资助这项工作是由ORWH SCOR P50 DK064540提供，RO1 DK 051406，RO1 AI 108749-01，F32 DK 101171，和F32 DK 104516-01。

伦理声明:华盛顿大学动物研究委员会批准所有的鼠标感染和程序的协议号20120216，这是批准2013年1月11日，截止2016年1月11日部分。整体护理的动物是有照顾和实验动物从国家研究委员会和美国农业部的动物保健资源指南的指导使用一致的。安乐死程序与一致"的动物版本2013的安乐死AVMA准则。" 1.统一企业UTI协议，接种针准备（图S1） <ol><li>卸下地下30针帽。线程大约1英寸PE10管到针的轴。一夜之间紫外线消毒针头组件。插管针可以无限期储存在无菌培养皿。 </li></ol> 2.统一企业细菌接种准备<ol><li>准备UTI89接种（开始之前接种72小时化天） <ol><li>连胜UTI89到从冷冻股票的LB（卢里亚BERTANI）琼脂平板上。孵育过夜，在37℃。选择一个菌落，接种到10ml的LB在125毫升瓶中。在37℃下放置24小时，静态。 </li><li>接种10微升过夜培养到10ml的LB的在一个新125毫升烧瓶另外24小时，在37℃，静态。 </li><li>传输3毫升（将根据应变和所需的接种量有所不同）无菌1.5毫升管和离心在7000 XG 3分钟，悬浮颗粒在1X PBS离心第二次。重悬细菌沉淀在1ml的1×PBS。 </li></ol></li><li>测量细菌的光密度，并调整浓度至所需要的接种物密度。所用的接种物的标准浓度为10 7细菌;然而，这可能会发生变化，由于该研究的设计。 </li></ol> 3.细菌接种<ol><li>清洁用70％乙醇和c工作站在用吸水纸面积（或使用无菌流罩）。 </li><li>绘制高达0.9毫升制备的细菌接种物加到1毫升（TB）的注射器（去除气泡）。附加准备无菌接种针管PE10，到含接种注射器，无菌然后修剪聚乙烯管材。 注:保持1毫米管的针的顶端的上方，以避免刺穿囊。 </li><li>切一个1英寸的方片封口膜，并把手术润滑剂的DAB（一毛钱的大约尺寸）之上。 </li><li>通过把它们放在一个32盎司的玻璃罐内含有茶输液球泡用3毫升异氟醚或蒸发器室的棉球麻醉雌性C3H / HeN小鼠（以下生产协议），直到不省人事，但仍正常呼吸（1呼吸/秒） 。 注意:有些IACUC委员会不批准使用一个罐子或蒸发器。请按照你的机构的IACUC委员会的指示。 注:如果玻璃瓶用茶球和/或鼻锥与异氟醚soakd棉球时，动物必须要仔细观察，以避免过量麻醉而死亡。用一个蒸发器和麻醉室的优点在于，它提供受控的异氟烷施用，避免意外过量。注意:异氟醚是一种吸入麻醉剂。用在通风良好的地方，并尽量减少吸入。 </li><li>从罐子/汽化器除去小鼠并将其放置在其上的纸背巾和蔓延的腿。 </li><li>覆盖鼠标与鼻锥鼻或50毫升锥形管，用棉球含有少量（大约1-2毫升（连接到用来提供受控异氟烷剂量是配备在某些汽化器单元的蒸发器的管） ）异氟醚。 </li><li>轻轻地悸膀胱排尿诱导并确保空隙膀胱。擦拭用100％乙醇擦拭尿道周围区域。第一轻拍接种针/注射器，点，进手术润滑剂。 </li><li>通过经尿道插入接种针，约12毫米，并且压下针筒柱塞轻轻分配接种入膀胱轻轻（10微升/秒）接种每个小鼠用50μl细菌溶液。取出接种针从鼠标。 注:接种时开始接种时尿道口立即返回指示不正确或不完整的插入针头。 </li><li>从鼻锥取出鼠标，并返回到它的笼子。每只小鼠重复步骤3.3-3.8。当/如果切换接种条件/株，处理在批准的锐器容器的注射器和针头接种，并重新开始步骤3.2。 注:接种肾盂肾炎生物一般不会引起严重的疼痛症状。然而，在少数情况下，给予大剂量的病原体可能引起发热，减少食物和水的摄入量和行为异常。一个nimal健康应在整个实验进行监测。如果公开痛症状是通知，止痛剂，如丁丙诺啡（0.05-0.1毫克/千克皮下注射），都可以应用。程序应根据每个机构的IACUC。 </li></ol> 4.确定细菌负担<ol><li>准备个单独的5 mL管每个膀胱和肾脏对收获。添加的1X PBS /管/鼠标膀胱1毫升收获。添加0.8毫升1X PBS /管/双鼠标肾脏收获。标记每个管以对应于一个给定的鼠标。制备含有180微升的1×PBS中的各孔对1:10连续稀释在96孔板。 </li><li>清洁用70％乙醇的工作区，还包括与吸收性盖或纸巾的区域。 </li><li>麻醉小鼠异氟醚（见步骤3.4），直到鼠标停止呼吸约1分钟，然后将其放置在吸收盖或纸巾。 euthana的其它方法SIA还接受IACUC委员会，如二氧化碳，可被取代的异氟醚过量。 </li><li>通过快速颈椎脱位，由抑制所述颈部在头部的基部和拉动尾部水平远离身体，直到发生错位的牺牲小鼠。 注意:大多数IACUC委员会需​​要安乐死和颈椎脱位的辅助装置是一个常用的方法。然而，其它方法可用于如经IACUC委员会。 </li><li>将死老鼠在它的后面，并喷洒70％的乙醇在腹部。解剖小鼠，收获膀胱和肾脏，在为了尽量减少从血液以下肾脏解剖潜在污染。独立广场机关到准备好的5毫升管含有1x PBS（见步骤4.1）。 注:使用不同的剪刀为外部解剖和器官摘取，以减少污染。 </li><li>点击管，以确保器官在T他的1X PBS。每只小鼠重复步骤4.3-4.5。清洁剪刀和在每个小鼠后70％的乙醇钳子。 </li></ol> 5.细菌恢复<ol><li>均质收获膀胱和肾脏中5毫升管子用组织匀浆器，15秒每肾和每囊30秒。在1×PBS中，70％的乙醇和样品之间的1×PBS冲洗顺序均化。 </li><li>使串行1:10稀释出来至10 -8和板在LB琼脂平板各稀释为CFU（菌落形成单位）。