这些研究经费，一部分，由波士顿儿童医院泌尿外科捐赠收入基金和国家机构的医疗补助NIBIB P41-EB002520（卡普兰）; NIDDK的T32-DK60442（弗里曼）; NIDDK 1K99-DK083616（Mauney）。我们承认，从佛蒙特大学建立廔管安置和膀胱的技术援助医生彼得Zvara。

手术方法 1。术前准备和麻醉<ol><li>设立必要的手术器械无菌手术领域：剃剪，牙钳，细无创伤钳，细针驱动器，纱布，梅岑鲍姆剪刀，切断术剪刀，手术刀刀片，30号注射器针头，盐水填充1毫升注射器，四6-0聚丙烯缝线polyglactin缝合，7-0，4-0 polyglactin缝合。 </li><li>动物麻醉与异氟醚吸入诱导室。转移到手术领域之前，确认完整的动物感应。确保在适当的位置，有连续的麻醉吸入麻醉管。 </li><li>将动物仰卧在无菌的悬垂性。 [膀胱分析，请参阅下面的一段隧道廔导管。] </li><li>使用剃须剪删除从下腹部的皮毛。 </li><li>准备的是腹部tadine和70％的乙醇。 </li><li>前切口，如丁丙诺啡（0.05-0.1毫克/公斤）镇痛可皮下注射围手术期疼痛控制。 </li></ol> 2。膀胱切口和曝光<ol><li>做一个1-2厘米（动物的大小，取决于是否大鼠或小鼠）较低正中切口，通过皮肤的手术刀。深化在较低的部分通过的直肌采取小心，不要伤害底层肠或膀胱肌肉切口的切口。 </li><li>用齿镊，提升直肌和免费后表面的肌肉解剖与细梅岑鲍姆剪刀。 </li><li>切开的肌肉在整个长度的皮肤切口中线的其余部分。 </li><li>提供通过膀胱切口（ 图1）。膀胱通常是在骨盆最依赖的器官。 （在男性中，前列腺实际上是更加依赖和较大的比解压膀胱）。 </li><li>通过膀胱后壁缝合的地方之一逗留，然后通过另一个用6-0聚丙烯缝线膀胱前壁。横向放置额外的缝合。不要把这些缝合。当缝线拉紧，膀胱，将有一个方形的配置，测量约1厘米2（ 图2）。要小心，因为他们可以很容易地通过膀胱组织拉不会有太多这些缝线的张力。 </li><li>纵向切开膀胱，通过前约1厘米（1.5-2厘米）在大鼠膀胱膀胱壁在中线（只逊色于膀胱圆顶）。 </li></ol> 3。吻合脚手架<ol><li>用细剪刀，修剪丝绸脚手架膀胱缺损的近似面积。 </li><li>使用缝线7-0 polyglactin，开始在一个角落里的脚手架缝合膀胱，在连续运行时尚创建一个防水密封周围所有的缺陷的方式（ 图3）。 </li><li>测试用无菌生理盐水填充灌输它通过用30号注射器针头膀胱壁膀胱吻合的完整性。如果发现泄漏，这是可以关闭额外中断7-0 polyglactin的缝合以缩小差距。 </li><li>减少到腹部的重建膀胱后面。 </li></ol> 4。切口封闭<ol><li>关闭腹壁之前，注入局部麻醉下直肌和皮下组织（&lt;3毫克/千克为0.25％）与布比卡因。 </li><li>重新估算一个连续运行4-0 polyglactin的缝合直肌。 </li><li>关闭一个连续运行4-0 polyglactin的缝合皮肤。 </li><li>清洁和干燥的切口（ 图4）。 </li><li>转移到温暖，从麻醉中苏醒的清洁笼子的动物。 </li></oL&gt; 廔导管置入膀胱分析的步骤如下： 5。隧道廔导管<ol><li>设立必要的手术器械的无菌医疗器械领域：剃剪，牙钳，精细的无创伤钳，细针驱动器，纱布，梅岑鲍姆剪刀，切断术剪刀，小弯钳，手术刀刀片，6-0聚丙烯，4-0 3-0丝线缝合（4-0丝线缝合小鼠），18G针头，22G钝尖针头，25G针头，1 mL生理盐水填充注射器，50聚乙烯管材（PE-50）polyglactin缝合，切割长度为〜10厘米。 </li><li>耀斑的PE-50管，轻轻地暴露在火焰。小心融化年底关闭或闭塞的管腔（这可以通过连接到“非喇叭形”结束1 25G针注射生理盐水，确保流动验证）。作为一个锚，以保持膀胱内管（ 图5）。 </li><l&gt;以上步骤1.2麻醉动物。 </li><li>使用剃须剪消除双方之间的肩胛骨和小腹上ventrum动物背部的皮毛。 </li><li>准备方面的优碘和70％的乙醇。将动物俯卧悬垂。 </li><li>肩胛骨之间的背部做一个1厘米的切口。梅岑鲍姆剪刀，发展之间的皮肤和底层肌肉的飞机，在飞机上放置剪刀的技巧和传播他们创造一个隧道，周围腹腹部。 </li><li>调运动物仰卧。使你的腹部切口，暴露膀胱以上步骤2.1-2.4。减少膀胱背部到腹部。 </li><li>放置到你的皮下隧道在步骤5.6从你的背部皮肤切口开始创建的小卡子。