Name: assessing urinary tract junction obstruction defects by methylene blue dye injection
Uploaded: 2017-10-12T12:30:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

我感谢 Dr. 艾伦· o. Perantoni (CDBL/国立卫生研究院/NIH) 支持这份手稿的提交。我还要感谢 Dr. 迈克尔霍尔 (CDBL/国立卫生研究院/nih) 和尼玛拉夏尔马 (CDBL/国立卫生研究院/nih) 编辑这篇手稿。我很感谢赖昌星 (SAIC) 的出色的鼠标饲养。这项工作得到了国立卫生研究院、国家癌症研究所和癌症研究中心的支持。

鼠标 ( Wnt5a flox / flox 小鼠 (Wnt5a tm1.1Tpy ) 和 Dll1Cre 线, UVJO 小鼠模型) 7 是根据 NIH 关于实验室动物护理和使用的指导方针进行管理的。并根据由动物保护和使用委员会批准的议定书进行研究. 
 1. 亚甲基蓝染料溶液的制备 <ol> <li> 测量 0.1 g 亚甲蓝粉. </li>
	<li> 在10毫升生理盐水或1x 磷酸盐缓冲盐水 (PBS) 中完全由涡流溶解亚甲基蓝. </li>
	<li> 用注射器过滤器 (膜孔径0.45 和 #181; m) 过滤10毫克/毫升亚甲基蓝溶液, 以消除注射过程中的堵塞. </li>
	<li> 组装一个无菌头皮静脉集 (27GX3/4 和 #8243;) 与3毫升一次性注射器和填充注射器3毫升过滤亚甲蓝溶液. 
	注意: 为了防止静水压和随后的染料流动, 将针尖放在装有亚甲基蓝溶液的注射器上. </li>
</ol> 2。产前胚胎解剖 <ol> <li> 用70% 乙醇清洗解剖剪刀和镊子. </li>
	<li> 在 E18.5 或 E19.5 中收集围产期胚胎, 先使用 CO 2 吸入, 然后根据 NIH 指南执行宫颈脱位. 
	注意: 怀孕的雌性老鼠通常从 E18.5 开始分娩, 但是, 较大的肾脏更容易操作。因此, 在 E19.5 接近出生的肾脏更容易进行这一分析。然而, 有双侧尿路阻塞的幼崽在出生后死亡。根据实验小鼠的特点, 应该经验地确定适当的收集日/时间. </li>
	<li> 在腹腹表面喷洒70% 乙醇, 然后用解剖剪刀和镊子打开腹腔腹. </li>
	<li> 抬起整个子宫, 用解剖剪刀在子宫角上切开, 将它与身体分开. </li>
	<li> 在培养皿中用 1x PBS 冲洗整个子宫. </li>
	<li> 用解剖剪刀切开子宫分段, 用解剖钳切除胎盘蜕膜, 将胚胎暴露在卵黄囊中. </li>
	<li> 先取下蛋黄囊, 然后用解剖钳取出羊膜, 释放围产期胚胎. </li>
	<li> 用解剖剪刀斩首胚胎。用无菌纱布擦拭多余的血液。在解剖显微镜上, 用数码相机进行成像, 将胚胎从腹面上固定下来。如果需要, 收集它的基因分型的尾巴. 
	注: 一次进行一次胚胎注射. </li>
	<li> 通过撕裂皮肤小心地打开一个胚胎的腹腔。然后, 通过切割或拔出它们来暴露肾脏、输尿管和位于背的膀胱 ( 图 1A ), 小心地清除多余的器官和组织, 如肝脏、胃和肠道。如有必要, 从解剖胚中吸收多余的血液, 用无菌纱布垫. 
	注: 过量的血液会影响到对染料注射的肾盂识别. </li>
</ol> 3。注射亚甲基蓝染料到肾盂和监测染料流 <ol> <li> 通过将亚甲基蓝溶液从针尖上排出, 通过静水压来除去针头和油管中的气泡。提起含有染料溶液的注射器, 在针尖的水平以上开始流动, 然后放下注射器停止流动. </li>
	<li> 将针插入靠近输尿管近端的肾盂, 注意不要在放置后再将其干扰。在肾脏中进行染料注射以确定其尿路梗阻. 
	注: 将染料注射到任何异常的肾脏 7 首先遵循同样的方法来确定其尿路梗阻. </li>
	<li> 抬起注射器约20厘米, 以提供静水压, 并让15和 #181; l 60 和 #181; 染料溶液流动的 l。流速将是大约3和 #181; 如果注射器是上升到这个高度在胚胎之上. </li>
	<li> 监测染料的蓝色颜色首先在肾盂中开始, 然后在输尿管的长度, 最后在膀胱腔. 
	注: 如果将输尿管正确插入膀胱内, 则需要大约5秒才能看到在膀胱腔中出现微弱的染料颜色。如果没有染料的颜色出现在膀胱腔中, 允许染料在被堵塞的地方堆积十五年代. </li>
	<li> 将注射器放下以停止染料流并从肾脏中取出针头. </li>
	<li> 使用与显微镜连接的照相机和成像程序, 将肾脏、输尿管和膀胱的图像与染料溶液一起跟踪. </li>
	<li> 将肾脏、us 和染料溶液的最终位置记录在实验室笔记本中. </li>
	<li> 执行3.1 节中描述的对侧肾脏的注射) 到 3.5), 并允许染料流在二十年代总. 
	注: 膀胱腔内将充满染料溶液, 以强烈的蓝色表示, 如果输尿管被正确插入膀胱腔。经尿路结肠梗阻评估, 输尿管和膀胱可固定切片. </li>
</ol>

