Este trabajo fue apoyado por subvenciones del Gobierno federal belga (IUAP P6/28), la Fundación de Investigación de Flandes (FWO) (G.0565.07 y G.0686.09), la Astellas European Foundation Award 2009 y el Consejo de Investigación de la KU Leuven (GOA 2009/07, EF/95/010 y PFV/10/006). PU y NOSOTROS somos los estudiantes de doctorado de la Fundación de Investigación de Flandes (FWO). MB es una beca Marie Curie. DDR un compañero fundamental-clínica de la FWO.

1. Animales de Laboratorio <ol><li> Los animales (ratones, ratas) están alojados en una instalación de animales especializada con un 12-horas de luz-oscuridad ciclo y el acceso ad libitum al agua y pastillas estándar de alimentos. Tanto la edad y el sexo de los animales son parámetros importantes que deben ser estandarizados de acuerdo con las necesidades. Nos suelen realizar cistometría en 10 a 12 semanas de edad, las hembras 5,6. </li><li> Todos los experimentos con animales se realicen de conformidad con las directrices europeas en materia comunitarias Consejo y aprobado por el comité de ética local. </li></ol> 2. Anestesia <ol><li> Anestesia con isoflurano se utiliza para realizar pequeños procedimientos quirúrgicos, ya que es fácil de dosificar debido a su vida media corta. Gas de desecho adecuado de barrido es necesario cuando se utiliza el isoflurano. <ol class="lalpha"><li> Los animales se colocan en una caja cerrada, gaseados con isoflurano 5% en oxígeno puro (1 l / min). </li><li> Después de la inducción, anestesia en el l apropiadoevel es mantenida por una pequeña máscara (un corte jeringa 10 ml) en 0,8 - 1 l / min de oxígeno con 1 a 1,5% de isoflurano. </li></ol></li><li> Para los ratones, la anestesia de uretano se utiliza durante las grabaciones funcionales (cfr. infra). Anestesia con uretano se utiliza durante las grabaciones funcionales porque, en contraste con la mayoría de otros anestésicos, el reflejo de micción no se suprime por este agente anestésico 4. Uretano de 1,2 g / kg de peso corporal se administra por vía subcutánea (sc) 60 min antes del inicio de las grabaciones. Si es necesario, la dosis de uretano necesario para obtener la anestesia adecuada se pueden valorar mediante la administración de dosis adicionales (0,1 g / kg) por vía subcutánea. Cabe señalar, sin embargo, que la anestesia adicional puede alterar la eficiencia de la micción y por consiguiente la frecuencia de la micción 7,8. Por lo tanto, las grabaciones cistométricos deben ser estrechamente vigilados y se desecha cuando desviaciones significativas de la conducta de control se observan. Los anestésicos no deben administrarse mientras pruebacompuestos se aplican para evitar una interpretación errónea de los datos. </li></ol> 3. Procedimiento quirúrgico - Implantación del catéter vesical <ol><li> Los instrumentos quirúrgicos se esterilizaron en un autoclave antes de su uso. Afeitar el abdomen del animal, desinfectar con etanol 70% y realizar una laparotomía media baja para exponer la vejiga. </li><li> Bajo un microscopio quirúrgico, coloque una sutura en bolsa de tabaco en la cúpula de la vejiga usando un no-absorbible, sutura monofilamento (tamaño 6-0). </li><li> Realizar una cistostomía pequeña (que preferentemente usan una aguja 18 G) en el interior del cordón de bolsa e insertar un catéter de polietileno PE 50, con un pequeño manguito en el extremo (obtenido calentando con precaución el tubo) a través de este agujero. En nuestras manos, tubos más delgados (PE 10) tienen una mayor resistencia y más variable. El tubo puede ser desinfectado colocándolas en una solución de etanol al 70% o esterilizado en un autoclave. Antes de la implantación, el tubo debe ser lavada con solución salina. </li><li> Fije la bolsa strING alrededor del tubo con un nudo de cirujano. Tire del catéter ligeramente hacia fuera hasta que la punta en forma de campana se coloca justo debajo de la sutura. Empuje un pedazo de catéter G 18 hacia la sutura para evitar fugas. </li><li> Infundir un volumen pequeño de solución salina (100 - 200 l) suavemente para comprobar las fugas. Si hay fugas, una sutura adicional debe ser colocado. </li><li> Cerrar los músculos abdominales utilizando suturas de monofilamento, dejando un paso para el catéter en la parte superior de la incisión. </li><li> Túnel del tubo a la región interescapular, utilizando una barra de metal hueco, evitando así que los animales para morder el tubo durante el experimento. </li><li> La piel se cierra utilizando no absorbibles, las suturas de monofilamento. El catéter implantado se lava con solución salina para comprobar su permeabilidad. </li><li> Antes de que los animales de despertar, los analgésicos se administran subcutáneamente (buprenorfina, 0,05 mg / kg). </li></ol> 4. Configuración y cistometría <ol><li> Dependiendo de la experimental questien (y las especies utilizadas) cistometría se realiza despierto (moderación o el desenfreno) o anestesiados con uretano animales. Considerando que la cistometría es perfectamente factible en ratas conscientes, que tienden a mantener la calma en un entorno restringido, por lo general realizar cistometría bajo anestesia con uretano (1,2 mg / kg, por vía subcutánea) utilizando ratones 3,5,6,9. </li><li> En ratones anestesiados, la temperatura corporal se controla constantemente y se mantuvo a 36,5 ° C ± 0,2 ° C por medio de una sonda de temperatura y una lámpara de calentamiento. </li><li> La puesta a punto es calibrado antes de cada experimento de acuerdo con las instrucciones del fabricante. </li><li> El catéter implantado está conectado por un trozo luer a un grifo de 3-way, que está conectado al transductor de presión (Edwards