Name: in vivo luminal measurement of distension-evoked urothelial atp release in rodents
Uploaded: 2022-09-07T13:20:00
Description: Watch this Scientific Journal Video about In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents at JoVE.com

这项工作得到了国家糖尿病，消化和肾脏疾病研究所（NIDDK）对JMB（DK117884）的资助。

在啮齿动物中执行的所有程序必须遵守适用的准则，并得到当地机构伦理审查委员会的批准。为该手稿进行的实验是根据国家研究委员会的实验动物护理和使用指南进行的，并得到了匹兹堡大学医学院机构动物护理和使用委员会（IACUC）的批准。有关此协议中使用的标准啮齿动物膀胱测量设置的修改版本，请参见 图1 。
1. 实验动物
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<ol>
	<li>Burnstock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+signalling+in+the+urinary+tract+in+health+and+disease.">Purinergic signalling in the urinary tract in health and disease.</a> Purinergic Signalling. 10 (1), 103-155 (2014).</li><li>Birder, L. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+extracellular+ATP+signaling+in+bladder+urothelial+cells+from+patients+with+interstitial+cystitis.">Augmented extracellular ATP signaling in bladder urothelial cells from patients with interstitial cystitis.</a> American Journal of Physiology - Cell Physiology. 290 (1), 27-34 (2006).</li><li>Gill, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+ATP+as+an+indicator+of+infection+and+inflammation+of+the+urinary+tract+in+patients+with+lower+urinary+tract+symptoms.">Urinary ATP as an indicator of infection and inflammation of the urinary tract in patients with lower urinary tract symptoms.</a> BMC Urology. 15 (1), 7 (2015).</li><li>S&#228;ve, S., Persson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+and+P2Y+receptor+activation+induce+a+proinflammatory+host+response+in+the+human+urinary+tract.">Extracellular ATP and P2Y receptor activation induce a proinflammatory host response in the human urinary tract.</a> Infection and Immunity. 78 (8), 3609-3615 (2010).</li><li>Munoz, A., Smith, C. P., Boone, T. B., Somogyi, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overactive+and+underactive+bladder+dysfunction+is+reflected+by+alterations+in+urothelial+ATP+and+NO+release.">Overactive and underactive bladder dysfunction is reflected by alterations in urothelial ATP and NO release.</a> Neurochemistry International. 58 (3), 295-300 (2011).</li><li>Beckel, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pannexin+1+channels+mediate+the+release+of+ATP+into+the+lumen+of+the+rat+urinary+bladder.">Pannexin 1 channels mediate the release of ATP into the lumen of the rat urinary bladder.</a> The Journal of Physiology. 593 (8), 1857-1871 (2015).</li><li>Zhang, Y. 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大多数关于尿路上皮ATP释放的研究都是在培养细胞中进行的，使用永生化细胞系或啮齿动物尿路上皮细胞的原代培养物。虽然这些模型具有相对高通量的优点（即，一次培养/传代可以制作许多细胞板/培养皿），但由于以下原因，它们的生理相关性降低：1）尿路上皮细胞无法生长极化，除非它们在特殊载体上生长，2）难以将培养的细胞暴露于生理水平的拉伸/压力。处理这些限制的一种方法是从?...