<ol>
	<li>Rasouly, H. M., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+urinary+tract+development+and+disease.">Lower urinary tract development and disease.</a> Wiley Interdiscip Rev Syst Biol Med. 5, 307-342 (2013).</li><li>Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Box+Genes+in+the+Kidney+and+Urinary+Tract.">T-Box Genes in the Kidney and Urinary Tract.</a> Curr Top Dev Biol. 122, 245-278 (2017).</li><li>Bohnenpoll, T., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureter+growth+and+differentiation.">Ureter growth and differentiation.</a> Semin Cell Dev Biol. 36, 21-30 (2014).</li><li>Nicolaou, N., Renkema, K. Y., Bongers, E. M., Giles, R. H., Knoers, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic,+environmental,+and+epigenetic+factors+involved+in+CAKUT.">Genetic, environmental, and epigenetic factors involved in CAKUT.</a> Nat Rev Nephrol. 11, 720-731 (2015).</li><li>Tripathi, P., Wang, Y., Casey, A. M., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+canonical+Smad+signaling+in+ureteral+and+bladder+mesenchyme+causes+ureteropelvic+junction+obstruction.">Absence of canonical Smad signaling in ureteral and bladder mesenchyme causes ureteropelvic junction obstruction.</a> J Am Soc Nephrol. 23, 618-628 (2012).</li><li>Aoki, Y., 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=Id2+haploinsufficiency+in+mice+leads+to+congenital+hydronephrosis+resembling+that+in+humans.">Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans.</a> Genes Cells. 9, 1287-1296 (2004).</li><li>Yun, K., Perantoni, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydronephrosis+in+the+Wnt5a-ablated+kidney+is+caused+by+an+abnormal+ureter-bladder+connection.">Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection.</a> Differentiation. 94, 1-7 (2017).</li><li>Uetani, N., Bouchard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plumbing+in+the+embryo:+developmental+defects+of+the+urinary+tracts.">Plumbing in the embryo: developmental defects of the urinary tracts.</a> Clin Genet. 75, 307-317 (2009).</li><li>Murawski, I. J., Watt, C. L., Gupta, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+urinary+tract+defects+in+mice:+methods+to+detect+the+presence+of+vesicoureteric+reflux+and+urinary+tract+obstruction.">Assessing urinary tract defects in mice: methods to detect the presence of vesicoureteric reflux and urinary tract obstruction.</a> Methods Mol Biol. 886, 351-362 (2012).</li><li>Weiss, A. C., 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=Nephric+duct+insertion+requires+EphA4/EphA7+signaling+from+the+pericloacal+mesenchyme.">Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.</a> Development. 141, 3420-3430 (2014).</li></ol>

