Todas las ratas se utilizaron de acuerdo con los protocolos aprobados por el Comité Experimental de Animales del Hospital de la Amistad de Beijing, Universidad Médica de la Capital.
1. Preparación quirúrgica, anestesia y técnicas quirúrgicas
NOTA: Un total de 40 ratas Wistar hembras, con un peso de 200-220 g, fueron obtenidas comercialmente para el presente estudio. De las 40 ratas, 10 fueron seleccionadas al azar como el grupo de control, y el resto fueron tratadas como el grupo de prueba. Todos los animales fueron alojados en un ambiente estéril en las instalaciones para animales del Hospital de la Amistad de Beijing, Universidad Médica Capital.
<ol><li>Realizar anestesia general administrando pentobarbital sódico por vía intraperitoneal (40 mg/kg). Alternativamente, inducir la anestesia usando isoflurano al 3% -4% y mantenerlo al 1% -3% (inhalado). Aplique ungüento oftálmico en los ojos para prevenir la sequedad. Luego, coloque la rata en la plataforma quirúrgica y proporcione soporte térmico. NOTA: Administrar analgésicos como buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 mL al inicio del procedimiento.</li>
	<li>Verifique la profundidad de la anestesia por la falta de respuesta al pellizco del dedo del pie. Afeite el pelaje de toda la zona de la espalda con una navaja de afeitar.</li>
	<li>Esterilice el sitio quirúrgico con al menos 3 ciclos de un exfoliante de dos etapas, como clorhexidina o povidona yodada seguido de alcohol isopropílico. Asegure las extremidades con cinta quirúrgica y haga una incisión mediana de unos 3 cm en la espalda con tijeras quirúrgicas.</li>
	<li>Profundice la incisión a través de los tejidos subcutáneos con tijeras quirúrgicas y corte los músculos unidos a la columna vertebral.</li>
	<li>Identifique visualmente y exponga la13ª costilla (el espacio intervertebral conectado a esa costilla es el intervalo T13-L1). Marque la13ª costilla con una sutura.</li>
	<li>Después de la identificación, reseque cuidadosamente los músculos unidos a la columna vertebral y exponga la columna vertebral. Resecar el ligamento supraespinoso y el ligamento interespinoso para una identificación precisa de la columna vertebral. Exponga el nivel de L4\u2012L5 con tijeras quirúrgicas y fórceps. NOTA: El ligamento supraespinoso se puede identificar fácilmente debido a la presencia de tejido subcutáneo delgado. Después de la resección del ligamento supraespinoso, el ligamento entre el proceso espinoso es el ligamento interespinoso.</li>
	<li>Diseccionar cuidadosamente el proceso espinoso vertebral L4-L5 y partes del proceso transversal usando pinzas Kelly para exponer la médula espinal (Figura 1).</li>
	<li>Exponga completamente el cono medular al nivel L4-L5 y transecte el cono medular totalmente con tijeras de iridectomía. Inserte un poco de taponamiento de tejido para bloquear la recuperación de la médula espinal.</li>
	<li>Cierre el músculo suprayacente y la piel en la capa externa de la piel con una sutura no absorbible 4-0.</li>
	<li>Para el grupo de control, realice los pasos 1.1\u20121.7 y deje intacto el cono medular. Cierre la incisión según el paso 1.9.</li>
</ol>2. Recuperación animal
<ol><li>Mantener a las ratas en una incubadora de temperatura controlada (37 °C) durante la primera hora después de la operación y controlarlas hasta que estén esternales o en movimiento activo. NOTA: Se tarda aproximadamente media hora para la recuperación total.</li>
	<li>Transfiera al animal a una jaula limpia con suficiente comida y agua. Mantenga a las ratas en jaulas separadas. NOTA: El éxito de la transección se indica cuando las ratas en el grupo de prueba se mueven solo con la ayuda de las patas delanteras, mientras que las ratas en el grupo de control podrían caminar normalmente.</li>
</ol>3. Gestión postoperatoria
<ol><li>Inyecte penicilina G, un antibiótico (50,000 U/ml por animal) por vía intraperitoneal. Administrar analgésicos como buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 mL cada 6-12 horas durante 48 horas postoperatorio.</li>
	<li>Comprima la vejiga urinaria en el hipogastrio para ayudar con la micción. Realice esto dos veces al día a la misma hora (8 am y 8 pm) durante seis semanas. NOTA: La pérdida de la constricción normal del detrusor es el símbolo de DU.</li>
	<li>Aloje a todas las ratas en jaulas metabólicas, cada una con un embudo de recolección de orina colocado sobre un papel absorbente previamente pesado para controlar la micción y la incontinencia.</li>
	<li>Recoja y anote el cambio de peso del papel absorbente, que indica el volumen vaciado (VV) y el volumen de orina residual por separado.</li>
</ol>4. Ensayos urodinámicos
<ol><li>A las seis semanas después de la operación, realice un cistometrograma, utilizando un equipo de medición urodinámica de la siguiente manera.<ol><li>Anestesiar ratas inyectando hidrato de cloral al 10% en la cavidad peritoneal (3 mL/kg).</li>
		<li>Comprima la vejiga para la micción, luego fije la rata a la plataforma quirúrgica con una cinta.</li>
		<li>Inserte el catéter epidural (3F) en la vejiga y conecte el equipo de medición urodinámica, el catéter epidural y la bomba de infusión mediante el tubo de tres extremidades.</li>
		<li>Bombee solución salina fisiológica a una velocidad de 0,2 mL/min para medición urodinámica (ver Tabla de materiales). Registre el MCC y DOP, y la distensibilidad de la vejiga (calculada dividiendo δ volumen de la vejiga con δ presión del detrusor).</li>
	</ol></li>
</ol>5. Análisis estadístico
<ol><li>Realizar análisis estadísticos utilizando software disponible comercialmente.</li>
	<li>Utilice la prueba de Kolmogorov-Smirnov para probar la normalidad de los datos.</li>
	<li>Expresar las variables normalmente distribuidas como valores medios con desviaciones estándar. Utilice las pruebas t de Student pareadas de dos colas para comparar los parámetros del cistometrograma en ambos grupos. NOTA: p < 0,05 indica que la diferencia tuvo significación estadística.</li>
</ol>