用你的手指，以保护通过腹壁进入腹部钳提示腹腔内的内容，皮尔斯。 </li><li>抓住了Smooth年底的PE-50管钳，拉回到通过背侧切口。确保不拉过去的腹壁（ 图6），bulbed年底。 </li></ol> 6。配售的廔管<ol><li>提供通过膀胱切口。从这个角度上，应处理膀胱细钳，膀胱，可能导致损坏或炎症，以防止外伤可以歪斜你的的膀胱结果或结果在动物手术后的不适。 </li><li>请注意廔管的建议网站。它应该被放置在膀胱圆顶（优越的扩充段）。这将防止扭结管或闭塞。 </li><li>用6-0聚丙烯缝线，放置1 pursestring针在膀胱圆顶以下列方式：第一掷，通过纵向的膀胱壁外侧廔管的选址。一个松散年底离开小钳，使缝合时不小心拉，一路过关斩将。放置在横向方向的下一个罚球，开始先进入膀胱壁略横向到你的第一个罚球的出口。 </li><li>不要拉缝线拉紧。你未来的缝合平行放置到你的第一个（纵向）进入膀胱刚刚头侧到你最后的出口，横向缝合网站的。第四次罚球将开始横向到最后缝合和结束对你的第一罚球入口旁的出口。做正确的，这个网站（ 图7）拟议导管周围形成环形广场。 </li><li>使用18G的针头刺破膀胱壁的中心的pursestring缝合。要小心，不要刺入太深（刚好是腔内）。放置在开放的罚款钳提示，轻轻蔓延扩大的洞。 </li><li>插入在缺陷的导管bulbed的膀胱，直到它是腔内。拉导管周围的pursestring缝合紧密配合下来。这应该肚带保持它在的地方（ 图8）的导管周围的膀胱壁。 </li><li>采取一端缝合和环绕导管一次和配合下，进一步巩固导管。 </li></ol> 7。测试的导管和关闭腹部切口<ol><li>用1毫升注射器和25G的针头，针插入油管慢慢注入生理盐水胀大的膀胱。观察周围的导管漏水。一旦你看到从尿道泄漏，吸生理盐水再次解压膀胱。 </li><li>以上步骤4.1-4.4关闭腹部切口。 </li></ol> 8。关闭背切口，将导管（老鼠） <ol><li>调运动物容易。 </li><li>削减的皮肤用剪刀水平导管管。插入22G钝钛带够针进油管。 </li><li> 4-0 polyglactin在运行方式，在油管关闭的皮肤。离开枢纽针从皮肤挤出。 </li><li>将钝针的静脉线帽。用3-0丝线缝合，保护皮肤的导管尖端（ 图9）。 </li><li>清洁切口。转移到温暖，从麻醉中苏醒的清洁笼子的老鼠。 </li></ol> 8。*闭幕背切口，将导管（小鼠或大鼠） <ol><li> *扭结或用火焰融化结束闭塞的导管末端。 </li><li> *线圈结束的油管，并留在皮下囊动物的背（不要切断油管缩短）（ 图10）。 </li><li> *关闭了油管的皮肤，在运行方式的4-0 polyglactin（ 图11）。 </li><li> *在的膀胱一天，准备用优碘和70％的乙醇背侧切口。麻醉下打开背侧切口，去除皮下袋的连续油管。关闭切口。从麻醉中醒来动物和执行时，它是完全清醒的膀胱。 </li></ol> 9。代表结果 - 手术方法重建膀胱应该尽可能防水，以避免一个重大的尿漏（ 图3）有关的并发症。疼痛或不适通常发抖或刮伤，并在腹部切口啃的清单。这可以皮下注射美洛昔康（0.5-1.0毫克/千克皮下）非甾体类抗炎等日常管理。通常情况下，动物只需要注射手术后第3天。这可与阿片类药物丁丙诺啡（0.05-0.1毫克/公斤皮下注射，每8-12小时），如需要补充。应监测动物，每日3次，手术后第3天，总重量手术后3-5天每天冰然后每天此后，疼痛，感染的迹象，充足的伤口愈合，活动，梳洗，和皮肤膨评估。抗生素（Baytril，按5mg/kg皮下量不超过0.1毫升每24小时）的第一个72小时的手术后，对手术预防感染。正常复苏的迹象是行走和活动的正常水平，适当的喂食和饮水，疼痛或痛苦的情况下（不发声）和与cagemates正常的社会。应前膀胱分析给出一个至少5-7天的恢复时间，使膀胱愈合，减少炎症，这可能会影响结果。 膀胱分析 10。醒来膀胱分析<ol><li>安装与数据采集和分析与LabChart V6（ADInstruments）和输液哈佛22注射泵（哈佛Appara描述与MLT844 ADInstrumentsHolliston，MA），土族，虽然其他类似的系统（ 图12）。 </li><li>校准基于两个音量膀胱系统的规格和压力。 </li><li>将暂停以上规模的代谢笼（与丝网地板的笼子）的动物。连接到传感器的规模。 </li><li>清除任何气泡系统，并确保从输液泵的连续流。 </li><li>连接计算机数据采集系统，并观察数据的踪迹。