0 不可吸收的单丝缝合线放置钱包线。</li>
		<li>使用微型剪刀，在钱包绳内创建一个小的膀胱造口术，并打一个孔，直到尿液流出。</li>
		<li>抓住导管的圆端并将其插入孔中。一旦管子的尖端穿过孔，就把钱包线缝合在管子周围。然后，轻轻地向外拉管子，直到在缝合线下感觉到尖端。</li>
		<li>从管的另一端缓慢注入 1 mL 生理盐水以扩张膀胱。检查导管周围是否有任何泄漏。如果存在渗漏，请再缝合一根。</li>
		<li>盐水从尿道流出后，取出盐水以减压膀胱。</li>
	</ol></li>
	<li>EUS 电极植入（图 1）<ol><li>使用手术剪刀将腹部切口向上延伸到骨盆底。</li>
		<li>与膀胱一致，将肌肉和膜移动到管，并找到尿道和括约肌。小心不要伤害髂尾中血管和神经。</li>
		<li>使用包含电极的针头在距离尿道口 1 cm 处穿刺双侧皮肤。</li>
		<li>用镊子小心地抓住钩尖，然后轻轻地将针头从皮肤上拉开。</li>
		<li>使用电极的尖端，小心地双侧钩住 EUS 肌肉。避免打得太深，因为这可能会伤害肌肉，从而导致可能的漏尿。</li>
		<li>使用 3-0 不可吸收的单丝缝合骨盆和腹部肌肉和皮肤。</li>
	</ol></li>
	<li>皮肤防水<ol><li>在电极出口的皮肤上涂上一层薄薄的氰基丙烯酸酯胶，以将电极固定到位。</li>
		<li>将氰基丙烯酸酯胶涂抹在尿道口周围 1 厘米处，并进一步延伸至腹部和缝合区域 3 厘米处。为避免接触胶水，请用镊子小心地将尿道口向上支撑。</li>
		<li>使用 0.5-10 μL 微量移液器抽出促进剂液体以干燥胶水。 注意： 促进剂液体是一种可燃液体。</li>
		<li>添加促进剂液以确保适当的胶粘剂反应。这将有助于更快地干燥胶水并确保其牢固凝固。</li>
	</ol></li>
	<li>尿动力学准备<ol><li>准备一个长、宽、深 4.5 厘米的倒置聚苯乙烯称重船。将其切成一个底部为 4 厘米的三角形，将鼠标的尿道口放在这个空间里。将一次性底模 37 mm x 24 mm x 5 mm 放在收集尿液的空间下方。</li>
		<li>将鼠标重新定位为俯卧位置，然后小心地将其移动到装有防毒面具的定制板上。</li>
		<li>确保尿道口正确定位在凹槽内。用胶带轻轻束缚鼠标的头部和肢体，并将板放在加热垫上，直到鼠标完全恢复意识（图 2）。</li>
		<li>仅在小鼠完全清醒时进行膀胱测压，即从麻醉中恢复后至少 40 分钟。</li>
	</ol></li>
</ol>7. 膀胱测压和 EUS-EMG 记录准备
<ol><li>根据制造商的说明设置和校准输液泵。</li>
	<li>取直径为 19.05 mm 的 20 mL 注射器，在室温下填充无菌 0.9% NaCl。将注射器固定到输液泵上。将输注速度设置为 0.01 mL/min。</li>
	<li>通过 PE-30 管将注射器连接到三通连接器的一侧。将膀胱导管连接到压力传感器的另一侧。在连接膀胱导管之前，请务必去除所有气泡。</li>
	<li>将压力传感器固定在与鼠标膀胱相同的水平。压力传感器通过放大器连接到数据采集系统。</li>
	<li>将一个接地线钩连接到皮肤，另一个连接到电极连接器部位。在软件中记录压力。</li>
	<li>启动软件后，检查膀胱内压 （IVP） 和 EUS-EMG 信号。保存样品名称并设置时间。</li>
	<li>开始泵输液。记录信号。</li>
</ol>

