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

Agradezco a Dr. Alan O. Perantoni (NIH/NCI/CDOBLE) para apoyar la presentación de este manuscrito. También agradezco el Dr. Michael Hall (NIH/NCI/CDOBLE) y Nirmala Sharma (NIH/NCI/CDOBLE) para la edición de este manuscrito. Agradezco a Lai Thang (SAIC) para su cría de ratón excelente. Este trabajo fue apoyado por los institutos nacionales de salud, Instituto Nacional del cáncer y centro de investigación del cáncer.

ratones (Wnt5a flox / flox ratones (Wnt5a tm1.1Tpy) y la línea Dll1Cre, modelo UVJO) 7 fueron manejados según pautas de NIH para el cuidado y uso de animales de laboratorio y estudió con un protocolo aprobado por el NCI Frederick Animal cuidado y uso.
 1. preparación de la solución de colorante azul de metileno <ol><li>medir 0,1 g de azul de metileno en polvo.</li>
	<li>Disolver el azul de metileno en solución salina normal 10 mL o 1 x de tampón fosfato salino (PBS) completamente por Vortex.</li>
	<li>Filtro con un filtro de jeringa (tamaño del poro de la membrana 0.45µm) para eliminar la obstrucción durante la inyección de la solución de azul de metileno 10 mg/mL.</li>
	<li>Montar un estéril cuero cabelludo vena set (27GX3/4 &#8243;) con una jeringa desechable de 3 mL y llene la jeringa con 3 mL filtrada azul de metileno solución. 
	Nota: Para evitar la presión hidrostática y el tinte posterior flujo, coloque la punta de la aguja sobre la jeringa llenada de solución de azul de metileno.</li>
</ol> 2. Disección de embriones prenatales <ol><li>limpio de disección, tijeras y pinzas con etanol al 70%.</li>
	<li>Para recolectar embriones perinatales en E18.5 o E19.5, eutanasia ratones embarazadas primero mediante inhalación de CO 2 y llevar a cabo la dislocación cervical según las pautas de NIH. 
	Nota: El ratón hembra embarazado generalmente da nacimiento partida en E18.5, sin embargo, los riñones más grandes son más fáciles de manipular. Por lo tanto, son más fáciles de realizar este análisis en riñones en E19.5 más cercano al nacimiento. Sin embargo, cachorros con troquel de obstrucciones urinarias bilateral después del nacimiento. Dependiendo de las características de los ratones experimentales, un día/hora de recogida apropiado debe determinarse empíricamente.</li>
	<li>Rociar etanol al 70% en la superficie ventral abdominal y luego abrir la cavidad abdominal ventral mediante disección tijeras y fórceps.</li>
	<li>Levantar el útero entero y separado del cuerpo cortando con tijeras en las puntas de los cuernos uterinos de disección.</li>
	<li>Lavar el útero entero con PBS 1 x en una placa Petri.</li>
	<li>Cortar el útero segmentally con tijeras de disección y retirar decidua placentaria con disección pinzas para exponer los embriones en sacos vitelinos.</li>
	<li>Primero retire el saco vitelino y después retire la membrana amniótica con fórceps para liberar los embriones perinatales de disección.</li>
	<li>Decapitar a un embrión con tijeras de disección. Limpie el exceso de sangre con gasas estériles. Precisar el embrión con su superficie ventral hacia arriba en un microscopio de disección equipado con una cámara digital para la proyección de imagen. Recoger la cola para genotipificación si es necesario. 
	Nota: Realizar un embrión las inyecciones a la vez.</li>
	<li>Abra cuidadosamente la cavidad abdominal del embrión con pinzas por el desgarramiento de la piel. Entonces, Remueva cuidadosamente el exceso órganos y tejidos como el hígado, el estómago y el intestino con fórceps por cortarlos o tirando de los mismos para exponer los riñones, los uréteres y la vejiga que se encuentran dorsalmente ( figura 1A). Absorber el exceso de sangre de embrión disecado con gasas estériles si fuera necesario. 
	Nota: Sangre exceso interfiere con la identificación de la pelvis renal para la inyección del tinte.</li>
</ol> 3. Inyección de colorante azul de metileno en la Pelvis Renal y el control de flujo de tinte <ol><li>quitar burbujas en la aguja y el tubo por expulsión de solución de azul de metileno de la punta de la aguja de presión hidrostática. Levantar la jeringa que contiene la solución de colorante por encima del nivel de la punta de la aguja para iniciar el flujo y luego baje la jeringa para detener flujo.</li>
	<li>Insertar la aguja en la pelvis renal cerca de uréter proximal, procurando no para lo una vez colocado molestar a. Realizar la inyección de colorante en un riñón para determinar la obstrucción del tracto urinario. 
	Nota: Ejecutar el tinte inyección en cualquier renal anormal 7 primera siguiendo la misma forma para determinar la obstrucción del tracto urinario.</li>
	<li>Levante la jeringa hasta unos 20 cm para proporcionar presión hidrostática y dejar 15 μl - 60 μL del flujo de la solución colorante. La tasa de flujo será aproximadamente 3 μl/s si la jeringa se eleva a esta altura el embrión.</li>
	<li>Monitorear el color azul del colorante a partir de primero en la pelvis renal, entonces en la longitud del uréter y finalmente en el lumen de la vejiga. 
	Nota: Tarda cerca de 5 s para ver un color de tinte débiles emergen dentro del lumen de la vejiga si el uréter se inserta correctamente en la vejiga. Permitir que el tinte que se acumulan en el sitio bloqueado cerca de 15 s si no hay color de tinte en el lumen de la vejiga aparece.</li>
	<li>Coloque la jeringa hasta la parada de tinte flujo y retire la aguja del riñón.</li>
	<li>Tomar imágenes de los riñones, el uréter y la vejiga trazado con solución colorante usando la cámara y programa la proyección de imagen conectan a un estereomicroscopio.</li>
	<li>Hacer un registro de hidronefrosis del riñón, hydroureter y la posición final de la solución de colorante en un cuaderno de laboratorio.</li>
	<li>Realizar la inyección del riñón contralateral como se describe en las secciones 3.1) a 3.5) y permitir el flujo del tinte cerca de 20 s en total. 
	Nota: La luz de la vejiga se llenará con solución colorante, indicado por un color azul fuerte, si el uréter se inserta adecuadamente en el lumen de la vejiga. Después de la evaluación de obstrucción de la Unión de las vías urinarias, los uréteres y la vejiga pueden ser fijados para seccionamiento.</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>