孵育所述板在37℃过夜并计数菌落第二天。 </li></ol> 6. IBC枚举<ol><li>无菌取出膀胱，并将其放置在1×PBS中的6孔板用硅氧烷底部。板可使用有机硅弹性体包和浇注大约1cm硅氧烷的每个孔中来制备，见生产商的说明。切用剪刀膀胱一半。 </li><li>用小金属针轻轻的张开BLAD明镜使膀胱内腔朝上。确保将销在膀胱部分的最外边缘，以确保尿路上皮的最大量被暴露。 </li><li>张开后，轻轻用1X PBS洗一次膀胱。通过吸管取出1X PBS。通过加入足够的3％多聚甲醛，以覆盖整个叉开膀胱修复膀胱。注意:多聚甲醛有致癌性。适当的防护用品，包括手套，应该在任何时候都可以穿。处置应遵循机构准则。 </li><li>在室温下孵育1小时。删除多聚甲醛溶液。与1X PBS洗一次。 </li><li>与LacZ的洗涤液（2毫米氯化镁2，0.01％脱氧胆酸钠和0.02％诺乃-1X p40in PBS）三次，每洗5分钟，洗净。 </li><li>除去洗涤溶液，并添加足够的LacZ染色（9.5毫升LacZ的洗涤溶液中加入0.4ml 25mg / ml的X-gal的和0.1ml的100mM K-亚铁氰化物/ K - 铁氰化物），以覆盖所述囊。孵育在30℃overniGHT避光。 </li><li>从孵化器中取出水囊张开观察中型散货箱，作为与可视化解剖范围，40-60X倍率显示为蓝色的泪点。 注:有时，鼠标可能小便前尿道下管的位置。在这种情况下，等待15-20分钟尝试再次收集尿液之前。 </li></ol> 7.收集尿液的细菌尿CFU枚举（不适用于CAUTI） <ol><li>收集尿液前或后感染握住尾巴，并把它放在平坦的表面升高（鼠笼顶部）轻轻地抑制鼠标。 </li><li>保持无菌的1.5mL管中鼠标下尿道。轻轻按压在小鼠背部的尾部附近向下施加压力，以膀胱。赶上尿1.5 ml管。 </li><li>使串行1时10稀释出10-7和板每个稀释适当LB。孵育所述板在37℃过夜并计数菌落第二天。 </li></ol> 8. CAUTI示范协议，导管针准备CAUTI模型（图S2） <ol><li>卸下地下30针帽。切7mm的一块PE10管材和5mm一块硅胶管，如RenaSIL的。螺纹与PE10管针，直到管顶住针的基部。 </li><li>然后馈送5mm的片上含PE10针。 UV消毒隔夜针组件。 注:当加载硅胶管，留下大约1mm过去针尖延长管。 </li></ol> 9. E. OG1RF 粪细菌接种物制备方法<ol><li>制备OG1RF接种开始之前接种一天48小时。条纹OG1RF到从冷冻的储存在BHI（脑心脏浸液）板。 </li><li>选择一个菌落，接种于10ml BHI并在37℃成长吧静态18小时。离心培养并在1ml体积无菌的1×PBS的洗涤沉淀（3次）。 </li><li>悬浮细菌沉淀到1X PBS。测量细菌光密度和浓度调整到所需的接种量。所用的接种物的标准浓度为10 7;然而，这可能会发生变化，由于该研究的设计</li></ol> 10.导管植入<ol><li>清洁用70％乙醇的工作站，并盖上一个吸收覆盖的区域（或使用无菌流罩）。 </li><li>连接导管针到空的1毫升（TB）的注射器。切一个1英寸的方片封口膜，并把手术润滑剂的DAB（一毛钱的大约尺寸）之上。 注:两种不同的针被用于在导管的沉积和对接种到最小偶然接种由于所需导管植入所述机械操纵。这还允许制备较少接种针的便利性。 </li><li>通过把它们放在一个32盎司的玻璃罐内含有茶麻醉C57BL / 6小鼠-infuser球浸在3毫升异氟醚或蒸发器室的棉球（以下生产协议），直到不省人事，但仍正常呼吸（1呼吸/秒）。 </li><li>然后把鼠标放在其在纸巾上，并回传到腿部。覆盖鼠标与鼻锥或50ml锥形管与含有少量（大约1-2毫升）的异氟烷棉球的鼻子。 注意:异氟醚是一种吸入麻醉剂。用在通风良好的地方，并尽量减少研究员吸入。 </li><li>轻轻地悸膀胱排尿诱导并确保空隙膀胱。擦拭尿道周围区，配有100％的乙醇擦拭。 </li><li>消毒与使用棉尖涂药10％聚维酮碘溶液尿道周围区域。第一轻拍接种针/注射器，点，进入手术润滑剂。 注:Povidine碘被用于导管植入，这是比醇更强无菌溶液。导管植入需要更多的机械操纵插入导管，从而污染更大的潜力。 </li><li>将导管插入尿道口。一旦进入，使用镊子推（7毫米PE10）长片向膀胱，从而推动（5毫摩硅）小导管并沉积到膀胱。取出针，用7毫米的管线还连着，立即执行。 </li></ol> 11.细菌接种<ol><li>按照细菌接种协议第3条，用准备好的接种从9节从鼻锥取出鼠标，并返回到它的笼子</li></ol> 12.在牺牲的每一个时间点<ol><li>准备5毫升管膀胱和肾脏（对于机关按照步骤4.1）和1.5毫升离心管的导管。加入1毫升1×PBS到1.5毫升Eppendorf管用于导管样本</li><li>按照步骤4.3至4.5。解剖鼠标，收获膀胱，肾脏和导管，解放军独立地CING组织成含有1x PBS中，导管进入1.5毫升的Eppendorf（见步骤12.1）5毫升管中。 注:使用不同的剪刀为外部解剖和器官摘取和导管恢复，以减少污染</li><li>然后按照步骤4.6。 </li></ol> 13.细菌恢复<ol><li>对于机关，按照步骤5.1和5.2。细菌回收从导管，涡旋以最大速度为30秒，超声处理导管样品用5分钟的水浴超声，然后涡旋以最大速度进行另外30秒。 </li><li>使串行1:10稀释出10-8和板每个稀释度对BHI板CFU枚举。孵育所述板在37ºC过夜并计数菌落第二天。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