使用氰基丙烯酸酯皮肤屏障对清醒小鼠进行精确尿动力学测量

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作者没有什么可披露的。

这种尿动力学技术描述了一种改进的程序，用于测量清醒和受束缚小鼠的尿量和 EUS-EMG 信号。尿道口和腹部周围存在的毛皮会吸收尿液，从而干扰 VV 测量的准确性。尽管尿道口和腹部周围的毛皮在手术前已被仔细剃除，但这些区域和皮肤内剩余的小毛皮仍然吸收尿液，通常在记录后在腹部留下潮湿区域。由于尿道口与周围皮肤之间的距离极短，这个问题在雌性啮齿动物中尤为明显18<...

尿液的储存和释放取决于膀胱和尿道外括约肌 （EUS） 的协调活动。在某些病理中，例如神经源性膀胱，膀胱逼尿肌和括约肌都可能功能障碍，导致严重的膀胱问题，尤其是在创伤性脊髓损伤 （SCI） 之后1。
小型啮齿类动物通常用作研究下尿路 （LUT） 临床前功能的实验模型2。充盈膀胱测压法 （FC） 和 EUS 肌电图 （EUS-EMG） 记录技术可以提供精确的客观信息，具体取决于方法的选择、准确的测量和结果的解释3.尿动力学测试通常用于评估排尿量 （VV）、排尿效率 （VE） 和膀胱容量4。VE 测量膀胱自行排空的效率。它的计算方法是将排尿量除以排尿量和残余尿量之和 （VV+RV）。另一方面，膀胱容量的计算方法是将 VV（排尿时排出的尿液量）与 RV（排尿后留在膀胱中的尿液量）相加5。因此，VV 和 RV 的测量是推导出其他参数的关键。
在尿动力学测试期间精确测量小鼠的 VV 存在各种挑战。啮齿动物的尿液在身体上被束缚在俯卧位时，由于重力的影响，往往会通过腹壁向下吸入6。这种现象会导致尿液被腹部毛皮和皮肤吸收，进而低估尿液排泄量。考虑到小鼠产生的尿液量很小，这种吸光度对结果准确性的影响更为明显7。此外，在 SCI 模型中，由于逼尿肌括约肌协同障碍 （DSD） 的影响，VV 通常低于正常小鼠，这增加了泄漏点压力和毛皮吸收尿液的风险8。这些因素对结果有重大影响。因此，在小鼠终末尿动力学研究期间准确测量 VV 和 RV 至关重要9。目前，已发表文献中提供的方法中缺乏有关如何在小鼠模型中准确测量尿量的细节。
氰基丙烯酸酯胶粘剂是一种胶水，由于其快速有效的粘合特性，常用于人类和动物模型的外科手术 10,11,12。这种胶粘剂特别适用于闭合伤口和撕裂伤，因为它在涂抹到皮肤上时会形成牢固而灵活的粘合13。此外，它可以成为防止尿液和湿气与毛皮和伤口接触的巨大屏障11。
在本文中，我们开发了一种新颖且具有成本效益的技术，该技术利用氰基丙烯酸酯粘合剂在清醒小鼠的膀胱测压和 EUS-EMG 记录中获得精确的结果。这种方法将有助于了解膀胱功能障碍的根本原因和为 LUT 疾病设计更有效的治疗方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accelerator</td>
			<td>BOB SMITH INDUSTRIES</td>
			<td>BSI-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate&#160;</td>
			<td>TED PELLA, Inc</td>
			<td>14478</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable base mold</td>
			<td>TED PELLA, Inc</td>
			<td>27147-4</td>
			<td></td>
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			<td>Infusion pump</td>
			<td>Harvard Apparatus PHD ULTRA</td>
			<td>70-3006</td>
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			<td>Isoflurane</td>
			<td>Henry Schein Inc</td>
			<td>1182097</td>
			<td></td>
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		<tr>
			<td>PIN</td>
			<td>World Precision Instruments</td>
			<td>5482</td>
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		<tr>
			<td>Polyethylene Tubing 30</td>
			<td>Braintree Scientific Inc</td>
			<td>PE30</td>
			<td></td>
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		<tr>
			<td>Sterile Weighing Boat</td>
			<td>HEATHROW SCIENTIFIC</td>
			<td>797CK2</td>
			<td></td>
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		<tr>
			<td>Windaq/Lite&#160;</td>
			<td>DATAQ INSTRUMENTS</td>
			<td>249022</td>
			<td></td>
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</table>

