Name: a decentralized (ex vivo) murine bladder model with the detrusor muscle removed for direct access to the suburothelium during bladder filling
Uploaded: 2019-11-28T12:30:00
Description: Watch this Scientific Journal Video about A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling 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资助DK41315的支持。

本手稿中描述的涉及动物的所有程序均按照国家卫生研究院《实验室动物护理和使用指南》和内华达大学机构动物使用与护理委员会进行。
注：此处介绍的模型包括去除除精肌肉，而尿道和SubU/LP保持完好（图1B），使调查人员在膀胱填充过程中直接访问SubU/LP。
1. 解剖无除气囊准备
<ol><li>将分离的?...

<ol>
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M., Fry, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+release+from+freshly+isolated+guinea-pig+bladder+urothelial+cells:+a+quantification+and+study+of+the+mechanisms+involved.">ATP release from freshly isolated guinea-pig bladder urothelial cells: a quantification and study of the mechanisms involved.</a> BJU International. 115, 987-993 (2015).</li><li>Birder, L. A., Apodaca, G., de Groat, W. C., Kanai, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic-+and+capsaicin-evoked+nitric+oxide+release+from+urothelium+and+afferent+nerves+in+urinary+bladder.">Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder.</a> American Journal of Physiology Renal Physiology. 275, F226-F229 (1998).</li><li>Birder, L. A., Kanai, A. J., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO:+effect+on+bladder+afferent+neurons+and+nitric+oxide+release.">DMSO: effect on bladder afferent neurons and nitric oxide release.</a> Journal of Urology. 158, 1989-1995 (1997).</li><li>Birder, L. A., 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=Vanilloid+receptor+expression+suggests+a+sensory+role+for+urinary+bladder+epithelial+cells.">Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells.</a> Proceedings of the National Academy of Sciences U S A. 98, 13396-13401 (2001).</li><li>Birder, L. A., 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=Beta-adrenoceptor+agonists+stimulate+endothelial+nitric+oxide+synthase+in+rat+urinary+bladder+urothelial+cells.">Beta-adrenoceptor agonists stimulate endothelial nitric oxide synthase in rat urinary bladder urothelial cells.</a> Journal of Neuroscience. 22, 8063-8070 (2002).</li><li>Durnin, L., 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=An+ex+vivo+bladder+model+with+detrusor+smooth+muscle+removed+to+analyse+biologically+active+mediators+released+from+the+suburothelium.">An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium.</a> Journal of Physiology. 597, 1467-1485 (2019).</li><li>Yoshida, 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=Non-neuronal+cholinergic+system+in+human+bladder+urothelium.">Non-neuronal cholinergic system in human bladder urothelium.</a> Urology. 67, 425-430 (2006).</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> Journal of Physiology. 593, 1857-1871 (2015).</li><li>Collins, V. 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=OnabotulinumtoxinA+significantly+attenuates+bladder+afferent+nerve+firing+and+inhibits+ATP+release+from+the+urothelium.">OnabotulinumtoxinA significantly attenuates bladder afferent nerve firing and inhibits ATP release from the urothelium.</a> BJU International. 112, 1018-1026 (2013).</li><li>Daly, D. M., Nocchi, L., Liaskos, M., McKay, N. G., Chapple, C., Grundy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+afferent+pathways+and+urothelial+function+in+the+male+mouse+bladder.">Age-related changes in afferent pathways and urothelial function in the male mouse bladder.</a> Journal of Physiology. 592, 537-549 (2014).</li><li>Durnin, L., Hayoz, S., Corrigan, R. D., Yanez, A., Koh, S. D., Mutafova-Yambolieva, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+purine+release+during+filling+of+murine+and+primate+bladders.">Urothelial purine release during filling of murine and primate bladders.</a> American Journal of Physiology Renal Physiology. 311, F708-F716 (2016).</li><li>Gonzalez, E. J., Heppner, T. J., Nelson, M. T., Vizzard, M. 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膀胱有两个功能：尿液的储存和排空。这些功能的正常运行需要通过膀胱壁中的细胞对宫内体积和压力和信号进行适当的机械感应，以调节突生肌肉的兴奋性。膀胱粘液（尿毒症）被认为通过释放SubU/LP中影响膀胱壁中多种细胞类型的多种信号分子来调节膀胱兴奋性。目前，大多数对泌尿原介质的表征尝试涉及使用不繁殖生理膀胱填充的膀胱制剂（例如，扁平膀胱壁片、膀胱壁条或培养细胞）。膀...