单纯性社区获得性尿路感染是一种常见的和昂贵的感染占每年46几万元的初级保健访问。此外，CAUTIs是一种常见的医疗获得性感染，已成为极其昂贵的医疗保健提供商医疗保险和医疗补助服务中心不再报销提供商用于治疗从医院获得45 CAUTI会带来额外的成本。 UTI，既单纯性膀胱炎和CAUTI的小鼠模型中，在这些协议中描述提供了宝贵的工具用于理解所必需的启动，维持和调节的几种形式的UTI的结果的宿主和病原体相互作用的分子基础。这些模型允许强大的细菌和小鼠遗传学，免疫学，微观，生物化学和化学遗传工具的应用UTI 23,47-49分析。此外，该协议此处概述提供了一个平台为发展和新的治疗和预防策略的测试，包括细菌的毒力的小分子抑制剂和新颖的疫苗接种目标和策略50-55。 有迹象表明，必须保持对单纯性膀胱炎模型的成功应用几个关键条件。如同所有的鼠标麻醉程序，必须小心，以防止小鼠的死亡率由于过度麻醉。当制备接种导管，硅胶管必须足够长，以覆盖所述针的尖端，但没有限制，只要破坏上导尿膀胱壁。另外，由于阴道口的附近和大小，必须小心以插入接种导管插入尿道，而不是阴道。在膀胱张开对于IBC枚举，至关重要的是，囊尽可能快张开尽可能以下解剖和组织进行固定的Approximately 1小时，但不可能一蹴而就。对于IBC枚举通过的LacZ染色，还需要有问题的细菌中的尿路上皮细胞定居产生的LacZ。当使用其中的LacZ酶不产生应变，它仍然是可能的使用免疫荧光显微镜用抗大肠杆菌中型散货枚举大肠杆菌抗体或GFP表达细菌。这种模型的一个特定特征是疾病进展和严重程度由不同近交小鼠品系呈现的不同的图案和原型UPEC菌株33,56的变化的致病潜力。这些差异都是一个实力和这种技术的限制，他们概括主机易感性和病原体的多样性已在临床和表型研究中观察到的，而复杂的实验设计和解释57,58的自然变异。这些变化提供了比较，允许主机和细菌mechan的阐发来源调节主义尿路感染并发症的33,56的发病机制。不过，他们也表示，由于鼠标线尿路感染的易感性不同程度，最大限度必须注意选择正确的鼠标线和统一企业的应变为解决特定问题的调查员询问。例如，C57BL / 6小鼠是急性感染的优良模型，具有高细菌滴度和大量IBC的早期时间点呈现。这株然后迅速解决与严重的肾脏感染和细胞内的静态储33,34产生的唯一持久定植3和7之间DPI感染。在C57BL / 6模型可以准确地代表最常见的结局对于许多病人尿路感染中出现，然后症状缓解。相反，C3H /纯鼠标背景表现出高度的敏感性，以慢性膀胱炎使他们的理想株为研究复发性尿路感染。其他UTI车型也已经确立在CBA，DBA和C3H / HeJ小鼠的背景证明为研究UTI 33,59的特定方面非常有用。初级实验缺点由单纯性膀胱炎模型显示出是多重感染的瓶颈限制的大规模基因筛选方法的应用所必需的急性，慢性和复发性膀胱炎60新的细菌因子的发现的存在。 关键的考虑因素为单纯性膀胱炎模型中的所有适用于有两个额外的关注植入基于模型CAUTI。第一，必须小心，以确保植入物的插入发生在一个无菌的方式，由于增加的易感性膀胱感染由植入物所赋予的。第二，它是必不可少的导管可用来代替除去植入针的期间保持。该CAUTI模型的主要限制是，它具有降低的范围内使用的主机遗传背景中，公关opensity用于导管损失随着时间的推移和无法收集尿液由于膀胱功能障碍由注入所产生的。为了补偿种植体损失和无法监测经过纵向尿液的感染，这种模式通常需要更高数目的插管小鼠购买的时间点和小鼠组的，必须在每个期望的时间点被牺牲掉。从禁止频率在其中一些小鼠品系，如C3H / HeN系，失去植入有限宿主遗传背景的结果（第Guiton，个人通信，2010）。其结果是，上述的CAUTI协议已经完全被优化在C57BL / 6小鼠品系。然而，在现有小鼠品系的CAUTI的限制是不幸的，许多基因变化，已经确立了在C57BL / 6背景和CAUTI模型通过改变膀胱环境被生物体便于殖民扩张过程中尿路感染细菌多样性可供研究，是否则非致病性在尿路感染的小鼠模型。这样的一个例子，是最近发现纤维蛋白原的期间膀胱在大肠杆菌导管诱发的炎症释放的作用CAUTI 42 粪中生长，生物膜的形成和持续性。本实施例说明在阐明不仅细菌分子发病机理也是宿主因子的感染过程中的作用CAUTI的植入物模型的功率。所有这些特性有助于理解这个相关临床感染，先前已经充分研究。 除了以上所述的实验方法，单纯性膀胱炎和CAUTI的小鼠模型是适合于大量的方法修改。在单纯性膀胱炎模型的情况下，这些修改可以包括减少接种量和接种物的滴注期间施加的力，以减少细菌数引入到肾脏61，不同的感染的持续时间，在时间点牺牲早1 HPI和使用庆大霉素保护测定，以评估在何种程度上的uropathogen已经侵入膀胱上皮细胞62。从小鼠中被显示出来气囊处死6至24 HPI也可切片并用抗体与各种主机和/或细菌的表位，并在荧光或共聚焦显微镜观察，观察IBC结构，细菌助熔和成丝和宿主组织状况25,33 ，63,64。 uropathogen感染和/或植入在主机的局部和全身免疫反应的影响，可以使用的BioPlex式微球系统或基于ELISA的测定法来确定在尿或血清分别33,65的细胞因子水平进行评估。最后，膀胱组织，位置和定植细菌的形态和生物膜的结构的条件沉积在导管可通过各种成像技术，包括电子显微镜25,41进行评估。 感染的这些基本模型也可以适用于模仿其他临床相关现象，包括复发性尿路感染和经由多重感染获得性免疫应答的发展。在此背景下，重复C57BL / 6小鼠感染导致阻力增大，以急性膀胱炎，而在C3H /女性模型，慢性膀胱炎其次是感染的间隙通过抗菌药物的管理的优先发展敏感性这些小鼠发展为慢性膀胱炎在随后的接种66。另外的风险因素尿路感染发展的分子机制可以检查。糖尿病和肥胖都被证明能增加尿路感染的频率和严重程度。 2型糖尿病可以遗传模型中的小鼠在FVB / NJ背景和化学诱导的1型糖尿病的模型已经alreadÝ被证明能增加尿道感染引起的两个UPEC和K.严重性肺 67,68。 最后，这些模型系统代表一个理想的试验场治疗方式旨在治疗或预防泌尿道感染和CAUTIs的开发和优化。通过这些模型提供的主机病原体相互作用的分子机制已经促进了有效的疫苗接种战略，旨在针对重要的致病因子51-55发展。此外，UPEC粘附的小分子抑制剂已被证明是有效的在无并发症的急性和慢性尿路感染的治疗以及CAUTIs引起UPEC 12,69,70。无论是在这些协议中描述的简单的UTI和CAUTI模型提供了实验工具解剖在尿路宿主 - 病原体相互作用的分子细节。这些工具可以在很多方面扩大我们的相互理解加以利用克这样的络合组条件并促进更有效的治疗干预的发展。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52892/establishment-and-characterization-of-uti-and-cauti-in-a-mouse-model">Establishment and Characterization of UTI and CAUTI in a Mouse Model</a>. The Protocol section has been updated.
The ethics statement has been updated from:
Ethics statement: The Washington University Animal Studies Committee approved all mouse infections and procedures as part of protocol number 20120216, which was approved 01/11/2013 and expires 01/11/2016. Overall care of the animals was consistent with The guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Euthanasia procedures are consistent with the &#8220;AVMA guidelines for the Euthanasia of Animals 2013 edition.&#8221;
to:
Ethics statement: The Washington University Animal Studies Committee approved all mouse infections and procedures as part of protocol number 20150226, which expires 12/10/2018. Overall care of the animals was consistent with The Guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Euthanasia procedures are consistent with the &#8220;AVMA guidelines for the Euthanasia of Animals 2013 edition.&#8221;
Step 3 has been updated from:
3. Bacterial Inoculation
<ol>
	<li>Clean the workstation with 70% ethanol and cover area with absorbent paper (or use sterile flow hood).</li>
	<li>Draw up to 0.9 ml of the prepared bacterial inoculum into a 1 ml (TB) syringe (remove air bubbles). Attach a prepared sterile inoculation needle with PE10 tubing, onto the syringe containing the inoculum, then sterilely trim the polyethylene tubing. 
	NOTE: Leave 1 mm of tubing above the tip of the needle to avoid puncturing the bladder.</li>
	<li>Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.</li>
	<li>Anesthetize female C3H/HeN mice by putting them in a 32 ounce glass jar containing a tea-infuser ball with cotton balls soaked with 3 ml of isoflurane or vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec). 
	NOTE: Some IACUC committees do not approve the use of a jar or vaporizer. Please follow the indication of the IACUC committee of your institution. 