尿 ATP 被认为在控制排尿中起显着的感觉作用1.例如，人们认为ATP从尿路上皮释放以响应膨胀，它可以作用于膀胱传入神经上的受体以增加其兴奋性，从而导致饱腹感2。因此，人们还认为尿ATP可能是膀胱病变发展的重要参与者。为了支持这一假设，患有膀胱过度活动症（OAB）3，膀胱疼痛综合征/间质性膀胱炎（BPS / IC）4或尿路感染（UTI）的患者的尿ATP浓度显着增加5，6，所有情况的特征都是增加的紧迫性，频率和有时疼痛。相反，患有膀胱活动不足（UAB）的患者，其特征是无法排空膀胱，有时可能包括感知膀胱充盈的能力下降，已被证明尿ATP浓度降低7。实验上，尿ATP浓度的操作可以改变大鼠的膀胱反射;通过阻断膀胱腔中的内源性ATP酶来增加ATP浓度可以增加排尿频率，而通过将外源性ATP酶注入膀胱来降低ATP浓度会降低排尿频率8。因此，尿ATP对膀胱功能的重要性是显而易见的。
鉴于尿ATP对膀胱病理学的明显重要性，准确测量尿路上皮ATP释放是了解控制释放机制的重要一步。许多研究已经使用不同的实验模型来完成测量尿路上皮ATP的释放。其中最重要的是细胞培养物，原代培养物或细胞系。然而，培养的尿路上皮细胞的使用很复杂，因为尿路上皮细胞不会呈现其生理极化形态，除非它们生长在特殊的可渗透膜上（例如Transwell技术[孔插入]）9。因此，很难将测量的任何ATP释放与生理学联系起来。在孔插入物上生长的尿路上皮细胞可以极化并形成类似于 体内看到的屏障;然而，完全分化的尿路上皮的生长可能需要数天或数周。此外，虽然可以将孔插入室安装到Ussing室中并向顶端侧施加压力以引起拉伸，但在病理期间很难施加足够的压力来模拟膀胱内的情况（即，压力为30cm H2O或以上）。整个膀胱组织也可以安装在Ussing室中进行拉伸实验，但这会将膀胱与维持尿路上皮细胞健康的营养因子一起从生物体中移除，从而维持尿路上皮屏障功能。因此，研究ATP响应拉伸或压力从尿路上皮释放的最生理相关方法是 体内。建立实验所需的手术技术与动物膀胱测量中常用的技术相同，因此，任何熟悉该技术的人都应该很容易进行。
在该协议中，我们将描述用于检查体重约200-250g的雌性Sprague Dawley大鼠管腔ATP的技术，因为下面描述的经尿道导管插入术在雌性中要容易得多;然而，经尿道导尿术也可以在雄性啮齿动物中进行10。由于经尿道导管插入术现在已经在两性小鼠中进行11，因此根据研究小组的需要，这些实验可以很容易地适用于小鼠或任何性别或不同大小的大鼠。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>amplifier</td>
			<td>World Precision Instruments (WPI)</td>
			<td>SYS-TBM4M</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP assay kit</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>FLAA-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>data acquisition system/ software</td>
			<td>DataQ Instruments</td>
			<td>DI-1100</td>
			<td>Software included, requires Windows-based computer</td>
		</tr>
		<tr>
			<td>Hexamethonium bromide</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>H0879</td>
			<td>20 mg/kg dose</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus North America</td>
			<td>29404</td>
			<td></td>
		</tr>
		<tr>
			<td>lidocaine</td>
			<td>Covetrus North America</td>
			<td>2468</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock plugs</td>
			<td>Fisher Scientific</td>
			<td>NC0455253</td>
			<td></td>
		</tr>
		<tr>
			<td>luminometer (GloMax 20/20)</td>
			<td>Promega</td>
			<td>E5311</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene (PE50) tubing</td>
			<td>Fisher Scientific</td>
			<td>14-170-12B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump 33 DDS syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>703333</td>
			<td></td>
		</tr>
		<tr>
			<td>pressure transducers</td>
			<td>World Precision Instruments (WPI)</td>
			<td>BLPR2</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical instruments (scissors, hemostats, forceps, etc.)</td>
			<td>Fine Science Tools</td>
			<td>multiple numbers</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical lubricant</td>
			<td>Fisher Scientific</td>
			<td>10-000-694</td>
			<td></td>
		</tr>
		<tr>
			<td>Sur-Vet I.V. catheter&#160;</td>
			<td>Covetrus North America</td>
			<td>50603</td>
			<td>20 G x 1 inch</td>
		</tr>
		<tr>
			<td>tiltable surgical table (Plas Labs)</td>
			<td>Fisher Scientific</td>
			<td>01-288-30A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing connectors</td>
			<td>Fisher Scientific</td>
			<td>14-826-19E</td>
			<td>allows Luer-Lock connectors to attach to tubing</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>U2500</td>
			<td>0.5 g/mL conc., 1.2 g/kg dose</td>
		</tr>
	</tbody>
</table>

in vivo luminal measurement of distension-evoked urothelial atp release in rodents