相应地调整规模。膀胱压力和尿量将连续记录。 </li><li>访问与通过T型管连接压力传感器和输液泵1 27G针头耻骨上导管。 12.5微升/分钟的鼠标和100μL/分钟大鼠在开始输注生理盐水。 </li><li>允许的排尿模式，跟踪稳定（膀胱压力上升，由一个void）。这通常需要大约mately 10-20分钟。记录排尿周期为45-120分钟，或至少3-4排尿周期。 </li><li>观察实时并发症，这将导致工件（即导管扭结，阻塞等;见下文讨论）来解决整个过程。 </li><li>停止输液，导管从系统中断开，并返回笼子里的动物。 </li></ol> 11。昏迷的膀胱分析（无耻骨上导管） <ol><li>麻醉与聚氨酯（1-2克/公斤）腹腔注射（IP）的动物。 </li><li>暴露的膀胱以上步骤1.3-2.4。 </li><li>在步骤9.2校准系统。准备步骤9.4-9.5系统。 </li><li>插入27G的针通过一个T型管连接压力传感器和膀胱侧面输液泵。 </li><li>记录排尿周期为45-90分钟。 </li><li>停止输液，从膀胱中取出针和安乐死的动物。&lt;/ LI&gt; </ol> 12。代表性成果 - 膀胱分析尿动力学轨迹，然后可以分析得出作废卷，合规性，峰值排尿压力，除收缩区间，排尿周期后无效残留量等参数。 cystometrogram可分为馅和排尿阶段。一个正常的灌浆阶段是排尿周期的部分在膀胱膀胱内压力的变化很小罢了。正常排尿阶段的跟踪，由相应的收缩，膀胱内的压力在稳步上升。最高压力达到期间跟踪排尿阶段被称为高峰排尿压力。高峰排尿压力，可以建议梗阻性排尿模式，hypercontractile膀胱或在SP导管扭结。可以计算出符合收购量注入杜里比例吴灌装阶段和压力（标准=ΔV/△P）的变化。一个hypocompliant膀胱之一，是无法容纳足够的尿量低的压力。 intercontraction间隔可以计算分析上的cystometrogram看到两收缩时间。短intercontraction间隔是暗示膀胱刺激。排尿周期时间是指整个灌装和排尿阶段完成，并可以很容易地跟踪分析确定所需的时间。在膀胱的结论，后残余尿量（PVR），可以得到。这是通过吸气后完成一个收缩的耻骨上导管。这些参数有助于客观的调查研究膀胱动态填充和清空膀胱。 <img alt="图1" src="/files/ftp_upload/3981/3981fig1.jpg" /> 腹部手术切口的照片图1。膀胱和挤压。 <img alt="图2" src="/files/ftp_upload/3981/3981fig2.jpg" /> 图2。膀胱膀胱腔暴露的切口。 <img alt="图3" src="/files/ftp_upload/3981/3981fig3.jpg" /> 图3。植入到膀胱壁的整合。 <img alt="图4" src="/files/ftp_upload/3981/3981fig4.jpg" /> 图4。封闭切口的照片。 <img alt="图5" src="/files/ftp_upload/3981/3981fig5.jpg" /> 图5。扩口端的PE-50管。 <img alt="图6" src="/files/ftp_upload/3981/3981fig6.jpg" /> 图6。PE-50管（导管）通过背切口。 <img alt="图7" src="/files/ftp_upload/3981/3981fig7.jpg" /> 图7。pursestring缝合。 <img alt="图8" src="/files/ftp_upload/3981/3981fig8.jpg" /> 图8。保护导管到膀胱。 <img alt="图9" src="/files/ftp_upload/3981/3981fig9.jpg" /> 图9。抵押导管枢纽。 <img alt="图10" src="/files/ftp_upload/3981/3981fig10.jpg" /> 图10。盘绕在皮下囊管。 <img alt="图11" src="/files/ftp_upload/3981/3981fig11.jpg" /> 图11。背切口倒闭。 <img alt="图12" src="/files/ftp_upload/3981/3981fig12.jpg" /> 图12例膀胱设置。 <img alt="图13" src="/files/ftp_upload/3981/3981fig13.jpg" /> 图13。代表膀胱跟踪。