	NOTE: If glass jar with tea-ball and/or nose cone with isoflurane-soakd cotton balls are used, the animal must be carefully observed to avoid anesthetic overdose and death. The advantage of using a vaporizer and anesthesia chamber is that it provides controlled isoflurane administration that avoids accidental overdoses. CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation.</li>
	<li>Remove the mouse from the jar/vaporizer and place it on its back on a paper towel and spread the legs.</li>
	<li>Cover the nose of the mouse with a nose cone (a tube connected to the vaporizer that provides a controlled isoflurance dose that comes equipped on some vaporizer units) or 50 ml conical tube with a cotton ball containing a small amount (approximately 1-2 ml) of isoflurane.</li>
	<li>Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with 100% ethanol wipe. Dab the inoculation needle/syringe, point first, into the surgical lubricant.</li>
	<li>Inoculate each mouse with 50 &#181;l of the bacteria solution by inserting the inoculation needle transurethrally, approximately 12 mm, and pressing down on the syringe plunger gently to dispense the inoculum into the bladder gently (10 &#181;l/sec). Remove the inoculation needle from the mouse. 
	NOTE: Immediate return of inoculum at the urethral opening when beginning to inoculate indicates improper or incomplete insertion of the needle.</li>
	<li>Remove the mouse from nose cone and return it to its cage. Repeat steps 3.3-3.8 for each mouse. When/if switching inoculum conditions/strains, dispose of the syringe and inoculation needle in an approved sharps container and start again step 3.2.&#160;&#160; 
	NOTE: Inoculation with uropathogenic organisms generally does not cause severe pain symptoms. However, in rare instances, administering large doses of pathogens may cause fever, reduction in food and water intake and abnormal behavior. Animal health should be monitored throughout the experiment. If overt pain symptoms are notice, an analgesic, such as Buprenorphine (0.05&#8722;0.1 mg/kg given subcutaneously), can be applied. Procedures should be in accordance with each institution&#8217;s IACUC.</li>
</ol>
to:
3. Bacterial Inoculation
<ol>
	<li>Clean the workstation with 70% ethanol and cover area with absorbent paper (or use sterile flow hood).</li>
	<li>Draw up to 0.9 ml of the prepared bacterial inoculum into a 1 ml (TB) syringe (remove air bubbles). Attach a prepared sterile inoculation needle with PE10 tubing, onto the syringe containing the inoculum, then sterilely trim the polyethylene tubing. 
	NOTE: Leave 1 mm of tubing above the tip of the needle to avoid puncturing the bladder.</li>
	<li>Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.</li>
	<li>Anesthetize female C3H/HeN mice by putting them in a vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec). 
	NOTE: Some IACUC committees do not approve the use of a vaporizer. Please follow the indication of the IACUC committee of your institution. 
	CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation.</li>
	<li>Remove the mouse from the vaporizer and place it on its back on a paper towel and spread the legs.</li>
	<li>Cover the nose of the mouse with a nose cone (a tube connected to the vaporizer that provides a controlled isoflurance dose that comes equipped on some vaporizer units) to maintain anesthetization.</li>
	<li>Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with 100% ethanol wipe. Dab the inoculation needle/syringe, point first, into the surgical lubricant.</li>
	<li>Inoculate each mouse with 50 &#181;l of the bacteria solution by inserting the inoculation needle transurethrally, approximately 12 mm, and pressing down on the syringe plunger gently to dispense the inoculum into the bladder gently (10 &#181;l/sec). Remove the inoculation needle from the mouse. 
	NOTE: Immediate return of inoculum at the urethral opening when beginning to inoculate indicates improper or incomplete insertion of the needle.</li>
	<li>Remove the mouse from nose cone and return it to its cage. Repeat steps 3.3-3.8 for each mouse. When/if switching inoculum conditions/strains, dispose of the syringe and inoculation needle in an approved sharps container and start again step 3.2.&#160;&#160; 
	NOTE: Inoculation with uropathogenic organisms generally does not cause severe pain symptoms. However, in rare instances, administering large doses of pathogens may cause fever, reduction in food and water intake and abnormal behavior. Animal health should be monitored throughout the experiment. If overt pain symptoms are notice, an analgesic, such as Buprenorphine (0.05&#8722;0.1 mg/kg given subcutaneously), can be applied. Procedures should be in accordance with each institution&#8217;s IACUC.</li>
</ol>

