作者没有任何致谢。

所有与动物有关的程序, 包括麻醉的方法和持续时间, 以及术后护理, 都与兽医进行了讨论, 并得到了机构动物护理和使用委员会 (IACUC) 的批准。附属机构。在这份手稿中, 所有的程序都是用8到15周大的 C57BL/6 雄性小鼠进行的, 该协议遵循 IACUC 在科罗拉多大学批准的动物保育指南. 
 1. 油管的制备 注: 为了灌输接种, 一根针连接到聚乙烯 (PE) 油管 ( 图 1A ) 或一个小的量规 (和 #60; 24 克) angiocatheter 可以使用 ( 图 1G )。使用 angiocatheter 不需要除绝育以外的70% 乙醇 (乙醇)。接种是被灌输/注入/沉积到泌尿道中的物质。这可以是一种含有细菌、病毒或化学物质的溶液。这是提前准备的时间和研究的协议。例如, 如果研究的目的是评估 大肠杆菌 感染对下尿路的影响, 研究者将需要准备含有细菌的接种, 该菌可以在下尿路接种/灌输. 
<ol> <li> 用于 pe 导管的针的制备简单, 包括以下内容: 根据靶器官和动物的年龄选择 pe 导管的尺寸。对于年龄小于12周的动物, 使用 PE-10 管 (外径为0.024 英寸, 内径为0.011 英寸)。PE-10 导管建议适用于 (1) 在前前列腺 (AP) 和精囊 (SV) 的接种的一次播种, 或 (2) 接种在一个小于12周龄的动物的膀胱中的一次播种。pe 管材的直径较大的 pe 50 (外径可达0.038 英寸, 内径可达0.023 英寸) 可用于接种在较年长动物的膀胱中的一次播种. </li>
	<li> 切割大约2.5 英寸的 PE 油管. </li>
	<li> 选择将适合所选 PE 直径的针。27克针能装入 PE-10 管的流明, 并且 22 g 针适合 PE-50 管的流明. 
	<ol> <li> 在将针的锋利边缘与商业可用的油石 ( 图 1A -b ) 进行摩擦之前, 先准备针插入 PE 管, 使其变钝, 或使用商业上可用的钝针. </li>
	</ol> </li> <li> 将钝针 ( 图 1C ) 引入 PE 管的流明中。通过确保针头保持在流明中心, 避免对流明或进入假腔的损伤。( 图 1D -e )。如果 PE 管在插入钝针时损坏, 请切断损坏的端部并重新启动过程. </li>
	<li> 将针附着 PE 导管放置在70% 的乙醇中以进行灭菌, 直到其用于程序。冲洗70% 乙醇通过针 pe 油管, 以消毒腔, 并确保没有从 PE 油管泄漏的过程中, 插入针。如果有泄漏, 丢弃的针 PE 管. 
	注: 在插管前, PE 导管的尖端需要平滑。为避免将 pe 导管引入尿道的问题, 建议在导管插入前立即切断 pe 导管的尖端 ( 图 1F )。建议的 PE 管的最小长度是1.5 英寸, 其中包括针周围的部分. </li>
</ol> 2。插管过程 <ol> <li> 在诱导麻醉之前, 清空鼠标的膀胱。
	<ol> <li> 对鼠标下腹应用柔和的压力和按摩。这些演习通常导致自发排尿。一个完整的膀胱可能导致稀释或立即泄漏任何灌输接种. 
		注: 一个额外的选项, 以确保一个空的膀胱在老鼠是剥夺动物的水至少1小时之前开始的经尿道灌注手术。作为动物议定书的一部分, 该机构应批准水资源匮乏. </li>
	</ol> </li> <li> 使用带有异氟醚流 (2%) 的麻醉机来诱导麻醉。
	<ol> <li> 一旦该动物被麻醉, 将它放在温暖的电热毯上, 用一个蓝色的垫子盖在一个 nosecone 内, 用于维持连续麻醉. </li>
		<li> 通过检查踏板反射 (脚趾捏) 确认该动物正在接受麻醉. </li>
	</ol> </li> <li> 一旦踏板反射不存在, 通过缩回包皮进行操作. 
	<ol> <li> 用一只手的拇指和食指 ( 图 2A 和 2B ) 按耻骨区域, 用另一只手的拇指和食指拉出腺体的阳具 ( 图 2C )。当这是正确的完成, 包皮, 头阴蒂, 和生殖器保持暴露。( 图 2D 和 图 2F )。不要用钳子拔出腺体的阳具, 因为这会导致组织受损. </li>
	</ol> </li> <li> 在插入导管之前, 用不育的手术润滑剂润滑导管和头的顶端。这将促进 PE 导管的插入. 
	注: 个别包装的外科润滑剂的平方, 而不是一个大的管, 建议每一个使用, 以保持不育的条件. </li>
	<li> 用钝钳轻轻握住小弟弟的尖端, 轻轻地挤压阴蒂, 使尿道口张开. </li>
	<li> 用针附着的 pe 油管或 pe 导管将注射器固定, 并通过尿道口插入 pe 导管. 
	注意: 建议使用拇指、食指和中指的注射器的三脚架握针与 pe 油管或 pe 管材本身的稳定性. 
	注: 导管的路径应该是弯曲的, 这是由于尿道的自然曲率, 在成年雄性小鼠的头状生殖器和阳具中所包围, 如 图 3A 和 图 3B 所示。插管 pe 导管在插管过程中可能会遇到阻力, 因此建议通过将导管向上或向下倾斜来调整角度, 以帮助进一步向靶器官推进 pe 导管。如果需要, PE 管可以稍微旋转, 以帮助它机动通过各种尖锐的尿道。当有太多的阻力, 油管应拉回, 然后再次前进, 以最小的压力。完全拉动 PE 油管, 使用更多的润滑可能是有帮助的. </li>
	<li> 当 PE 导管到达所需的器官时, 通过推针柱塞来注入所需的接种 (5-200 和 #181; 建议采用 L 音量). 
	<ol> <li> 用于将接种注入膀胱, 将 pe 导管向前推进, 直到插入 pe 油管大约0.75 英寸 ( 图 3A ). 
		注: 对接种在 AP 和 SV, 一般, th 的灌输插入的导管一旦到达前前列腺的开口点, 就会遇到阻力。通过倾斜油管向上或向下调整角度, pe 导管将进一步向前推进, 直到插入了大约1英寸 pe 油管 ( 图 3B ). </li>