El autor no tiene nada que revelar.

Riñones de ratón son funcionales a partir de las E16.5 y una prueba de inyección de tinte es teóricamente posible a partir de este momento. Sin embargo, el riñón es demasiado pequeño para ser inyectada con la solución de tinte y fenotipos como hidronefrosis Hidrouréter no se observan claramente puesto que estos fenotipos son un efecto secundario de orina acumulado debido al transporte anormal de la orina. Estos fenotipos, de los obstáculos como UPJO o UVJO, son evidentes en E18.5 por distensión de los riñones...

El sistema urinario consiste en un par de riñones y uréteres comunes vejiga y uretra. La función principal del sistema urinario es mantener la homeostasis del cuerpo al administrar el equilibrio de agua y electrolitos de la sangre. Los riñones filtran la sangre para control las concentraciones de electrolitos y equilibrio acido-base y producen orina para excretar el exceso de agua y residuos incluidos los solutos y metabolitos. Orina es transportada a través del uréter de la pelvis renal del riñón a la bladderin una manera unidireccional donde se almacenan y finalmente eliminado a través de la uretra1.
Los uréteres son tubos rectos que se origina en el conducto nephric. Después de la brotación del conducto nephric en el día embrionario 10.5 (E10.5) en el ratón, el tallo del uréter se alarga y distingue a una estructura de varias capas llamada urotelio que es impermeable entre ratón E12.5 y E16.5. Las células mesenquimales que rodea el tallo del uréter también se diferencian en tres capas que consiste de internas células del estroma, células de grosor intermedio del músculo liso y fibroblastos adventitial externos. Se propagan las ondas peristálticas ureterales iniciando en la pelvis renal a través de la capa de músculo liso de la pared del uréter a la vejiga para el transporte de orina2,3, que se produce a partir de E16.5 en el ratón1.
Anomalías congénitas del riñón y vías urinarias (CAKUT) se encuentran entre las enfermedades genéticas más frecuentes, presentes en aproximadamente el 1% de fetos humanos1,4y compuesto de una variedad de fenotipos como hidronefrosis e Hidrouréter. La acumulación anormal de orina en el riñón y del uréter produce hydronephrotic del riñón e Hidrouréter formación. Una causa de hidronefrosis o Hidrouréter formación es una obstrucción de las vías urinarias. Obstrucción de la UUP (UPJO) es causada por el flujo de orina aberrantes debido a una obstrucción entre el uréter proximal y la pelvis renal, dando por resultado hydronephrosis y estrechamiento proximal Hidrouréter con forma angular o persistente plegable5, 6. Además, la inserción ectópica del uréter distal en la pared de la vejiga o el tracto reproductivo se denomina obstrucción ureterovesical (UVJO). UVJO también puede inducir la hidronefrosis y hydromegaureter de7,de formación8. Un defecto de unión (UVJ) ureterovesical adicional es el reflujo vesicoureteral (RVU). VUR se caracteriza por flujo retrógrado de orina desde la vejiga hacia el riñón en UVJ. En comparación con UVJO, perinatales embriones con RVU no claramente muestran un riñón hydronephrotic dilatado o Hidrouréter severa fenotipo9.
En el ratón de laboratorio, flujo de orina puede ser examinado por la inyección de un colorante, como azul de metileno, en la pelvis renal9. La solución de azul de injectedmethylene traza la trayectoria de la orina desde la pelvis renal a través del uréter y la vejiga. La hidronefrosis puede ser reconocida por una expansión del tinte en el riñón. UPJO puede ser detectado como una obstrucción del flujo del tinte en el uréter proximal con pelvis renal dilatada5. Cualquier dilatación del uréter indicada un diámetro ensanchado muestra un ejemplo de Hidrouréter. Finalmente, acumulación de tinte en la pared de la vejiga o en el sitio del tracto reproductivo indica UVJO con riñón hydronephrotic distendido y dilatado hydromegaureter7,10. Para detectar el RVU, la solución de tinte se inyecta en la vejiga y el posterior flujo retrógrado es monitoreado en el riñón9.
Aquí, se presenta un protocolo para la inyección de tinte de azul de metileno en la pelvis renal de un embrión perinatal. Este protocolo permite el rastreo de flujo de la orina desde la pelvis renal a través del uréter y la vejiga y verifica posibles obstrucciones de cruce vías urinarias tales como UPJO o 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