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52892/establishment-and-characterization-of-uti-and-cauti-in-a-mouse-model">Establishment and Characterization of UTI and CAUTI in a Mouse Model</a>. The Protocol section has been updated.

Erratum: Establishment and Characterization of UTI and CAUTI in a Mouse Model

尿路感染（尿路感染）是最常见的细菌感染中的一个，并可以分为基于采集，社区和医院获得性泌尿道感染的机制两类。社区获得性尿路感染常发生在其他方面健康的妇女，研究显示妇女大约50％将具有至少一个UTI在其一生中1。此外，复发是一个重大问题。谁拥有一个初始急性感染的妇女，尽管有适当的抗生素治疗，许多妇女继续有反复发作2具有半年内第二次感染的25-40％的机会。引起这类感染的细菌也日益抗生素有抗药性的进一步混杂的治疗方案3-6。每年UTI影响数以百万计的个人耗资约2.5十亿美元的医疗保健相关支出在美国，强调了疾病的影响和流行1,7 .Nosocomial收购尿路感染主要与外国机构，如留置导尿管的存在有关。导管相关尿路感染（CAUTI）仍然获得尿路感染最常见的院内，占〜这种感染8的70-80％。此外，CAUTI与增加的发病率和死亡率相关联，并且它是次要血流感染9的最常见原因。 尿路感染收购相关的统一企业社会被认为是由通过机械操纵引进细菌进入膀胱从水库在胃肠道性交，卫生条件差或不同主机之间的其它微生物种群动态过程中造成的生态位10。一旦膀胱内，UPEC雇用许多毒力因子，包括胶囊，铁采集系统，毒素，毒力质粒的tRNA，致病岛和已被证明在发病中发挥作用定居因子&lt;SUP&gt; 11-14。关键要建立UPEC殖民化，统一企业也编码多种类型的胶粘剂伴侣迎来通路（CUP）菌毛识别受体的特异性立体15。 1型菌毛，放倒与FimH粘附，是由统一企业表达并结合甘露糖基uroplakins 16和α-1，β-3整联17，其被表达在人和小鼠囊18的腔表面。这些FimH介导的互动有利于细菌的定植和入侵的浅表上皮细胞19,20。一旦进入细胞，UPEC可以逃逸到细胞质其中单个细菌可以快速分裂，以形成细胞内细菌群落（IBC），其在成熟化，可以含有约10 4细菌21。 IBC形成已被证明在至少六个不同的小鼠品系，C3H / HeN系，C3H / HeJ小鼠，C57BL / 6，CBA，FVB / NJ和BALB / c，和与多种不同UPE的ç株和其他肠杆菌科22-24。然而IBC形成时空差异可以根据鼠标背景和感染UPEC应变而变化。在C3H /感染的典型UPEC HeN小鼠品系UTI89或CFT073，IBC形成可以早在3 HPI（小时后感染）可视化细菌的生物量小。该社区的不断扩大和达到的发展约6 HPI一个"中点"当棒状细菌占据终末分化肤浅伞细胞的细胞质空间的大百分比这些早期中型散货形式与显示类似的尺寸多数相对同步的方式和形态。 〜8 HPI的细菌从一个杆菌IBC变化球菌形态。中型散货箱是一过性的。因此，从12-18 HPI导致细菌群体的不断扩大IBC成熟，其次是他们的成丝和扩散出细胞的Wi随后日蔓延到相邻小区23。因此，IBC利基允许快速的细菌生长在从宿主免疫应答和抗生素25保护的环境。被UPEC感染所看到小鼠不同的阶段也观察到在人中，如中型散货和成丝，支持尿路感染的小鼠模型，可用于模拟UTI在人类22,26-28一个有益的工具。 虽然大多数女性体验UTI在其一生中，这些感染的结果可以从急性自限性感染无复发，频繁复发膀胱炎。此外，研究表明UTI强烈家族性发生，这表明遗传成分有助于UTI易感性29。我们已发现，看到在诊所的不同的UTI结果可以通过实验UPEC感染之间的近交小鼠品系30的不同的结果进行镜像。例如，C3H /母鸡，CBA，DBA和C3H / HeOuJ小鼠易感，在感染性剂量依赖性方式，以持久，慢性膀胱炎特征在于持久性，高滴度菌（&gt; 10 4集落形成单位（CFU）/ ml）的高滴度细菌膀胱负担在牺牲&gt;后4周感染（WPI），慢性炎症，和尿路上皮坏死。这些小鼠也显示，IL-6，G-CSF，KC，和IL-5的前24 HPI内升高的血清水平是作为生物标志物用于治疗慢性膀胱炎的发展。这可准确地表示UTI的自然过程在一些妇女，作为安慰剂的研究表明，妇女经历后膀胱炎的最初症状的UTI将保留在他们的尿高水平细菌数周的大百分比，如果不给予抗生素治疗31 32。此外，使用C3H / HeN小鼠中，我们发现，慢性膀胱炎的历史是用于随后的严重反复感染一个显著危险因素。复发性尿路感染是最SIUTI和C3H /纯鼠标gnificant临床表现是目前唯一研究模式，概括先前曝光后增加的倾向。第二UTI结局概括在C57BL / 6小鼠急性那里感染统一企业是自限性，与膀胱炎和菌大约在一个星期内解决。有趣的是，在这种模式下，UPEC容易形成内膀胱组织静止的细胞内储从中UPEC能够从休眠状态出现重新发起活性UTI，可能解释一种机制用于 ​​同一菌株复发性尿路感染在人中33,34。 除了上尿路感染易感性的遗传影响，引入导管插入膀胱大大增加具有感染以及增加的细菌能够引起感染的范围的可能性。已经证实，人类导尿引起组织学和由于机械应力，结果在一个健壮的炎症反应，剥脱，固有层和submucusa，尿路上皮变薄的水肿，尿路上皮和肾脏35,36的粘膜损伤的免疫学变化膀胱。此外，该导管提供用于细菌附着从而产生由几个物种用来使CAUTI的环境中的表面。而统一企业仍然是一个主要因素， 粪肠球菌占这些CAUTI 37的15％。E.粪变得对抗生素耐药越来越具有耐万古霉素的出现，构成了严重的健康问题38。E.肠球菌具有许多毒力因子，包括毒素和必要的附件二者导管和上皮38粘附。期间导尿，宿主是易受微生物附着，繁殖和传播，在泌尿道39,40。E. faeca利斯形成在导管生物膜作为一个机构的一部分，以坚持在膀胱和传播对肾脏，这是再生在小鼠CAUTI模型41。最近，已经在导尿所示，纤维蛋白原（纤维蛋白原）被释放到膀胱的炎症反应的一部分。蛋白原积聚在膀胱，大衣的导管，是必需成分为E.粪肠球菌生物膜的形成，运作作为附件支架。在CAUTI的C57BL / 6小鼠模型中，我们发现， 大肠杆菌肠球菌生物膜形成的导管，并因此持久性膀胱，是依赖于EBP菌毛 ​​，特别是其前端粘附EBPA。我们发现，EBPA的N-末端结构域特异性结合FG涂覆导管。此外，人们发现， 大肠杆菌利用粪作为纤维蛋白原期间感染源 ​​代谢产物，从而提高生物膜形成42。 小鼠模型已经证明关键understanding以及预测和UTI 41 CAUTI临床表现。在这篇文章中，我们证明接种物制备的膀胱炎UPEC的隔离UTI89和经尿道接种C3H / HeN小鼠的。此外，我们展示了一个协议，用于导管插入大肠杆菌的C57BL / 6小鼠和接种OG1RF 粪应变。这两种技术导致一致的和可靠的UTI或CAUTI小鼠。我们还显示用于急性膀胱炎和尿液收集用于治疗慢性或复发性膀胱炎的分析，观察IBC形成技术。 C3H / HeN小鼠已被用于研究统一企业发病机制方面，包括最初的细菌的入侵，形成IBC，成丝和慢性膀胱炎23,33,43的发展。这些毒力参数也已研究了在各种其他小鼠背景22,33。对于CAUTI的C57BL / 6模型允许异物植入，随后细菌共同膀胱电离，从而可以维持7天感染后41。这些模型已经用于评估细菌毒力机制，宿主对尿路感染和机制，以颠覆宿主反应，其中大部分已被随后概括或临床人群中观察到的有用。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material for catheter and needle preparation:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30ga needles</td>
			<td>BD Precision Glide</td>
			<td>305106</td>
			<td>30Gx &#189; (0.3 mm x 13mm)</td>
		</tr>
		<tr>
			<td>PE10 polyethylene tubing</td>
			<td>BD</td>
			<td>427400</td>
			<td>Inside diameter -0.011 in (0.28 mm); outside diameter &#8211; 0.024 in (0.61 mm)</td>
		</tr>
		<tr>
			<td>RenaSIL 025 platinum cured silicon tubing</td>