	</ol> </li> </ol> 3。 在体内 下尿路和肾脏的影像学 注意: 在本手稿中, 活体 成像的目的是: (1) 确定接种的空间定位; (2) 评估接种的反流发生率肾脏.为此, 可以使用麻醉或安乐死的鼠标. 
<ol> <li> 对成年雄性小鼠下尿路进行可视化, 进行下腹中线剖腹探查, 以暴露膀胱、前列腺和精囊炎. 
	<ol> <li> 用剪刀将中线上的皮肤从耻骨切割到剑过程的下方. </li>
		<li> 将皮下组织的皮肤抬高. </li>
		<li> 确定位于中线的缺血性式阿尔巴, 并将其切开以进入所有脏器所在的腹膜腔. </li>
		<li> 通过可视化蓝色染料或其他合适的跟踪代理来确认空间定位. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lee, S., Yang, G., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostatic+inflammation+induces+urinary+frequency+in+adult+mice.">Prostatic inflammation induces urinary frequency in adult mice.</a> PLoS One. 10 (2), e0116827 (2015).</li><li>Wong, L., Hutson, P. R., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostatic+inflammation+induces+fibrosis+in+a+mouse+model+of+chronic+bacterial+infection.">Prostatic inflammation induces fibrosis in a mouse model of chronic bacterial infection.</a> PLoS One. 9 (6), e100770 (2014).</li><li>Elkahwaji, J. E., Zhong, W., Hopkins, W. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+bacterial+infection+and+inflammation+incite+reactive+hyperplasia+in+a+mouse+model+of+chronic+prostatitis.">Chronic bacterial infection and inflammation incite reactive hyperplasia in a mouse model of chronic prostatitis.</a> Prostate. 67 (1), 14-21 (2007).</li><li>Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+bacterial+inflammation+of+the+mouse+prostate.">Acute bacterial inflammation of the mouse prostate.</a> Prostate. 72 (3), 307-317 (2012).</li><li>Bjorling, D. E., Wang, Z. Y., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+inflammation+of+the+lower+urinary+tract.">Models of inflammation of the lower urinary tract.</a> Neurourol Urodyn. 30 (5), 673-682 (2011).</li><li>Thai, K. H., Thathireddy, A., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transurethral+induction+of+mouse+urinary+tract+infection.">Transurethral induction of mouse urinary tract infection.</a> J Vis Exp. (42), (2010).</li><li>Bjorling, D. E., Wang, Z. Y., Boldon, K., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cystitis+is+accompanied+by+increased+peripheral+thermal+sensitivity+in+mice.">Bacterial cystitis is accompanied by increased peripheral thermal sensitivity in mice.</a> J Urol. 179 (2), 759-763 (2008).</li><li>Hopkins, W. J., Gendron-Fitzpatrick, A., Balish, E., Uehling, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+host+responses+to+Escherichia+coli+urinary+tract+infection+in+genetically+distinct+mouse+strains.">Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains.</a> Infect Immun. 66 (6), 2798-2802 (1998).</li><li>Dinis, P., Charrua, A., Avelino, A., Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesical+resiniferatoxin+decreases+spinal+c-fos+expression+and+increases+bladder+volume+to+reflex+micturition+in+rats+with+chronic+inflamed+urinary+bladders.">Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders.</a> BJU Int. 94 (1), 153-157 (2004).</li><li>Cayan, S., 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=Botulinum+toxin+type+A+may+improve+bladder+function+in+a+rat+chemical+cystitis+model.">Botulinum toxin type A may improve bladder function in a rat chemical cystitis model.</a> Urol Res. 30 (6), 399-404 (2003).</li><li>Jerde, T. J., Bjorling, D. E., Steinberg, H., Warner, T., Saban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+mouse+bladder+inflammatory+response+to+E.+coli+lipopolysaccharide.">Determination of mouse bladder inflammatory response to E. coli lipopolysaccharide.</a> Urol Res. 28 (4), 269-273 (2000).</li><li>Saban, M. R., 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=LPS-sensory+peptide+communication+in+experimental+cystitis.">LPS-sensory peptide communication in experimental cystitis.</a> Am J Physiol Renal Physiol. 282 (2), F202-F210 (2002).</li><li>Schilling, J. D., Mulvey, M. A., Vincent, C. D., Lorenz, R. G., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+invasion+augments+epithelial+cytokine+responses+to+Escherichia+coli+through+a+lipopolysaccharide-dependent+mechanism.">Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism.</a> J Immunol. 166 (2), 1148-1155 (2001).</li><li>Saban, M. R., 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=Discriminators+of+mouse+bladder+response+to+intravesical+Bacillus+Calmette-Guerin+(BCG).">Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG).</a> BMC Immunol. 8, 6 (2007).</li><li>Bjorling, D. E., 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=Acute+acrolein-induced+cystitis+in+mice.">Acute acrolein-induced cystitis in mice.</a> BJU Int. 99 (6), 1523-1529 (2007).</li><li>Sadler, K. E., Stratton, J. M., Kolber, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+bladder+distention+evoked+visceromotor+responses+as+a+model+for+bladder+pain+in+mice.">Urinary bladder distention evoked visceromotor responses as a model for bladder pain in mice.</a> J Vis Exp. (86), (2014).</li></ol>