ATP 因膀胱扩张而从尿路上皮释放，被认为在控制排尿中起显著的感觉作用。因此，在生理环境中准确测量尿路上皮ATP释放是研究控制膀胱嘌呤能信号传导机制的重要第一步。研究机械诱发尿路上皮ATP释放的现有技术利用接种在柔性支架上的培养细胞或固定在Ussing室中的膀胱组织;然而，这些技术中的每一种都不能完全模拟完整膀胱的状况。因此，开发了一种实验装置来直接测量啮齿动物膀胱管腔中的ATP浓度。
在这种设置中，麻醉啮齿动物的膀胱通过膀胱圆 顶和尿 道外口的导管灌注。通过盖住尿道导管，同时通过圆顶将无菌液体注入膀胱来增加膀胱中的压力。使用连接到膀胱圆顶导管的压力传感器实现膀胱内压力的测量，类似于用于膀胱测量的设置。一旦达到所需的压力，取下尿道导管的盖，并通过荧光素-荧光素酶测定收集液体用于ATP定量。通过这种实验装置，可以通过在灌注液中加入各种激动剂或拮抗剂或通过比较野生型和转基因动物之间的结果来询问控制尿路上皮ATP释放的机械和化学刺激的机制。

所描述的方案允许使用标准啮齿动物膀胱测量设置的修改版本准确测量膀胱 腔体内尿 路上皮ATP释放（见 图1）。这使得研究人员能够在生理环境中检查药物对拉伸介导的ATP释放的影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64227/64227fig01.jpg"> 图 1：实验设置。 （A） 显示<...

Watch this Scientific Journal Video about In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents at JoVE.com