optimizing mouse urodynamic techniques for improved accuracy

准确测量清醒小鼠的尿液参数对于了解下尿路 （LUT） 功能障碍至关重要，尤其是在神经源性膀胱创伤后脊髓损伤 （SCI） 等情况下。然而，在小鼠中进行膀胱测压记录存在显着挑战。当小鼠在记录过程中处于俯卧和受限位置时，尿液往往会被皮毛和皮肤吸收，导致排尿量 （VV） 被低估。本研究的目的是提高清醒小鼠膀胱测压和尿道外括约肌电图 （EUS-EMG） 记录的准确性。我们开发了一种独特的方法，利用氰基丙烯酸酯胶粘剂在尿道口和腹部周围形成防水皮肤屏障，防止尿液吸收并确保精确测量。结果表明，施用氰基丙烯酸酯后，VV 和 RV 的总和与注入的盐水量保持一致，实验后没有观察到潮湿区域，表明成功防止了尿液吸收。此外，该方法同时稳定了与尿道外括约肌 （EUS） 相连的电极，确保了稳定的肌电图 （EMG） 信号，并最大限度地减少了因醒来的小鼠的运动和实验者的操作而引起的伪影。讨论了方法学细节、结果和意义，强调了改进尿动力学技术在临床前研究中的重要性。

The storage and release of urine are dependent on the coordinated activity of the urinary bladder and external urethral sphincter (EUS). In some pathologies such as neurogenic bladder, both the bladder detrusor muscles and the sphincter can become dysfunctional, leading to significant bladder problems, especially after traumatic spinal cord injury(SCI)1.
Small rodents are commonly used as an experimental model to study the preclinical function of the lower urinary tract (LUT)2. Filling Cystometry (FC) and EUS electromyography (EUS-EMG) recording techniques can provide precise objective information depending on the choice of methods, accurate measurement, and interpretation of results3. Urodynamic tests are commonly used to evaluate the voiding volume (VV), voiding efficiency (VE), and bladder capacity4. VE measures how effectively the bladder can empty itself. It is calculated by dividing the voided volume by the sum of voided and residual volumes (VV+RV). On the other hand, bladder capacity is calculated by adding the VV (the amount of urine expelled during urination) to the RV (the amount of urine left in the bladder after urination)5. Therefore, the measurement of VV and RV are the keys to deducing other parameters.
Precisely measuring VV in mice during urodynamic tests presents various challenges. The urine of rodents, when physically restrained in a prone position, tends to be drawn downwards through the ventral abdominal wall due to the influence of gravity6. This phenomenon can lead to the absorption of urine by the abdominal fur and skin, which, in turn, underestimates the volume of urine excreted. Considering the small amount of urine produced by mouse, the impact of this absorbance on the accuracy of results is even more pronounced7. Furthermore, in models of SCI, VV is often lower than in normal mice due to the impact of the detrusor sphincter dyssynergia (DSD), which increases the risk of leak point pressures and urine absorption by the fur8. These factors have a significant impact on the results. Therefore, accurate measurement of VV and RV during terminal urodynamic studies in mice is crucial9. Currently, there is a lack of details in the methodologies provided in published literature about how to measure urine volume accurately in mouse models.
Cyanoacrylate adhesive is a type of glue that is commonly used in surgical procedures in human and animal models due to its quick and effective bonding properties10,11,12. This adhesive is particularly useful for closing wounds and lacerations, as it forms a strong and flexible bond when applied to the skin13. Moreover, it can be a great barrier against urine and wetness that may come into contact with fur and wounds11.
In this article, we have developed a novel and cost-effective technique that utilizes cyanoacrylate adhesive to achieve precise results in cystometry and EUS-EMG recordings in awake mice. This method will be beneficial in understanding the underlying causes of bladder dysfunction and devising more effective treatments for LUT disorders.