作者没有什么可透露的。

本手稿详细介绍了经尿道灌注法在成年雄性小鼠中的应用。采用不同直径的 PE 管材, 可达到差异主播种面积。PE-50 是建议成功达到膀胱腔, 而较大的直径 PE-10 是用来达到 AP 和 SV (图 3A-b)。除了选择 PE 管的大小和靶器官的接种外, 其他因素, 如注入量的接种, 也发挥了作用的最终位置和分布的解决方案。我们的实验证明, 经尿道灌注100µL 的蓝色染料使用 PE-10 或 PE-50...

在以往的研究中, 采用经尿道灌注法作为建立膀胱疾病模型的方法之一1,2,3,4 , 可用于评估当地提供的治疗小鼠的效果。虽然动物模型不能完全重述人类的病症, 但在动物研究中的基本机制的识别更能更好地理解人类的查找障碍, 如间质性膀胱炎/膀胱疼痛综合症, 神经原膀胱炎, 自身免疫性膀胱炎, 前列腺炎症5。
在成年雄性小鼠身上进行的经尿道灌注手术比在成年雌性老鼠6中的膀胱灌注更具有技术挑战性。男性尿道的自然弯曲解剖和它的小直径使它在技术上难以完成经尿道插入导管。因此,详细说明对经尿道感染的小鼠泌尿道传染病的诱导6和查找源性炎症7,8,910,12,13,14,15通过膀胱插入导管之前只对雌性小鼠进行了概述。这份手稿的目的是提供一个 step-by 一步的描述的技术, 在男性小鼠经尿道注入的物质, 包括视频剪辑, 图像和插图。经尿道灌注手术的成年雄性小鼠可以执行的变量接种类似于以前所描述的雌性鼠标7和合并的其他技术, 如肌电图记录visceromotor 响应 (VMR)16。

<table><tbody><tr><td>BD INTRAMEDIC Polyethylene Tubing/ PE 10</td><td>BD Medical</td><td>63019-004</td><td></td></tr><tr><td>BD INTRAMEDIC Polyethylene Tubing/ PE 50</td><td>BD Medical</td><td>63019-048</td><td></td></tr><tr><td>BD Insyte-N Autoguard/ Angiocath</td><td>BD Medical</td><td>311411</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>BD Needle Only 22 Gauge (G) 1 inch</td><td>Becton Dickson</td><td>205155</td><td></td></tr><tr><td>BD Needle Only 27 Gauge (G) 1/2 inch</td><td>Becton Dickson</td><td>305129</td><td></td></tr><tr><td>Luer stub, 22 G X 0.5 in / Blunt Needle</td><td>Instech</td><td>LS22</td><td></td></tr><tr><td>Luer stub, 27 G X 0.5 in / Blunt Needle</td><td>Instech</td><td>LS27</td><td></td></tr><tr><td>6&quot; Long x 2&quot; Wide x 1&quot; Thick, Aluminum Oxide Sharpening Stone / Oil Stone</td><td>Norton</td><td>40204034</td><td></td></tr><tr><td>Surgical Lubricant Sterile Bacteriostatic Folilpac</td><td>Surgilube</td><td>281020543</td><td></td></tr><tr><td>Isoflurane</td><td>Fluriso</td><td>13985-528-60</td><td></td></tr><tr><td>SomnoSuite Low-Flow Anesthesia System</td><td>Kent Scientific</td><td>SS-01</td><td></td></tr><tr><td>Dissecting Forceps, Curved</td><td>vwr</td><td>82027-392</td><td></td></tr><tr><td>BD 1 ml Syringe</td><td>BD Medical</td><td>309626</td><td></td></tr><tr><td>India Ink Solution., 0.2% in PBS buffer</td><td>Alfa Aesar</td><td>J61007</td><td></td></tr><tr><td>8~12 weeks old male C57B/6 Mice</td><td>Jackson Lab</td><td>#000664</td><td></td></tr></tbody></table>