Lifesciences) en un lado y una bomba de infusión por la otra (Harvard Apparatus). El transductor de presión está conectado a través de un amplificador (78534c monitor, Hewlett Packard) para el sistema de adquisición de datos (DI-730-USB, DATAQ instruments) y un computer. Windaq / Lite software puede ser utilizado para la grabación. </li><li> La bomba de infusión se inicia, lo que permite la infusión de solución salina o PBS a, por ejemplo, 100 l / min (en ratas) o 20 l / min (en ratones). Como tal, la vejiga se llena a una velocidad constante, mientras que la presión intravesical se registró. </li><li> La Figura 5 representa una traza de presión típico: durante la infusión de fluido, hay una acumulación de presión lenta en la vejiga, hasta un cierto volumen / presión se alcanza, el umbral de micción. Una vez que se alcanza este umbral, la vejiga se contrae y, posteriormente, el esfínter urinario se abrirá, permitiendo el paso de la orina a través de la uretra. Como tal, se vacía la vejiga, la contracción se detiene y la presión caerá de nuevo a la &quot;basal&quot; nivel. </li><li> A medida que la rata se coloca en una jaula metabólica y por encima de una balanza digital, el volumen evacuado se pueden medir simultáneamente, proporcionando información sobre el volumen evacuado. Debido a los pequeños volúmenes anulados en mice, puede ser muy difícil de usar este sistema. Por lo tanto, volumen vacío se determina utilizando pequeños papeles de filtro pesados ​​antes y después de la micción. </li><li> Típicamente, se deja que el sistema se equilibre durante 30 min, seguido por un período de registro de 30 min. Entonces, los medicamentos pueden ser administrados sistémicamente o por vía intravesical y otros 30 min se registran. </li><li> Los datos registrados se pueden exportar como archivos csv. Que se pueden importar en un software especializado para el análisis cuantitativo. Nos suelen utilizar Origin (OriginLab Corporation, MA, EE.UU.). </li><li> Después de los experimentos, los animales se sacrificaron por dislocación cervical (ratones) o CO 2 intoxicación (ratas). </li></ol> 5. Los resultados representativos <img alt="Figura 1" src="/files/ftp_upload/3869/3869fig1.jpg" /> Figura 1. Panorama laparotomía. A) Coloque la rata en la posición supina. B) Shave y antisépticamente preparar el sitio quirúrgico. C) La incisión de la piel. D) Incisión oe los músculos abdominales y la exposición vejiga. <img alt="Figura 2" src="/files/ftp_upload/3869/3869fig2.jpg" /> Figura 2. Una sutura en bolsa de tabaco. <img alt="Figura 3" src="/files/ftp_upload/3869/3869fig3.jpg" /> Figura 3. Implantación de un catéter. <img alt="Figura 4" src="/files/ftp_upload/3869/3869fig4.jpg" /> Figura 4. Excavación de túneles del catéter. Ejemplos de mediciones de la presión intravesical obtenidos durante la perfusión de solución salina en una rata consciente y un ratón anestesiado se muestra en la Figura 5. Múltiples parámetros pueden ser extraídos de la señal de presión (por ejemplo, el intervalo de intercontractile, la presión de referencia y la presión de umbral). Para obtener una descripción completa de estos parámetros, consulte Andersson et al. (Ref. 4) y Yoshiyama et al. (<html> ref 10). Recientemente hemos utilizado cistometría para identificar las dianas moleculares de aceite de mostaza (MO), un compuesto muy reactivo que se ha utilizado durante mucho tiempo en los modelos experimentales de inflamación y la hiperalgesia de los órganos viscerales, tales como la vejiga urinaria 11,12. Infusión intravesical de 10 mM MO indujo un fuerte aumento de la frecuencia miccional (disminución del intervalo intercontractile) en ratones de tipo salvaje (Figura 6A, B) y una disminución del volumen vaciado 6. Curiosamente, MO indujo cambios similares en ratones deficientes del receptor TRPA1 MO. Por el contrario, MO inducido cambios mucho más débiles en los parámetros cistométricos en TRPV1 ratones KO que en ratones WT y fue sin ningún efecto en Trpa1/Trpv1 ratones KO. Junto con las mediciones de la liberación de la calcitonina Gene Related Peptide (CGRP) 6, estos datos indican demostrar que TRPV1 puede jugar un papel clave en la irritación visceral inducida por MO. ve_content &quot;&gt; <img alt="Figura 5" src="/files/ftp_upload/3869/3869fig5.jpg" /> Figura 5. Trazas representativas de la presión intravesical registrado en una rata consciente hembra (A) y en un ratón anestesiado hembra (B). La presión más baja se define como la &quot;presión de línea de base&quot; (flechas rojas). La presión al final de la fase de llenado está marcado con flechas azules. El volumen de líquido infundido entre estos puntos, dividido por la diferencia de presión, permite el cálculo de la conformidad de la pared de la vejiga (compliance = volumen infundido / (presión umbral -. Presión basal) El &quot;intervalo intercontractile&quot; (ICI) es el tiempo entre dos contracciones miccionales. <img alt="La figura 6" src="/files/ftp_upload/3869/3869fig6.jpg" /> Figura 6. Efectos de la aplicación intravesical de aceite de mostaza en el patrón cistometría de tipo salvaje y ratones TRPA1, TRPV1 y Trpa1/Trpv1 nocaut.(A) Los ejemplos representativos de los cambios de presión intravesical registradas en WT, KO TRPA1, KO TRPV1 y ratones KO Trpa1/Trpv1 en respuesta a la infusión de solución salina y 10 mM MO. (B) Evolución temporal de la frecuencia miccional promedio instantánea antes y durante la infusión intravesical de MO. Para todos los ratones, los datos se normalizaron a la frecuencia media obtenida durante la infusión salina. Estos datos son una adaptación de Everaerts et al. (Ref. 6), con permiso de Elsevier.