作者聚焦：用于神经源性膀胱清醒小鼠精确尿液测量的增强型尿动力学方法

优化小鼠尿动力学技术以提高准确性 

采用膀胱测压和 EUS-EMG 活动追踪分析数据。连续膀胱测压法包括将生理盐水注入膀胱，同时测量膀胱中的压力和体积变化。为了测量 VV，以 0.01 mL/min 的速度注入 0.4 mL 生理盐水，并在盖子中收集尿液超过 40 分钟。排尿后残留尿量 （PVR） 可以通过导管吸出生理盐水来获得。在没有胶水的正常小鼠中，VV 和 RV 的总和通常小于 0.4 mL。实验后，由于吸收尿液，腹部和鼻道周围的皮毛是湿的（<strong class=...

该协议提供了用氰基丙烯酸酯对皮肤进行防水以防止尿液被毛皮和皮肤吸收的指南。它包括将胶水涂抹在皮肤上、植入膀胱导管以及用于清醒小鼠膀胱测压和尿道外括约肌电图记录的电极的说明。

Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions ...

optimizing-mouse-urodynamic-techniques-for-improved-accuracy

Research

JoVE Journal

Neuroscience

Optimizing Mouse Urodynamic Techniques for Improved Accuracy

378 次观看.  Indiana University School of Medicine. 本研究的目的是改进尿动力学技术以提高尿液测量的准确性。准确测量清醒小鼠的尿液参数对于了解下尿路功能障碍至关重要，尤其是在神经源性膀胱、创伤后脊髓损伤等情况下。我们开发了一种独特的方法，利用氰基丙烯酸酯胶粘剂在腹部周围形成防水皮肤屏障，防止尿液吸收并确保精确测量排尿量。我们将使用这种更准确的测定法来评估脊...

视频: 作者聚焦：用于神经源性膀胱清醒小鼠精确尿液测量的增强型尿动力学方法

Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions like neurogenic bladder post-traumatic spinal cord injury (SCI). However, conducting cystometry recordings in mice presents notable challenges. When mice are in a prone and restricted position during recording sessions, urine tends to be absorbed by the fur and skin, leading to an underestimation of voided volume (VV). The goal of this study was to enhance the accuracy of cystometry and external urethral sphincter electromyography (EUS-EMG) recordings in awake mice. We developed a unique method utilizing cyanoacrylate adhesive to create a waterproof skin barrier around the urethral meatus and abdomen, preventing urine absorption and ensuring precise measurements. Results show that after applying the cyanoacrylate, the sum of VV and RV remained consistent with the infused saline volume, and there were no wet areas observed post-experiment, indicating successful prevention of urine absorption. Additionally, the method simultaneously stabilized the electrodes connected with the external urethral sphincter (EUS), ensured stable electromyography (EMG) signals, and minimized artifacts caused by the movement of the awakened mouse and manipulation of the experimenter. Methodological details, results, and implications are discussed, highlighting the importance of improving urodynamic techniques in preclinical research.

This protocol provides a guide for waterproofing the skin with cyanoacrylate to prevent urine absorption by fur and skin. It includes instructions for applying the glue to the skin, implanting a bladder catheter, and electrodes for cystometry and external urethral sphincter electromyography recordings in awake mice.