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gel+spun+silk-based+biomaterials+in+a+murine+model+of+bladder+augmentation.">Evaluation of gel spun silk-based biomaterials in a murine model of bladder augmentation.</a> Biomaterials. 32, 808-818 (2011).</li><li>Persson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+and+peripheral+mechanisms+contributing+to+hyperactive+voiding+in+spontaneously+hypertensive+rats.">Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats.</a> Am. J. Physiol. 275, 1366-1373 (1998).</li><li>Yaksh, T. L., Durant, P. A., Brent, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micturition+in+rats:+a+chronic+model+for+study+of+bladder+function+and+effect+of+anesthetics.">Micturition in rats: a chronic model for study of bladder function and effect of anesthetics.</a> Am. J. Physiol. 251, 1177-1185 (1986).</li><li>Pandita, R. K., Fujiwara, M., Alm, P., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+evaluation+of+bladder+function+in+non-anesthetized+mice+with+and+without+bladder+outlet+obstruction.">Cystometric evaluation of bladder function in non-anesthetized mice with and without bladder outlet obstruction.</a> J. Urol. 164, 1385-1389 (2000).</li><li>Soler, R., Fullhase, C., Lu, B., Bishop, C. E., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+in+a+new+mutant+mouse+model+with+increased+superoxide--lack+of+nitric+oxide.">Bladder dysfunction in a new mutant mouse model with increased superoxide--lack of nitric oxide.</a> J. Urol. 183, 780-785 (2010).</li><li>Matsuura, S., Downie, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anesthetics+on+reflex+micturition+in+the+chronic+cannula-implanted+rat.">Effect of anesthetics on reflex micturition in the chronic cannula-implanted rat.</a> Neurourol. Urodyn. 19, 87-99 (2000).</li><li>Andersson, K. E., Soler, R., Fullhase, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+urodynamic+investigation.">Rodent models for urodynamic investigation.</a> Neurourol. Urodyn. 30, 636-646 (2011).</li><li>Soler, R., Fullhase, C., Santos, C., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bladder+dysfunction+in+a+rat+model+of+dopaminergic+brain+lesion.">Development of bladder dysfunction in a rat model of dopaminergic brain lesion.</a> Neurourol Urodyn. 30, 188-193 (2011).</li><li>Streng, T., Santti, R., Andersson, K. E., Talo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+rhabdosphincter+in+female+rat+voiding.">The role of the rhabdosphincter in female rat voiding.</a> BJU Int. 94, 138-142 (2004).</li><li>Malmgren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometrical+evaluation+of+bladder+instability+in+rats+with+infravesical+outflow+obstruction.">Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction.</a> J. Urol. 137, 1291-1294 (1987).</li><li>Smith, P. P., Kuchel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+uroflow+cystometry+in+the+urethane-anesthetized+mouse.">Continuous uroflow cystometry in the urethane-anesthetized mouse.</a> Neurourol. Urodyn. 29, 1344-1349 (2010).</li><li>Cannon, T. W., Damaser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anesthesia+on+cystometry+and+leak+point+pressure+of+the+female+rat.">Effects of anesthesia on cystometry and leak point pressure of the female rat.</a> Life Sci. 69, 1193-1202 (2001).</li><li>Smith, P. P., Hurtado, E., Smith, C. P., Boone, T. B., Somogyi, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cystometric+methods+in+female+rats.">Comparison of cystometric methods in female rats.</a> Neurourol. Urodyn. 27, 324-329 (2008).</li></ol>