			<td>Braintree Scientific, Inc</td>
			<td>SIL 025</td>
			<td>inside diameter-0.012 x outside diameter 0.025, 25 ft coil</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material for infections:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflourane &#8211; Isothesia</td>
			<td>Butler Schein</td>
			<td>29405</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Clear Glass Straight-Sided Jar</td>
			<td>Kimble Chase</td>
			<td>5413289V 21</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Tea Infuser</td>
			<td>Schefs-Amazon</td>
			<td></td>
			<td>Premium Loose Leaf Tea Infuser By Schefs - Stainless Steel - Large Capacity -</td>
		</tr>
		<tr>
			<td>Non-sterile cotton balls</td>
			<td>Fisherbrand</td>
			<td>22-456-880</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Falcon tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotec 3 -vaporizer</td>
			<td>Ohmeda</td>
			<td>1224478</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear punch</td>
			<td>Fisher Scientific</td>
			<td>13-812-201</td>
			<td>(when necessary)</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Betadine solution</td>
			<td></td>
			<td>10% Povidie-iodine topical solution</td>
		</tr>
		<tr>
			<td>Q-tips</td>
			<td>Fisher Scientific</td>
			<td>22-037-924</td>
			<td>6 in</td>
		</tr>
		<tr>
			<td>Diapers for bench</td>
			<td>Fisherbrand</td>
			<td>14206 63</td>
			<td>Absorbent Underpads (20&#8221;X36&#8221;mats)</td>
		</tr>
		<tr>
			<td>Surgical lubricant</td>
			<td>Surgilube</td>
			<td>0281-0205-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor</td>
			<td>Fine Science tools, INC</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine Science tools, INC</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>Tuberculin slip tip</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td>4 in x 125 FT</td>
		</tr>
		<tr>
			<td>Eppendorf rack</td>
			<td>Fisherbrand</td>
			<td>05-541-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>MIDSCI</td>
			<td>AVX-T-17-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Harvesting catheters, bladders and kidneys:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>PRO Scientific INC</td>
			<td>Bio-Gen Pro 200</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polypropylene round-bottom tube</td>
			<td>BD</td>
			<td>352063</td>
			<td>for organ homogenization</td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td>Georgia-Pacific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-AAPER</td>
			<td>11100020S</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Costar&#8482; Clear Polystyrene 96-Well Plates</td>
			<td>Corning</td>
			<td>3788</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phosphate sodium saline</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>BRANSONIC Ultrasonic cleaner 1210</td>
			<td>Branson Ultrasonics Corporation</td>
			<td>1210</td>
			<td></td>
		</tr>
		<tr>
			<td>IBC materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture test plate</td>
			<td>Techno Plastic Products</td>
			<td>92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside)</td>
			<td>Invitrogen</td>
			<td>15520-034</td>
			<td>Ultrapure</td>
		</tr>
		<tr>
			<td>N, N-Dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 (Magnesium chloride)</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet-P40</td>
			<td>Roche</td>
			<td>11754599001</td>
			<td>Octylphenolpoly(ethyleneglycolether)n</td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(II) trihydrate (K-ferrOcyanide)</td>
			<td>Sigma Aldrich</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(III) (K-ferrIcyanide)</td>
			<td>Sigma Aldrich</td>
			<td>60299</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment and characterization of uti and cauti in a mouse model