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Los autores no tienen nada que revelar.

El uranio empobrecido es una causa común de síntomas del tracto urinario inferior tanto en hombres como en mujeres. Es una constelación compleja de síntomas con pocas opciones de tratamiento que pueden disminuir significativamente la calidad de vida (CdV) de los afectados18. Aunque se cree que el uranio empobrecido es multifactorial, la comprensión de su patogénesis sigue siendo rudimentaria. Los estudios han demostrado que la patogénesis del uranio empobrecido podría estar relacionada con...

La hipoactividad del detrusor (UD) es una disfunción típica del tracto urinario inferior que ha permanecido poco estudiada. A pesar de que el DU ha sido definido por la International Continence Society (ICS)1, se utilizan numerosas terminologías diferentes para referirse a esta enfermedad, por ejemplo, "insuficiencia del detrusor", "vejiga contráctil", "arreflexia del detrusor"2. El uranio empobrecido, según lo definido por la Sociedad Internacional de Continencia (ICS) en 2002, es una contracción de fuerza y duración reducidas, lo que resulta en un aumento prolongado del tiempo para el vaciado de la vejiga, lo que resulta en la imposibilidad de lograr el vaciado completo de la vejiga dentro de un período normal.
El uranio empobrecido puede afectar al 48% de los hombres y al 12% de las mujeres (de >70 años)3 con síntomas del tracto urinario inferior. Parece ser multifactorial, y no existe un tratamiento eficaz. Se informa que el uranio empobrecido es ubicuo en pacientes con disfunción neurogénica de la vejiga, como la esclerosis múltiple4, la diabetes mellitus5, la enfermedad de Parkinson6 o el accidente cerebrovascular7. El uranio empobrecido también puede ser causado por daño del nervio iatrogénico, como histerectomía laparoscópica, prostatectomía u otras intervenciones quirúrgicas en la pelvis delgada8. Los cambios fisiopatológicos y los tratamientos disponibles para el uranio empobrecido siguen siendo confusos debido a la falta de un modelo animal apropiado para el estudio.
El reflejo miccional está controlado por vías espino-bulboespinales que combinan el centro miccional pontino, el núcleo parasimpático sacro y los centros de corteza más antiguos9. La activación y el mantenimiento del reflejo miccional dependen principalmente del transporte regular de señales sensoriales desde la vejiga a los centros de la corteza más antiguos. Se puede postular que la disfunción sensorial contribuye al uranio empobrecido.
La mayoría de los estudios experimentales en animales relacionados con disfunciones del tracto urinario inferior se han centrado en modelos de vejiga hiperactiva (VH)10. Estos modelos proporcionan una comprensión razonable de la fisiopatología y el pronóstico de la VH. Sin embargo, solo se han descrito unos pocos modelos de uranio empobrecido, p. ej., lesión supraespinal (lesiones locales, descerebración y oclusión de la arteria cerebral media), lesión por transección o contusión de la médula espinal, administración sistémica (p. ej., ciclofosfamida) o intravesical de agentes irritantes o inflamatorios (p. ej., ácido, acroleína y lipopolisacárido)11,12,13,14 . Entre estos métodos, solo se puede usar el método de transección de la médula espinal o lesión por contusión para establecer un modelo animal de uranio empobrecido13. Los intentos que involucraron la lesión del centro miccional pontino y los centros de la corteza superior fueron abandonados debido al trauma severo. Por lo tanto, se está prestando mayor atención para encontrar una ubicación precisa en el centro reflejo miccional para inducir el uranio empobrecido con efectos secundarios mínimos.
Como se mencionó anteriormente, uno de los mecanismos de inducir uranio empobrecido es lesionar la médula espinal para dañar la vía de señalización del reflejo miccional. El método de pérdida de peso de Allen fue desarrollado para establecer animales de laboratorio con médula espinal lesionada15. Sin embargo, no hay más datos experimentales disponibles sobre este método. Además, dado que partes de los animales recuperaron la función espinal después de un accidente cerebrovascular sin uranio empobrecido, no puede considerarse como un método perfecto para generar un modelo animal de uranio empobrecido16.
En 1987, Bregman excogitó un proceso de transección de la médula espinal para generar el modelo animal de uranio empobrecido y adquirió datos experimentales17. Sin embargo, este método no se aplicó para establecer el modelo animal de uranio empobrecido. En ese momento, los investigadores todavía estaban confundidos acerca de la patogénesis del uranio empobrecido. Como las ubicaciones en la médula espinal asociadas con la inducción de VH o UE son adyacentes entre sí, no pudieron encontrar el sitio preciso de daño a la médula espinal para inducir uranio empobrecido17. OAB y DU se introdujeron juntos o por separado por este método. Por lo tanto, aunque este método introdujo el uranio empobrecido, era impreciso y no podía utilizarse para comprender la ocurrencia y el procesamiento del uranio empobrecido.
Como se indicó anteriormente, la falta de un modelo animal adecuado de uranio empobrecido es uno de los principales obstáculos para el estudio del uranio empobrecido. Los investigadores buscan continuamente un modelo preciso y manejable que pueda simular la patología del uranio empobrecido. Incluso las opciones de tratamiento para el uranio empobrecido no han mejorado significativamente durante los últimos 20 años. Colectivamente, existe una gran necesidad de describir un protocolo estándar para establecer un modelo animal de uranio empobrecido.
Entonces, en este artículo, describimos un método para establecer con éxito un modelo de rata de DU por transección del cono medularris. La transección se realizó en el nivel L4-L5 para separar el cono medular. La capacidad cistométrica máxima (CCM), la presión de apertura del detrusor (DOP) y la distensibilidad de la vejiga se registraron y analizaron para validar el protocolo. El protocolo que se indica a continuación combina tanto la viabilidad como la fiabilidad de una manera estandarizada para establecer el modelo animal de uranio empobrecido, simulando la ocurrencia y el procesamiento del uranio empobrecido. El protocolo se puede utilizar como una técnica para el estudio adicional del uranio empobrecido.