没有利益冲突的声明。

生物材料配置膀胱后植入膀胱扩大术在小动物模型的评价，表示验证确定在临床情况下使用的结构优化和矩阵设计的机械特性的重要一步。在这项研究中，我们描述手术方法进行膀胱增强小鼠和大鼠以及膀胱技术工程器官功能评估，以确定尿动力学性能。我们利用这些技术涉及多个实验小鼠和大鼠中，每30无显著问题+的啮齿动物组成的实验。我们的研究实验室是一个基本的科学家和医生外科医生的多元化企业集团，并具有至少5-6年毕业后手术培训的外科医生进行这些实验的程序方面。 无论生物材料的使用，主要DI型在大鼠与小鼠膀胱之间增强fference是膀胱的大小。由于到膀胱规模较小，解剖和生物材料掺入更多的鼠标在技术上困难。为了帮助在可视化，手术显微镜可以使用。由于在大鼠膀胱的大小越大，它更适合有多个程序的情况下要对膀胱（如隆胸和安置廔导管）执行。此外，协议上面介绍了利用大鼠13的PE-50管，但是，更大的大小导管，尤其是长期研究14 PE-100已经使用。在小鼠实验中，如体育-10油管的小口径可以利用15,16，但应牢记，更小，更柔韧的管可能没有压力的变化传送到传感器准确。此外，确保导管的替代方法的背（*以上步骤8）在MICE由于其体型较小，钝尖的针和四帽是太麻烦了。这个缺点是需要提取年底前膀胱导管在皮下囊麻醉。 有研究表明，在最初的第一天（0-4天）后放置导管，膀胱透露与高膀胱排尿量低的压力和过度。这些研究结果出现周围第六稳定第七天14,17，因此，可能是膀胱评价的理想时机。然而，在文献中的大多数报告进行膀胱导尿18第3天，在上述时间相对参数变化的户口本内。离开了超过3天的持续时间更长的耻骨上导管带有并发症，如结石的风险，移位，感染，血尿和闭塞的导管碎片。 &lt;类p =的“jove_content”&gt;不同输注速率在 ​​膀胱已经从1-3mL/hr为15,16小鼠和大鼠13,19,20 10-11mL/hr。超生理输注速率可引起假性升高的压力14。我们使用输注率12.5μL/分钟（0.75毫升/小时）小鼠和100微升/分钟（6毫升/小时）大鼠在我们的设置，但较低的利率还可以利用。生理盐水的温度至少应为室温，虽然温水（37℃）生理盐水是为了避免膀胱过度挑起与灌输冷解决方案更优化。在清醒的膀胱，关键是要稳定的排尿模式使动物成为调整的笼子里，在我们的经验需要一段时间约10-20分钟在此之后，定时排尿周期可以记录45-120分钟，或至少3-4排尿周期。动物应实时观察，因为动物是自由舞动g和并发症，如导管扭曲或扭结可以改变膀胱分析。环境噪声限制在膀胱希望减少动物运动和随后的文物。无意识的膀胱不会有清醒膀胱随之而来的问题，但多个麻醉药已被证明能抑制自发性膀胱收缩。这种抑制作用直接对应的麻醉药品的行动预计持续，即当麻醉效果消退，恢复自发性收缩14。此外，膀胱溢出时，测量的压力，在麻醉大鼠统计学更大，都活着和验尸报告，表明对膀胱壁的被动遵守属性的效果。这种效果被视为与21巴比妥，氯胺酮和氯醛糖IM / IP协议，除了吸入氟烷和鞘内nesacaine 14的 。一个各种麻醉药confir的更广泛的研究M这个发现在温和的麻醉水平17（同时吸入异氟醚和甲氧）和巴比妥（苯巴比妥和thiobutabarbital）麻醉药抑制排尿反射。这种效应甚至轻或镇静麻醉药物，如芬太尼，氟哌利多和氯胺酮地西泮水平观察，在以往的研究中，麻醉效果消退，所以没有抑制17。此过程中，乌拉坦腹腔注射可以使用，因为它已被证明，反射排尿保留，同时也允许适当的麻醉17,22。此外，没有任何效果观察排尿压力23。耻骨上膀胱导管放置这里所描述的，因为尿道导管已被证明有较高的膀胱压力曲线和较低的流速相对膀胱出口梗阻24。此外，尿道插管，仅在麻醉动物是可行的，即使在当时，导管可以是困难的，尤其是雄性老鼠和小鼠。 总之，用于膀胱扩大术和/或膀胱分析该模型的选择是依赖于具体的研究目标。从技术角度大鼠模型清楚地持有上述原因的优势。然而，在小鼠模型可用于研究评估尿路疾病的特定基因编码的高端产品，由于其易感性基因操纵的角色。这不是一般在大鼠可行的。 清醒膀胱最准确地模仿这些动物接受他们的排尿周期正常的生理状态，所以，很可能给一个更可靠的膀胱功能的生理测定。此外，混杂变量的直接影响膀胱功能nesthetics是可以避免的。