尿路感染（UTI）是非常普遍的，发病的一个显著原因，并且是对治疗日益耐抗生素。女性不成比例地被折磨UTI:所有妇女的50％将在其一生中的UTI。此外，这些妇女谁有一个初步的UTI将遭受复发与一些患有复发频繁，在生活，疼痛和不适，日常活动中断的质量严重恶化的20-40％，医疗费用增加，以及一些治疗方案等较长期抗生素预防。尿路致病性大肠杆菌 （统一企业）是社区获得性尿路感染的主要病原体。导管相关UTI（CAUTI）是最常见的医院获得性感染占一百万出现在每年美国和戏剧性的医疗费用。而统一企业也是CAUTI的首要原因，其他病原体是增加的意义，包括肠球菌粪。在这里，我们利用这一概括许多这些人类疾病的临床特点两个成熟的小鼠模型。对于尿路感染，一个C3H /纯模式概括了许多致病统一企业在包括人类宿主反应，IBC的形成和丝状观察到的特征。对于CAUTI，使用C57BL / 6小鼠，其保留导管膀胱植入一个模型，已被证明是容易受到与粪膀胱感染。这些代表车型被用来获得惊人的新的见解尿路感染的发病机制，这是导致新的治疗和管理或预防战略。

简单和导管相关UTI的膀胱内的模型提供了灵活的平台，阐明细菌发病机理，宿主组织上这些疾病的影响，以及发展和新的方法测试的分子机制来管理这些常见和昂贵的感染。根据小鼠品系和病原体，膀胱内接种可用于研究宿主-病原体相互作用阐明必要用于发起或调节急性因素（ 图1和3），慢性或复发性（ 图2）膀胱炎。在图1中所示的数据是代表急性期（24 HPI）尿路定植原型UPEC膀胱炎的隔离UTI89 44。接种后，将感染任其发展24小时处死小鼠在该点与膀胱和肾组织匀浆。该组织匀浆是连续dilut埃德和镀细菌定植的枚举。慢性统一企业定植和膀胱炎可以建立并保持在感染剂量和uropathogen依赖性一些鼠标线（特别是C3H /女性）。慢性膀胱炎被定义为持续性的高滴度菌（&gt; 10 4 CFU / ml）的，膀胱炎和高滴度细菌膀胱负担（&gt; 10 4 CFU /膀胱）在牺牲33。在图2所示的数据是C3H / HeN小鼠的接种2×10 7 CFU的UTI89的后发生4周代表的CFUs。在这些实验条件下，受感染的小鼠的20-50％发展成慢性膀胱炎，而小鼠的余下50-80％解决感染。结果这对偶是依赖于在第一24 HPI内决定的宿主 - 病原体的检查点。在细菌滴度的观察双峰分布，开始体现在检查站的结果激活（或不）尿在3 dpi的（天感染后），并在膀胱在处死28 dpi的（ 图2）33。小鼠慢性膀胱炎病史的挑战时都显著倾向于反复发作的慢性膀胱炎后，初次感染被清除与抗生素治疗33。同样，UTI的历史是在人类复发性尿路感染的一个已知危险因素。因此，这是用于研究复发性尿路感染的发病机制中的唯一已知模型。当实验建模UTI在替代鼠标背景，它以确定该小鼠品系易受UTI，复发性尿路感染或CAUTI，因为某些菌株是或多或少耐显影这些综合症33是重要的。 急性膀胱炎的附加测量是IBC枚举。 IBC形成仅限于终末分化肤浅伞细胞。 IBC量化是一个内容丰富的措施细菌负担急性囊肿炎和高水平IBC的关联与发展为慢性膀胱炎21的风险。 IBC的形成只发生在疾病的急性阶段。以下浅表伞细胞的剥离，中型散货不再观察到。在C3H / HeN系UTI模型，中型散货可测6小时接种的UTI89〜1×10 7 CFU进入膀胱经尿道后。气囊张开与金属销上硅板和染色LacZ基因的表达。 LacZ基因，β半乳糖苷酶，是一种常用表达细菌酶能裂解半乳糖和其他糖之间的β链。这种酶可用于通过提供X-gal的，β型半乳糖苷类似物，其在切割时产生兰色，以检测细菌。染色后，该气囊在解剖显微镜用中型散货出现作为尿路上皮和中型散货punctae蓝色/紫色的点下成像的通过计数的点（ 图3A）被例举。尽管我的数每只动物形成的BC而变化，在C3H / HeN系背景的平均〜50中型散货每膀胱（ 图3B）被看见。中型散货增加以剂量依赖性方​​式。形成IBC的数目UPEC和鼠标背景品系之间变化。例如，在C57BL / 6，〜1×10 7 UTI89结果中的每膀胱几百中型散货形成的种菌。 除了收购UTI的简单社会仿照以上，相关健康保健尿路感染代表显著负担的卫生保健系统。尿路感染占所有卫生保健相关感染的约40％，最高达到95％，所有相关的医疗保健尿路感染是相关的45导尿管。在这里，我们证明CAUTI的膀胱植入小鼠模型，代表一个强大的系统为这个困难的临床问题的实验分析。在图4中呈现的数据代表了24小时的感染与模型E.粪 s<html>训练OG1RF。当1×10 7 OG1RF CFU的是膀胱内接种到未注入膀胱，感染开始明确由24 HPI（ 图4A）和由3 dpi的感染已解决41。相反，接种OG1RF成植入膀胱结果在这两个导管和膀胱的定植，用10倍的更高的膀胱定植水平比在未注入气囊观察24 HPI（ 图4A和4B）。