此模型的目的是使膀胱粘膜在膀胱填充的不同阶段能够直接进入下粘膜侧。
膀胱在填充过程中必须避免过早收缩，当达到临界体积和压力时，膀胱必须避免过早收缩。尿液异常的失禁或空位经常与膀胱填充过程中分度平滑肌（DSM）的异常兴奋性有关。DSM 的可兴奋性由平滑肌细胞固有的因素和膀胱壁内不同细胞类型产生的影响决定。膀胱壁由尿道（粘液）、亚尿体（SubU）/拉米纳利普里亚（LP）、德特鲁索平滑肌（DSM）和塞罗萨（图1A）组成。尿体由伞状细胞（即尿体最外层）、中间细胞和基底细胞（即尿体最内层）组成。各种类型的细胞，包括间质细胞、成纤维细胞、神经终端、小血管和免疫细胞都驻留在SubU/LP中。人们普遍认为膀胱尿毒症是一种感觉器官，通过将介质释放到影响SubU/LP和DSM1、2、3的细胞的亚粘体中，启动反射性粘动和节制。在大多数情况下，这些假设基于已经证明释放介质的研究：从暴露于静水压力变化的粘液片4，5;从培养的尿毒细胞暴露到拉伸6，7，低度引起的细胞肿胀7或阻力8;从分离的膀胱壁条受体或神经激活9，10，11，12，13，14;并在膀胱流明在膀胱填充结束15，16，17，18，19。虽然这些研究有助于证明在机械刺激膀胱壁段或培养的泌尿细胞时释放介质，但它们需要有直接证据支持，以释放通过复制膀胱填充的生理刺激引起的子粘膜中介质的释放。这是一项具有挑战性的任务，因为 SubU/LP 位于膀胱壁深处，妨碍在膀胱填充过程中直接进入 SubU/LP 附近。
在这里，我们演示一个分散（前体）鼠膀胱模型与去皮肌肉去除13，这是开发，以促进研究局部机制的中导转导，参与膀胱尿，DSM和膀胱壁的其他细胞类型之间的信号。这种方法优于使用扁平膀胱壁片、膀胱壁带或培养的泌尿细胞，因为它允许在SubU/LP附近直接测量尿毒源介质，这些介质是因膀胱中的生理压力和体积而释放或形成的，并避免细胞培养中潜在的表型变化。它可用于测量子U/LP中调解员在膀胱填充的不同阶段的可用性、释放、代谢和横射物传输（图1B）。该制剂还可用于检查过度活跃和不足的膀胱综合征模型中的泌尿信号和中导转导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>C79</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D16</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Dissecting pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Infusion Pump</td>
			<td>Kent Scientific</td>
			<td>GenieTouch</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P284</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>SCHOTT ACEI</td>
			<td></td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus SZX7</td>
			<td></td>
			<td>Flexible to use any scope</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>M33</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S671</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher</td>
			<td>S233</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Needles 25G</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Organ bath</td>
			<td>Custom made</td>
			<td></td>
			<td>Flexible source; We made it from Radnoti dissecting dish</td>
		</tr>
		<tr>
			<td>PE-20 tubing</td>
			<td>Intramedic</td>
			<td>427405</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Pressure transducer</td>
			<td>AD instrument</td>
			<td></td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps</td>
			<td>Fine Science Tools</td>
			<td>00632-11</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Software pressure-volume</td>
			<td>AD Instruments</td>
			<td>Power lab</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Nylon, 6-0</td>
			<td>AD surgical</td>
			<td>S-N618R13</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Suture Silk, 6-0</td>
			<td>Deknatel via Braintree Scientific, Inc.</td>
			<td>07J1500190</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Syringes 1 ml</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>Source flexible</td>
		</tr>
		<tr>
			<td>Water circulator</td>
			<td>Baxter</td>
			<td>K-MOD 100</td>
			<td>Source flexible</td>
		</tr>
	</tbody>
</table>

a decentralized (ex vivo) murine bladder model with the detrusor muscle removed for direct access to the suburothelium during bladder filling

先前的研究已经确定，在静水压力变化、拉伸、细胞肿胀或阻力时，在静水压力变化、拉伸、细胞肿胀或阻力时，以及气囊流明在填充结束时，从扁平膀胱粘液片中释放化学物质，暴露于静水压力或机械拉伸的变化中，并暴露于培养的泌尿细胞中。这些发现导致假设，这些介质也被释放在子尿层（SubU）/拉米纳丙酸（LP）在膀胱填充，其中他们影响细胞深在膀胱壁，最终调节膀胱兴奋性。此类研究中至少有两个明显的局限性：1） 这些方法均未提供有关子U/LP 中调解员存在的直接信息;2） 使用的刺激不是生理上的，并且不重述膀胱的真实填充。在这里，我们讨论一个程序，使直接访问膀胱粘液的亚尿道表面在膀胱填充过程中。我们创造的无鼠排骨制剂与完整膀胱的填充非常相似，允许在没有来自脊柱反射和分离肌的混淆信号的情况下，对膀胱进行压力体积研究。使用新型无分子膀胱模型，我们最近证明，在膀胱填充过程中，不能将介质的宫内测量用作 SubU/LP 中释放或存在的物的代理。该模型能够检查在膀胱填充过程中释放、代谢产生和/或输送到SubU/LP的泌尿衍生信号分子，以将信息传输到膀胱的神经元和平滑肌，并调节其在节制和气动过程中的兴奋性。

无鼠排叶的膀胱制剂壁完好无损，包含除 DSM 和 Serosa 外的所有层。原理证明研究表明，无DSM的膀胱壁包括尿道和SubU/LP，而图尼卡肌肉和serosa则不存在（图2）13。
无分子膀胱的填充近似于正常膀胱填充。图3显示了以不同填充速率、体积和宫内压力填充体外?...