作者没有什么要透露的。

老鼠肾脏是功能开始在 E16.5 和染料射入测试从理论上可能从这个时间点。然而, 肾脏太小, 不能被注射的染料溶液和表型, 如肾积水和 us 没有清楚地观察到, 因为这些表型是由于尿液转运异常而产生的二次作用。这些表型, 从障碍, 如 UPJO 或 UVJO, 是明显的 E18.5 肿胀肾脏和输尿管。E19.5 或新生幼崽的胚胎肾脏比 E18.5 或更年轻的胚胎更容易操作。然而, 有双侧尿路阻塞的幼崽在出生后死亡。因此, 根据?...

泌尿系统包括一对肾脏和输尿管和一个共同的膀胱和尿道。泌尿系统的主要功能是通过管理血液的水和电解质平衡来维持身体的稳态。肾脏过滤血液以控制电解质浓度和酸碱平衡, 并产生尿液排出过量的水和废物, 包括溶质和代谢物。然后, 尿液通过输尿管从肾脏的肾盂转移到 bladderin 的单向的方式, 它是储存和最终消除通过尿道1。
输尿管是来自肾导管的直管。在小鼠胚胎日 10.5 (E10.5) 的肾导管萌芽后, 输尿管柄拉长并分化成一种称为膀胱的多层结构, 在小鼠 E12.5 和 E16.5 之间是不透水的。输尿管柄周围的间充质细胞也被分为三层, 包括内基质细胞、中层粗平滑肌细胞和外外成纤维细胞。输尿管蠕动波起始于肾盂, 通过输尿管壁的平滑肌层传播至膀胱以传输尿液2,3, 它是在鼠标1中从 E16.5 开始生成的。
先天性异常的肾脏和泌尿道 (CAKUT) 是最常见的遗传性疾病, 目前在大约1% 的人类胎儿1,4, 并组成的各种表型包括肾积水和 us。肾脏和输尿管的尿液异常积累导致积水肾和 us 的形成。肾积水和/或 us 形成的一个原因是尿路梗阻。肾盂结肠梗阻 (UPJO) 是由于在近端输尿管和肾盂之间的堵塞造成的尿流异常, 导致肾积水和近端 us 狭窄与角度或持续折叠5, 6。此外, 远端输尿管的异位插入膀胱壁或生殖道称为输尿管结肠梗阻 (UVJO)。UVJO 还可诱发肾积水和 hydromegaureter 形成7,8。另外的输尿管结 (UVJ) 缺陷是膀胱回流 (VUR)。VUR 的特点是逆行尿流从膀胱向肾脏在 UVJ。与 UVJO 相比, 围产期胚胎与 VUR 不明显显示肿胀积水肾或严重 us 表型9。
在实验小鼠, 尿液流动可以通过注射染料, 如亚甲蓝, 进入肾盂9。injectedmethylene 蓝溶液将从肾盂经输尿管进入膀胱, 追踪尿液轨迹。肾积水可以通过在肾脏中的染料的膨胀来识别。UPJO 可被检测为堵塞的染料流在近端输尿管与肿胀肾盂5。经加宽的直径所示的输尿管的任何扩张都显示了 us 的一个例子。最后, 染料堆积在膀胱壁或在生殖道的站点表明 UVJO 与膨胀的积水肾脏和扩张的 hydromegaureter7,10。为了检测 VUR, 染料溶液注入膀胱, 随后的逆行流在肾脏的9中被监测。
在这里, 提出了一个协议的亚甲蓝染料注射到肾盂的围产期胚胎。这项协议允许追踪尿液从肾盂通过输尿管和进入膀胱, 并验证潜在的泌尿道交界处的障碍, 如 UPJO 或 UVJO。