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

该协议描述了在麻醉啮齿动物中测量膀胱腔中ATP浓度的程序。

体内 啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

我们的协议测量ATP，这是膀胱中的重要递质，这将使我们能够进一步询问控制其释放的机制。我们的技术直接从膀胱腔测量ATP，使其比使用培养细胞或膀胱组织的技术更具生理相关性。演示该程序的将是我实验室的研究技术人员Keara Healy和Stephanie Daugherty。首先，通过将动物置于用4%至5%异氟醚在氧气中气体的封闭盒中来诱导初始麻醉。皮下注射双侧聚氨酯，然后将动物放在笼子中，让聚氨酯生效近两个小时。等待聚氨酯生效后，用镊子捏住动物的脚来测试适当的麻醉平面。为了防止在实验过程中膀胱因膨胀而收缩，向动物注射神经节阻断剂六甲铵。接下来，将眼药膏涂抹在动物的眼睛上，以防止在实验过程中干燥。现在剃掉动物的腹部并进行中线剖腹手术以暴露膀胱。使用火焰，燃烧短长度PE50内医用管的一端，将22号针放入管的另一端，并填充克雷布斯溶液。在膀胱圆顶上放置一小圈 3-0 丝缝，并进行小膀胱造口术以插入导管的喇叭形端。在用一只手握住导管的同时，用另一只手拧紧缝合环以将导管固定到位。通过在缝合线上打两个结来完成导管固定。向后拉导管，直到喇叭头与膀胱壁接触。通过导管注入少量克雷布斯溶液来测试设置的泄漏。将静脉导管的末端浸入手术润滑剂中。用一对镊子轻轻握住尿道外道口，并将导管尖端沿尾部方向插入尿道口，如手稿中所述。将导管旋转 90 度并轻轻前进。插入导管，直到鲁尔锁毂距离尿道外开口远端 5 毫米。固定导管并防止导管周围泄漏，方法是在尿道外道口周围缠绕一小段 3-0 丝线并将其紧紧绑住。用胶带将导管固定在尾部，以防止其意外拉出。导尿后，通过耻骨上膀胱导管轻轻将克雷布斯溶液注入膀胱，并确认液体从尿道导管流出而不是在其周围。使用3-0丝线关闭膀胱上的腹部切口。将吸收垫放在加热垫上，以保持身体热量并吸收液体。将动物固定在倾斜板上，以帮助通过尿道导管排出膀胱内液体，然后打开加热垫。将耻骨上导管连接到三向止动旋塞，将导管连接到注射泵和压力传感器。通过放大器和数据采集系统将压力传感器连接到计算机。以每分钟 0.1 毫升的速度通过耻骨上导管输注 Krebs 溶液，并让液体通过尿道导管排出一小时，以洗掉导管植入期间释放的任何残留 ATP。在此冲洗期之后，使用鲁尔锁塞盖住尿道导管。测量膀胱中的压力，并寻找膀胱内压力缓慢上升至30厘米水的压力，而压力没有急剧增加，表明膀胱收缩。当压力达到30厘米水时，从尿道导管上取下塞子，以防止对膀胱造成损害。输注膀胱并从尿道导管中收集淋洗液。立即测试100微升等分试样的淋洗液的ATP或冷冻以进行以后的批量定量。为了测试膀胱扩张对管腔ATP浓度的影响，用塞子盖住尿道导管并监测膀胱压力，直到达到所需水平，然后打开尿道导管盖并收集淋洗液进行ATP测量或冷冻，如上所述。每次膨胀后，让膀胱休息并冲洗 10 到 15 分钟，然后再采集额外的样本。为了测试药物对ATP释放的影响，请将输注膀胱的克雷布斯溶液切换到含有所选药物的克雷布斯。如前所述，灌注药物10至15分钟以产生效果，然后从未膨胀和扩张的膀胱中收集样本。将100微升灌注液样品与50微升测定混合物放入光度计中以供读取。要从相对光单位转换为ATP浓度，请在克雷布斯溶液中以10倍稀释液连续稀释ATP，范围从1微摩尔到10皮摩尔，以创建标准曲线，并在光度计中读取它们。将结果读数绘制在图表上，并执行非线性回归以推断从动物身上采集的样品中的浓度。当尿道导管堵塞时，将溶液输注到膀胱中会导致膀胱内压力随着膀胱收缩而急剧上升。六甲铵给药后，观察到膀胱内压力更缓慢地升高，允许压力上升至30厘米的水而不会收缩。亮蓝色FCF显著降低了标准曲线的斜率，足以改变样品中ATP浓度的计算值。使用含有亮蓝色FCF的克雷布斯标准品计算样品中ATP的浓度可能导致ATP浓度被低估50% 膀胱膨胀会增加管腔ATP浓度，在NTPDase apvrase存在下可能会降低，或者在ARL67156抑制内源性NTPD ACE时升高。分析了牵拉诱导的尿路上皮ATP释放。膨胀诱发的ATP释放被潘克丁通道拮抗剂，亮蓝色FCF和碳氧松阻断，但不能被连接子通道拮抗剂18α-甘草次酸阻断。必须使用适当的标准曲线将所有光度计读数转换为ATP浓度，因为路西法荧光素酶反应极易受到多种不同试剂的干扰。我们的技术可用于研究膀胱腔内任何分子的膨胀诱发释放，假设存在该分子的合适测定。

Research

JoVE Journal

Medicine

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

科研

医学

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI for Cell Tracking

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

远程肢体缺血预适应：在啮齿动物的保护技术

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Using a Chemical Biopsy for Graft Quality Assessment

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

体内 啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量

体内啮齿动物膨胀诱发尿路上皮ATP释放的管腔测量