此外，这种感染保持在&gt; 10 6 CFU在膀胱直到导管损失约7 dpi的41。所有描述的实验模型是适合于实验的高度灵活性，包括但不排他，以各种方法对感染的组织和表面的宏观和微观成像，改变的接种物的大小和疾病倍，竞争性感染，和主机免疫分析。 ve_content"&gt; <img alt="图1" src="/files/ftp_upload/52892/52892fig1.jpg" /> 图1:24小时尿路定植女，7-8周龄C3H / HeN小鼠进行经尿道接种原型UPEC膀胱炎〜2×10 7 CFU隔离UTI89 24小时。从收获的膀胱，肾组织回收代表性的CFUs示中，n = 5，虚线表示检测（20 CFU）的下限。 <img alt="图2" src="/files/ftp_upload/52892/52892fig2.jpg" /> 图2:28天尿路定植女，7-8周龄C3H / HeN小鼠进行经尿道接种〜2×10 7 CFU UTI89。尿液收集在指定天感染后（DPI）和镀CFU枚举。代表28天膀胱和肾的滴度也显示，正的检测= 8尿CFU下限（1,000 CFU / ml）的被表示为虚线。虚线represents的子组织检测下限，20 CFU /组织。 <img alt="图3" src="/files/ftp_upload/52892/52892fig3.jpg" /> 图3:IBC枚举女，7-8周龄C3H / HeN小鼠进行经尿道接种〜1×10 7 CFU UTI89。膀胱收获6 HPI和张开的IBC染色。的LacZ染色后张开膀胱（A）图片。中型散装容器表示为蓝点。的插图图像是数字图像中的黑盒的部分的放大。 （ 二）形成C3H / HeN小鼠6 HPI IBC的代表数，N = 10。 <img alt="图4" src="/files/ftp_upload/52892/52892fig4.jpg" /> 图4: 大肠杆菌肠球菌导管相关UTI。C57BL / 6小鼠接种有或无导管与的〜1×10 7 CFU <em&gt;电子。粪应变OG1RF 24小时。 （A）E.粪中的存在或不存在导管的膀胱定植。 （B）的 大肠杆菌粪导管定植。曼-惠特尼U检验用于小鼠实验，P &lt;0.05为差异有统计学显著。 **，P &lt;0.005。 <img alt="图S1" src="/files/ftp_upload/52892/52892figS1.jpg" /> 图S1:UTI接种针制备图显示接种针制备步骤，包括（1）需要，就到了纫针导管的材料（2）和接种前修整管（3）。 <img alt="图S2" src="/files/ftp_upload/52892/52892figS2.jpg" /> 图S2:CAUTI接种针制剂图显示需要（1），线程接种导管针接种准备材料（2）和植入导管上到针（3）。

Watch this Scientific Journal Video about Establishment and Characterization of UTI and CAUTI in a Mouse Model at JoVE.com

Establishment and Characterization of UTI and CAUTI in a Mouse Model

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

establishment-and-characterization-of-uti-and-cauti-in-a-mouse-model

Nanyang Technological University

Research

JoVE Journal

Medicine

19.9K 次观看.  Washington University School of Medicine. The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work’s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

视频: Establishment and Characterization of UTI and CAUTI in a Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

建立小鼠重症急性胰腺炎模型，将牛磺胆酸钠逆行注射到胆胰管中

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.