<table border="1"><tbody><tr><td> 材料： </td><td> 说明/使用： </td></tr><tr><td>剃剪</td><td>准备手术鼠/鼠标</td></tr><tr><td>无菌单，优碘，70％的乙醇，无菌纱布</td><td>无菌医疗器械领域的制备</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 工具： </td></tr><tr><td>手术刀刀片</td><td>皮肤切口</td></tr><tr><td>镊子与牙齿</td><td>操纵皮肤</td></tr><tr><td>精细镊子</td><td>非创伤性（无齿），没有锯齿或细锯齿操纵</td></tr><tr><td>小针驱动程序</td><td>夏普组织清扫</td></tr><tr><td>梅岑鲍姆剪刀</td><td> bldder切口</td></tr><tr><td>切断术剪刀</td><td>对于回缩缝线和发展皮下ţunnel（廔导管） </td></tr><tr><td>小弯钳</td><td>皮下隧道（廔导管） </td></tr><tr><td></td><td></td><td></td><td colspan="2"> 缝合： </td></tr><tr><td> 6-0聚丙烯缝线</td><td>膀胱住宿缝线和pursestring缝合</td></tr><tr><td> 7-0 polyglactin缝合</td><td>膀胱吻合术支架</td></tr><tr><td> 4-0 polyglactin缝合</td><td>肌肉/皮肤闭幕</td></tr><tr><td> 3-0或4-0丝线缝合</td><td>确保导管尖端皮肤</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 针头和注射器： </td></tr><tr><td> 18号针头</td><td>刺入膀胱廔导管</td></tr><tr><td> 25日和30表针</td><td>泄漏测试膀胱</td></tr><tr><td> 1毫升生理盐水filled注射器</td><td></td></tr><tr><td> 22号钝尖针</td><td></td></tr><tr><td></td><td></td><td></td><td colspan="2"> 廔导管： </td></tr><tr><td>体育-50油管</td><td></td></tr><tr><td>打火机</td><td>迸出的PE-50管</td></tr><tr><td>小弯钳</td><td>发展皮下隧道</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 膀胱： </td></tr><tr><td> MLT844 ADInstruments数据采集和LabChart软件的</td><td>压力数据采集</td></tr><tr><td>哈佛大学22注射泵（哈佛仪器，Holliston，马） </td><td>输液泵</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 麻醉药（无意识膀胱）： </td></tr><tr><td>我soflurane </td><td>全身麻醉的诱导/维护</td></tr><tr><td>聚氨酯</td><td> unconconscious膀胱</td></tr><tr><td>布比卡因或同等学历</td><td>局部麻醉</td></tr><tr><td>美洛昔康</td><td>手术后的镇痛</td></tr><tr><td>丁丙诺啡</td><td>手术后的镇痛</td></tr></tbody></table>