<table><tbody><tr><td>0.9% saline</td><td>Wuhan Prosai Company</td><td>EY-C1178</td><td>pump for urodynamic measurement</td></tr><tr><td>10% chloral hydrate</td><td>Shandong Yulong Co., Ltd</td><td>H37022673</td><td>3mL/kg, administered intraperitoneally</td></tr><tr><td>Buprenorphine Hydrochloride Injection</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td><td>H12020275</td><td>0.05mg/kg subcutaneously 24h and 48h postoperation</td></tr><tr><td>Epidural Catheter</td><td>Shandong Xinghua Co, Ltd</td><td>VABR3L</td><td>for urodynamic measurement</td></tr><tr><td>Penicillin G</td><td>Alta Technology Co., Ltd</td><td>1ST5637</td><td>50,000 unit/ml per animal</td></tr><tr><td>pentobarbital</td><td>Beijing solabo Technology Co., Ltd</td><td>NK-WF0001</td><td>40 mg/kg, administered intraperitoneally</td></tr><tr><td>Suture line(4-0)</td><td>ETHICON</td><td>VCP422H</td><td>suture the injury</td></tr><tr><td>Three-limb tube</td><td>Shandong Xinghua Co, Ltd</td><td>VAB3T</td><td>for urodynamic measurement</td></tr><tr><td>Trace infusion pump</td><td>Zhejiang Smith Medical Instrument Co., Ltd</td><td>20162540335</td><td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td></tr><tr><td>Urodynamic measurement equipment</td><td>Medical Measurement SystemsB.V.</td><td>08-0467</td><td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td></tr><tr><td>Wistar Rats</td><td>HFK Biotechnology Co.Ltd,Beijing ,China</td><td>SCXK2012-0023</td><td>200-220g</td></tr></tbody></table>