Watch this Scientific Journal Video about A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling at JoVE.com

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

无分泌物的膀胱模型允许直接进入亚尿层，以研究在尿液储存和排空期间调节亚尿层/拉米纳尿体中生物活性介质可用性的本地机制。制备与填充完整膀胱非常相似，允许在无系统影响的情况下进行压力体积研究。

分散（Ex Vivo）的毛骨球模型，去除了除位肌肉，用于在膀胱填充期间直接进入苏布罗氦

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

我们的膀胱模型与去除的松弛肌肉是一种新的实验方法，同时研究在膀胱填充期间在拉米亚丙体和膀胱流明的尿皮中介的可用性。此模型可以提供新的见解，了解在填充过程中调节膀胱兴奋性尿素、亚尿素和德特鲁索肌肉之间的信号机制。首先将完整的穆林膀胱标本放入含有10摄氏度含氧KBS的解剖显微镜下解剖盘中，并清理膀胱周围的结缔和脂肪组织。将隔离膀胱圆顶的一小部分固定到远离肌部肌肉最内侧边缘的分离膀胱，以制成充满KBS的带状解剖盘，并固定尿管和膀胱颈部，以显示完整的膀胱制剂。使用细尖钳子，轻轻拉一块在尿尿道和膀胱体之间的角落的血清，并调整显微镜的光线，以提高透明度，并区分尿道肌肉下方的亚木科萨边缘。为了避免穿孔的制备壁，一定要切断德特鲁索肌肉只，而膀胱粘膜的横向边缘是清晰可见的。使用细尖剪刀，开始沿着德特鲁索肌肉层的内表面切割膀胱壁，同时轻轻地将切开部分从准备中拉开，同时注意可以观察到粘膜的侧缘，同时避免接触组织的边缘。将一小块德特鲁瑟肌肉留在膀胱圆顶顶部，以便在协议的剩余步骤中固定制备，并填充两厘米的 20 个聚乙烯管，并填充 37 摄氏度的氧化 KBS。将导管插入膀胱尿道的孔后，轻轻推动导管，直到导管尖端到达膀胱的近似中间。然后用双环6.0尼龙缝合线将导管固定在膀胱颈部周围组织。检查组织有无泄漏后，将排骨膀胱制剂放入37摄氏度水套管管的三毫升腔内，并固定导管到腔室的一侧，使制剂不会浮在灌注溶液表面之上。将膀胱导管连接到一块聚乙烯 20 管，连接到三向止动器和充满 37 摄氏度含氧 KBS 的输液注射器，然后启动输液泵，用 KBS 小心地对膀胱进行填充，以监测灌装过程中内压中的灌装量。为了检测制备过程中亚尿层中介质，在各适当的实验时间点收集浴液的等分，并按照计划的下游分析处理样品。切除膀胱准备壁包含尿素和亚尿素，但不是德特鲁索平滑肌。压力体积关系在完好无损的制剂中非常相似。测量在灌装过程中在亚尿素层状丙烯侧释放的尿素衍生介质的模型是否适用，可以通过测量紫菜介质释放到溶液中，使被消退制剂的亚尿素层状介质的释放情况得到测试。如所证明，在含有完整切除肌肉的隔离膀胱制剂的浴池中，可以检测到微不足道的紫菜;而从含有排骨膀胱制剂的浴池中收集的样本中，这些紫菜的量要高得多。值得注意的是，从流明和亚尿素的样品中，在填充结束时的紫菜和代谢物的分布差异很大。将 ATP 的一个高度荧光模拟 Atp 添加到无毒剂制备的亚三边，导致 Atp 的减少，Atheno ATP 产品的增加，atheno ADP、atheno AMP 和 atheno ADO 的添加。同样，在流明制备中加入 atheno ATP 会导致 atheno ATP 的减少，以及流明中 atheno ADP、atheno AMP 和 atheno ADO 的增加。此外，在德努德制剂的亚尿层层丙条一侧添加 atheno ATP 会导致 atheno AMP 和 atheno ADO 在流明中出现。同样，在流明中添加 atheno ATP 会导致 atheno AMP 和 atheno ADO 在亚尿素层中出现。按照这个程序，兴奋和抑制性调停剂的可用性可以在膀胱填充的不同阶段的拉米纳丙体和膀胱流明测量。也可以利用该模型研究调停器的双边横贯运输和调停器的代谢。使用这种新的膀胱模型将导致对内在，局部机械敏感信号之间的尿素和德特鲁索平滑肌肉，控制膀胱兴奋在健康和疾病的新的理解。

Research

JoVE Journal

Medicine

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Mouse Bladder Wall Injection

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

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

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

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

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 ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

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 ...

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

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 ...

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

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 ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...