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No hay conflictos de interés declarado.

La técnica de la cistometría aquí se presenta permite realizar mediciones in vivo de la función vesical en modelos animales. Las ratas son probablemente el modelo animal más utilizado. Los ratones son más difíciles de manejar, pero ofrecen la ventaja de la utilización de animales manipulados genéticamente. Debido a la dificultad técnica de la utilización de ratones conscientes, que tienden a ser muy activo dando como resultado el aflojamiento del catéter implantado y los cambios en las presiones int...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Nombre comercial, empresa, </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> uretano </td><td> Uretano, Sigma-Aldrich </td><td> 315419 </td><td> carcinógeno grupo 2B </td></tr><tr><td> isoflurano </td><td> Isoba, Schering-Plough Animal Health </td><td></td><td></td></tr><tr><td> catéter de polietileno </td><td> Polietileno tubo PE50 Intramedic, Becton Dickinson </td><td> 427411 </td><td></td></tr><tr><td> microscopio quirúrgico </td><td> Op-Mi 6, Carl Zeiss </td><td> Op-Mi 6 </td><td></td></tr><tr><td> sutura en bolsa de tabaco </td><td> Prolene 6/0, Ethicon </td><td> 8610H </td><td></td></tr><tr><td> fascia y sutura de la piel </td><td> Ethilon 4/0 o 5/0, Ethicon </td><td> 662G 661G o </td><td></td></tr><tr><td> analgésicos postoperatorios </td><td> Temgesic, Schering-Plough Animal Health </td><td></td><td> dosis para ratas: 0,05 mg / kg </td></tr><tr><td> amplificador </td><td> 78534c monitor, Hewlett Packard </td><td></td><td></td></tr><tr><td> balanzas analíticas y de balanza de software de adquisición de datos </td><td> FZ 300i, A &amp; D </td><td> FZ-300i </td><td></td></tr><tr><td> bombas de infusión </td><td> bomba 33, Harvard aparato </td><td> HA33 </td><td></td></tr><tr><td> cistometría sistema de grabación </td><td> Instrumentos DATAQ, DI-730 serie y Windaq / Lite </td><td> DI-730-USB Windaq / Lite </td><td></td></tr><tr><td> temperatura registro </td><td> Fluke 52 Termómetro KJ </td><td> 52 KJ </td><td></td></tr><tr><td> transductores de presión </td><td> Edwards Lifesciences, conjunto de control de presión </td><td> T322247A </td><td></td></tr></tbody></table>