evaluation of biomaterials for bladder augmentation using cystometric analyses in various rodent models

肾功能和尿尿失禁是严重依赖上，存储在低气压的尿液和排出精确策划收缩膀胱的正常功能。包括后尿道瓣膜，良性前列腺增生症，脊柱裂/脊髓损伤继发神经源性膀胱的先天和后天的泌尿系统异常可能导致病理组织重塑导致受损遵守和能力下降1。尿路梗阻功能性或解剖经常与这些条件，可能导致尿失禁和肾损害增加的储尿及排尿压力2。手术植入胃肠道段，扩大器官的能力，降低膀胱内压力表示为这些疾病的主要手术治疗选项时，医疗管理失败3。然而，这种做法是阻碍教育署提供捐助组织的限制，并与包括代谢扰动，慢性尿路感染，泌尿系结石的形成，和继发恶性肿瘤4,5显着的并发症。 膀胱组织工程的研究现状在很大程度上侧重于查明缺陷的网站，可以支持组织再生的生物材料配置。传统的3-D支架来自天然和合成聚合物，如小肠黏膜下层和聚乙醇酸的显示支持以及促进上皮和平滑肌再生器官在动物模型的存储容量增加，在一些短期的成功诊所6,7。然而，在支架的机械完整性和生物相容性的缺陷往往导致移植挛缩9，8，有害纤维化和钙化10，从而增加了植入失败的风险和需要FOŕ二次手术。此外，恢复正常排尿的特点，利用标准的生物材料的结构，增强膀胱成形术尚未实现，因此，研究和发展新型的矩阵可以发挥这种作用是必要的。 为了成功地开发和评价，为临床膀胱扩大术的最佳生物材料的研究，疗效必须首先进行标准化动物模型，利用详细的手术方法和功能结果评估。我们以前曾报道在小鼠膀胱扩大术模型使用基于丝素蛋白支架的潜力，以确定调解组织再生和功能性排尿的特点。11,12膀胱这种模式的分析显示，在结构和机械的植入性能的变化可以影响11,12的组织工程膀胱的尿流动力学特征。积极correlations之间的基质介导的组织再生的程度决定组织由膀胱 ​​功能合规性和能力评估，证明在这个模型中11,12。因此，这些结果表明，在啮齿类动物膀胱增强系统的生物材料配置功能的评价可能是一个有用的格式脚手架性能评估和建立之前，大量的动物实验研究和临床部署在体内的可行性。目前的研究中，我们将介绍使用蚕丝支架在小鼠和​​大鼠膀胱扩大术的各种手术阶段，并表现出的清醒和麻醉膀胱技术。

Watch this Scientific Journal Video about Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models at JoVE.com

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

在小鼠和大鼠模型，用三维支架膀胱扩大术的手术阶段。为了测试在膀胱扩大术中使用的生物材料配置的功效，膀胱清醒和麻醉技术。

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

evaluation-biomaterials-for-bladder-augmentation-using-cystometric

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Bioengineering

19.1K 次观看.  Harvard Medical School. 在小鼠和大鼠模型，用三维支架膀胱扩大术的手术阶段。为了测试在膀胱扩大术中使用的生物材料配置的功效，膀胱清醒和麻醉技术。

视频: Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

科研

生物工程

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生生物材料的生物相容性曲线

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

通过叶反射和叶绿素荧光分析同步测量的光合行为评价

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...