detrusor underactivity model in rats by conus medullaris transection

El objetivo del protocolo presentado fue establecer un modelo de hipoactividad del detrusor (DU) en la rata a través de la transección del cono medular. La laminectomía se realizó en un total de 40 ratas Wistar hembra (grupo control: 10 ratas; grupo de prueba: 30 ratas) con un peso de 200-220 g, y el cono medular se transectó en el nivel L4-L5 en el grupo de prueba. Todas las ratas fueron alojadas y alimentadas bajo las mismas condiciones ambientales durante seis semanas. En el grupo de prueba, la micción de orina se realizó dos veces al día durante seis semanas, y se registró el volumen residual medio de orina. Se realizó un cistometrograma en ambos grupos. Se registraron y calcularon la capacidad cistométrica máxima (CCM), la presión de apertura del detrusor (DOP) y la distensibilidad de la vejiga. El grupo de prueba mostró retención urinaria significativa después de la cirugía, tanto durante como después del shock espinal. Sin embargo, no se observó ninguna anomalía en el grupo control. En comparación con el grupo control, el CCM y la distensibilidad de la vejiga en el grupo de prueba fueron significativamente mayores que los del grupo de prueba (3,24 ± 2,261 ml versus 1,04 ± 0,571 ml; 0,43 ± 0,578 ml / cmH 2 O frente a 0,032 ± 0,016 ml / cmH 2 O), mientras que la DOP en el grupo de pruebafue menor que el control (20,28 ± 14,022 cmH 2 O versus 35 ± 13,258 cmH2 O). Este método de establecer un modelo animal de uranio empobrecido mediante la transección del cono medular ofrece una excelente oportunidad para comprender mejor la fisiopatología del uranio empobrecido.

Todo el procedimiento de la transección del cono medular puede completarse en 45 minutos por cirujanos experimentados. Nuestro laboratorio ha realizado más de 100 casos de cirugías de transección de cono medular. La tasa de éxito es superior al 95%, según lo definido por la supervivencia de las ratas y la inducción exitosa de uranio empobrecido. La prueba urodinámica confirmó la inducción de DU.
Según nuestra experiencia, la inducción de DU puede evaluarse preliminarmente por el vo...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Presentamos un método para establecer un modelo de subactividad del detrusor mediante transección del cono medular en ratas. La falta de actividad del detrusor se estimuló con éxito en estos animales. El modelo se puede utilizar para estudiar la función del tracto urinario.

Modelo de hipoactividad del detrusor en ratas por transección del cono medullaris

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K vistas.  Capital Medical University. Presentamos un método para establecer un modelo de subactividad del detrusor mediante transección del cono medular en ratas. La falta de actividad del detrusor se estimuló con éxito en estos animales. El modelo se puede utilizar para estudiar la función del tracto urinario.

Video: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

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 ...

Investigación

Bioingeniería

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

El uso de la cistometría en pequeños roedores: un estudio de la vejiga chemosensation

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Medicina

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Las respuestas de la Vejiga Urinaria Distensión Evocados visceromotor como modelo para la vejiga Dolor en ratones

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

Vejiga contractilidad del músculo liso de Gaza como un método para evaluar tracto urinario inferior Farmacología

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Preservación de los nervios de mitad de obstrucción uretral (NEMO) en femenino pequeños roedores

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Evaluación del procedimiento para realizar cistometría despierta en un modelo de ratón

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

Un modelo de vejiga murina descentralizada (Ex Vivo) con el músculo detrusor eliminado para el acceso directo al suburotelio durante el llenado de la vejiga

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Medición de esfínter uretral externo cistométrico en ratas despiertas con catéter implantado y electrodos para medidas repetidas

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Biología

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Inducción de estenosis uretral seguida de uretroplastia de injerto de mucosa bucal en un modelo de rata

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...

Efficient Rat Model for Urethral Stricture: Electric Knife Method

Iatrogenic Injury Recapitulated: Electroexcision Technique for Urethral Stricture Modeling in Rats

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for US, such as urethral dilatation and internal urethrotomy, fail to fully resolve the condition and are associated with high rates of recurrence and complications. 
Additionally, the pathogenesis of US is not well understood. To explore the pathogenesis of US and develop new therapeutic strategies, it is crucial to establish a standardized rat model that accurately reflects the clinical manifestations. This study outlines a straightforward and repeatable method for inducing US in rats using a high-frequency electric knife. The method involves making a longitudinal incision with the electric knife set to a unipolar mixed cutting mode at 4 W, which inflicts significant urethral damage. Histopathological analysis shows thickening of the urothelium, inflammatory infiltration, and disorganized collagen fibers. This model effectively replicates iatrogenic injury through electroexcision in the rat urethra. In summary, this study successfully establishes a new, efficient, and stable rat model of US that closely mimics the clinical scenario, providing a valuable tool for further research into the mechanisms and novel treatments for US.

In this study, we present a novel, efficient, and stable rat model of urethral stricture created through electroexcision of the rat urethra, which effectively simulates iatrogenic injury observed in clinical settings.

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for ...