the use of cystometry in small rodents: a study of bladder chemosensation

<p class="jove_content"&gt; Las tracto urinario inferior (LUT) funciona como un depósito dinámico que es capaz de almacenar la orina y para expulsar eficientemente que en un momento conveniente. Mientras que el almacenamiento de orina, sin embargo, la vejiga está expuesto durante períodos prolongados a los productos de desecho. Al actuar como una barrera apretado, el revestimiento epitelial de la LUT, el urotelio, evita la re-absorción de sustancias nocivas. Además, los productos químicos nocivos estimular inervación nociceptiva de la vejiga e iniciar la micción contracciones que expulsan los contenidos de la vejiga. Curiosamente, la sensibilidad de la vejiga para los productos químicos nocivos se ha utilizado con éxito en la práctica clínica, por vía intravesical infusión de la capsaicina TRPV1 agonista para tratar la hiperactividad de la vejiga neurogénica<sup&gt; 1</sup&gt;. Esto pone de relieve la ventaja de ver la vejiga como un órgano quimiosensorial y solicita investigación clínica adicional. Sin embargo, las cuestiones éticas limitan seriamente las posibilidades de llevar a cabo, en el ser humano, las mediciones invasivas que son necesarios para desentrañar las bases moleculares de la farmacología clínica LUT. Una manera de superar esta limitación es el uso de varios modelos animales<sup&gt; 2</sup&gt;. Aquí se describe la aplicación de la cistometría en ratas y ratones, una técnica que permite la medición de la presión intravesical en condiciones de perfusión vejiga controlada.</p&gt;<p class="jove_content"&gt; Después de la laparotomía, un catéter se implanta en la cúpula de la vejiga y subcutáneamente en la región interescapular. A continuación, la vejiga puede ser llenado a una velocidad controlada, mientras que la uretra se deja libre para la micción. Durante los ciclos repetitivos de llenado y vaciado, la presión intravesical se puede medir a través del catéter implantado. Como tal, los cambios de presión pueden ser cuantificados y analizados. Además, la medición simultánea del volumen miccional permite distinguir contracciones de vaciado de no miccionales contracciones<sup&gt; 3</sup&gt;.</p&gt;<p class="jove_content"&gt; Es importante destacar que, debido a las diferencias en el control de la micción entre roedores y humanos, las mediciones cistométricos en estos animales tienen sólo un valor limitado traslacional<sup&gt; 4</sup&gt;. Sin embargo, son bastante instrumental en el estudio de la fisiopatología de la vejiga y de la farmacología experimental en pre-clínicos configuración. Investigaciones recientes utilizando esta técnica ha puesto de manifiesto el papel clave de los nuevos jugadores moleculares en las propiedades mecano-y quimio-sensoriales de la vejiga.</p

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

Cystometry es una técnica eficaz para medir la función de la vejiga de animales pequeños<em&gt; In vivo</em&gt;. La vejiga se infundió continuamente a velocidades controladas a través de un catéter intravesical, mientras que la uretra se deja libre para la micción. Esto permite repetitivo de llenado y vaciado de la vejiga, mientras que la presión intravesical y volumen evacuado se registran.

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 ...

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Research

JoVE Journal

Medicine

22.3K vistas.  KU Leuven, Belgium. Cystometry es una técnica eficaz para medir la función de la vejiga de animales pequeños In vivo. La vejiga se infundió continuamente a velocidades controladas a través de un catéter intravesical, mientras que la uretra se deja libre para la micción. Esto permite repetitivo de llenado y vaciado de la vejiga, mientras que la presión intravesical y volumen evacuado se registran.

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

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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Limb remoto Preacondicionamiento Isquémico: una técnica neuroprotector en roedores

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

Realizar Subretinal Inyecciones en roedores administrar células de la retina epitelio pigmentario en Suspensión

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Evaluación de la eficacia de la<em&gt; H. pylori</em&gt; Proteína HP-PAN como herramienta terapéutica para el tratamiento de cáncer de vejiga en un modelo murino ortotópico

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

El uso de ultra-alta MRI Field en Modelos pequeño roedor de la enfermedad renal poliquística para<em&gt; En Vivo</em&gt; Fenotipificación de Drogas y Vigilancia

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

Uso de un Modelo de Cochinillo para el Estudio de la Neurotoxicidad de Desarrollo inducida por Anestesia (AIDN): Un Enfoque de Neurociencia Translacional

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

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 ...