Defectos de la obstrucción urinaria cruce son anomalías congénitas inducción de hidronefrosis e Hidrouréter. Defectos de obstrucción urinaria murino cruce pueden evaluarse mediante el seguimiento flujo de tinte de azul de metileno dentro del sistema urinario. Se inyecta azul de metileno en la pelvis renal de riñones embrionarios perinatales y flujo del tinte es monitoreado desde la pelvis renal del riñón por el uréter y en el lumen de la vejiga después de aplicar presión hidrostática. Acumulación de tinte será evidente en el lumen de la vejiga del tracto urinario perinatal normal, pero será limitada entre la pelvis renal y el punto final de un uréter anormal, si se producen obstrucciones del tracto urinario. Este método facilita la confirmación de obstrucción de vías urinarias cruce y visualización del hydronephrosis y del hydroureter. Este manuscrito describe un protocolo para la inyección de tinte de azul de metileno en la pelvis renal para confirmar obstrucciones de cruce de las vías urinarias.

Los riñones y sistema urinario inferior se encuentran dorsal a la mayoría de los otros órganos internos como el hígado y el intestino. Después de quitar estos otros órganos internos, un par de riñones y los uréteres y la vejiga de una sola son visibles (figura 1A). Sobre disección exitosa, fluirá el colorante inyectado en la pelvis renal del riñón en la vejiga vía el uréter. La pelvis renal es la forma de embudo parte proximal dilatada del urét...

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

Inyección de colorante azul de metileno en la pelvis renal facilita la evaluación de los defectos de obstrucción urinaria cruce durante el desarrollo embrionario las vías urinarias del ratón. Aquí, se describe un protocolo para la inyección de colorante azul de metileno en la pelvis renal.

Evaluación defectos de obstrucción urinaria cruce por la inyección de tinte de azul de metileno

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 vistas.  National Institutes of Health. Inyección de colorante azul de metileno en la pelvis renal facilita la evaluación de los defectos de obstrucción urinaria cruce durante el desarrollo embrionario las vías urinarias del ratón. Aquí, se describe un protocolo para la inyección de colorante azul de metileno en la pelvis renal.

Video: 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.

Vejiga ratón pared de inyección

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Investigación

Medicina

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).

La aplicación del 1% de tinte azul de metileno como una sola técnica en la biopsia de ganglio centinela de cáncer de mama

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 ...

Investigación sobre el cáncer

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.

Evaluación de Biomateriales para el aumento de la vejiga usando análisis cistométricos en modelos de roedores Varios

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

Bioingeniería

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.

Cistografía fotoacústica

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.

Intraoperatoria detección de endometriosis sutil: un nuevo paradigma para la detección y tratamiento del dolor pélvico asociado con la pérdida de la integridad Peritoneal

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.

Basada en sondeos endomicroscopía Láser Confocal del tracto urinario: la técnica

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.

Procedimiento transuretral instilación en ratón macho adulto

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

Inmunología e Infección

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).

Ultrasonografía del tracto urinario masculino adulto para pruebas funcionales urinarias

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.

Uso de microscopía de 2 fotones para cuantificar los efectos de la obstrucción ureteral unilateral crónica en los procesos glomerulares

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.

Un modelo murino inmaduro de obstrucción ureteral unilateral reversible

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