<table><tbody><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>Quality Biological Inc. NORMAL SALINE - 500ML</td><td>Fisherscientific</td><td>50-983-204</td><td></td></tr><tr><td>3 mL disposable syringe with BD Luer-Lok tip</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Acrodisc Syringe Filters with Supor Membrane</td><td>Pall corporation</td><td>4614</td><td></td></tr><tr><td>Exel Scalp Vein (Butterfly) Sets; 27G x 3/4&quot; 12&quot;&amp;nbsp;</td><td>EXELINT</td><td>26709</td><td></td></tr><tr><td>Delicate Operating Scissors</td><td>Roboz Surgical Instrument Co.</td><td>RS-6702</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>Roboz Surgical Instrument Co.</td><td>RS-5135</td><td></td></tr><tr><td>Dumont Tweezers; Pattern #5</td><td>Roboz Surgical Instrument Co.</td><td>RS-5045</td><td></td></tr><tr><td>BD PrecisionGlide Needles 30 G x 1/2&quot;</td><td>BD</td><td>305106</td><td></td></tr></tbody></table>

assessing urinary tract junction obstruction defects by methylene blue dye injection

尿路结肠梗阻是先天性异常, 诱发肾积水和 us。小鼠泌尿道结肠梗阻的缺陷可以通过跟踪在泌尿系统内的亚甲基蓝染料流动来评估。将亚甲蓝染料注入围产期胚胎肾脏的肾盂中, 在应用静水压后, 通过输尿管肾脏的肾盂和膀胱腔内的染料流进行监测。在正常围产期泌尿道的膀胱腔内, 染料堆积明显, 但在肾盂与输尿管异常的终点之间, 如果出现尿路梗阻, 则会受到限制。该方法可促进尿路结肠梗阻的确诊和肾积水和 us 的可视化。这份手稿描述了一个协议的亚甲蓝染料注射液进入肾盂, 确认泌尿道交界处的障碍。

肾脏和下泌尿系统位于背部的大多数其他内脏器官, 如肝脏和肠道。取出这些其他内脏器官后, 可看到一对肾脏和输尿管和一个膀胱 (图 1A)。成功解剖后, 注入肾盂的染料将通过输尿管从肾脏流向膀胱。肾盂是肾中输尿管的漏斗状扩张的近端部分。因此, 染料注射点附近的可见近端输尿管与肾脏连接, 用黑色箭头表示 (图 1B-1...

Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

亚甲蓝染料注射入肾盂有助于评估尿路结肠梗阻的缺陷, 在小鼠胚胎泌尿道发育。在这里, 一个协议的亚甲蓝染料注射到肾盂是描述。

亚甲蓝染料注射液评价泌尿道结肠梗阻的缺陷

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

assessing-urinary-tract-junction-obstruction-defects-methylene-blue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.6K 次观看.  National Institutes of Health. 亚甲蓝染料注射入肾盂有助于评估尿路结肠梗阻的缺陷, 在小鼠胚胎泌尿道发育。在这里, 一个协议的亚甲蓝染料注射到肾盂是描述。

视频: Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

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. ...

科研

医学

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

1%亚甲蓝染料作为单一技术在乳腺癌哨点节点活检中的应用

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

癌症研究

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

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

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 ...

生物工程

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

光声膀胱造影

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

术中的微妙的子宫内膜异位症的检测：A新型范式为相关联的与的的腹膜的完整性之亏损盆腔疼痛的的的的检测和治疗的

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

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 ...

免疫与感染

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 ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

使用2光子显微镜量化慢性单侧输尿管梗阻对肾小球过程的影响

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

可逆性单侧输尿管梗阻的未成熟小鼠模型

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...