transurethral instillation procedure in adult male mouse

经尿道灌注可用于提供不同的解决方案与活性成分 (例如,药物, 化学药品, 细菌和病毒) 当地进入膀胱, 要么诱发动物模型膀胱病理或评估膀胱灌注治疗的有效性。大多数啮齿动物模型的下尿路 (查找) 病理是诱导雌性小鼠, 由于容易膀胱灌注的物质通过女性尿道。然而, 由于女性与男性之间的解剖差异, 经尿道灌注的雄性小鼠被认为是一个非常具有挑战性的过程, 并没有事先描述。在这份手稿中, 我们提供了一个详细的描述如何准备聚乙烯 (PE) 油管后插入到尿道的雄性鼠标。此外, 我们还讨论了理想的 PE 管的类型, 要根据所需的接种地点使用。此外, 我们还描述了如何准备一个成功的经尿道灌注的动物, 以避免对尿道的伤害, 并确保提供的解决方案的理想地点。手术的开始是通过缩回包皮和头来暴露尿道口的开口。其次, 用钝 non-crushing 钳抓住阴蒂, 稳定小弟弟和 PE 管。PE 导管首先插入到与动物身体平行的尿道道中, 然后通过倾斜导管来调整它的角度来操纵它以遵循尿道的自然曲率。这项技术可用于诱导小鼠膀胱病理模型和/或评估的有效性, 在雄性老鼠的治疗。

通过 pe 50 导管经尿道注入蓝染料后, 将染料滴入膀胱 (图 4A), 并通过 pe 10 导管在 SV 和 AP (图 4B) 着色中分别进行。为了评价经尿道电切术是否使接种的即刻回流到肾脏, 肾脏和输尿管被观察到, 然后注入100µL 的蓝色染料和荧光染料 (图 4A-b)。没有使用两种不同尺寸的 PE 导管和两种不同的染料的?...

Watch this Scientific Journal Video about Transurethral Instillation Procedure in Adult Male Mouse at JoVE.com

Transurethral Instillation Procedure in Adult Male Mouse

经尿道灌注是一项具有挑战性的手术, 在文献中没有得到很好的描述。本手稿的目的是描述一种技术, 经尿道插入导尿管膀胱灌注液体的活性物质进入膀胱和/或前列腺的成年雄性小鼠。

成年雄性小鼠经尿道灌注术

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

transurethral-instillation-procedure-in-adult-male-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.5K 次观看.  University of Colorado School of Medicine. 经尿道灌注是一项具有挑战性的手术, 在文献中没有得到很好的描述。本手稿的目的是描述一种技术, 经尿道插入导尿管膀胱灌注液体的活性物质进入膀胱和/或前列腺的成年雄性小鼠。

视频: Transurethral Instillation Procedure in Adult Male Mouse

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

经尿道诱导小鼠尿路感染

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

科研

免疫与感染

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. ...

医学

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

用于尿功能测试的成人男性尿道超声检查

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

生物学

Modeling Orthotopic Bladder Tumor in Mouse Model: A Procedure for Intravesical Administration of Cancer Cells into Murine Bladder

Source: Tham, S. M. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55078" target="_blank">A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.</a>&#160;J. Vis. Exp.&#160;(2017)
In this video, we describe the intravesical procedure of delivering cancer cells into the bladder of a mouse model. This procedure helps generate an orthotopic bladder tumor model that can be used to develop therapeutic strategies.

小鼠模型中原位膀胱肿瘤建模:将癌细胞膀胱内给药到小鼠膀胱中的程序

Source: Tham, S. M. et al. A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.&#160;J. Vis. Exp.&#160;(2017)
In this video, we describe ...

JoVE Encyclopedia of Experiments

癌症研究

Urinary Tract Cancer

In vivo studies

Urine Depletion and Bladder Rinsing in Mouse Model: A Catheter-based Procedure to Empty and Wash Murine Urinary Bladder

Source: Yu, C. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/57649" target="_blank">Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation.</a> J. Vis. Exp. (2018)
In this video, we describe a catheter-based procedure to empty and rinse the urinary bladder of a mouse model. The prepped mouse can then be used for further experimental procedures.

小鼠模型中的尿液消耗和膀胱冲洗:一种基于导管的小鼠膀胱排空和清洗程序

Source: Yu, C. et al. Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation. J. Vis. Exp. (2018)
In this video, we describe ...

Transurethral Catheterization and Bacterial Inoculation to Generate a Urinary Tract Infection Model in Male Mice

Source: Zychlinsky Scharff, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54432">Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.

经尿道导管插入术和细菌接种在雄性小鼠中生成尿路感染模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunology

Immunopathology

Host-Pathogen Interaction Models

Establishment of a Urinary Tract Infection Model in Female Mice

Source: Zychlinsky Scharff, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54432">Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.