This work was supported by funding from the March of Dimes FY12-133 (S.K.E.) and the National Institutes of Health R01HD037831 (S.K.E.). We would like to thank Dr. Deborah Frank for critical review of the manuscript.

NOTA: Todos los animales procedimientos conforma a las directrices para el cuidado y uso de animales establecidos por los Institutos Nacionales de Salud. Todos los protocolos fueron aprobados por la Comisión del Estudio de animales en la Universidad de Washington en St. Louis. NOTA: Los materiales y equipos específicos para este protocolo se enumeran Materiales y Equipos mesa. 1. inseminación a tiempo fijo de ratones hembra <ol><li> Raza hembras adultas entre dos y seis meses de edad en una ventana de tiempo de 2 horas con los varones. Hr específico de cría no son necesarios, siempre y cuando se mantengan consistentes para la totalidad del experimento. </li><li> Confirmar el embarazo por la presencia de un tapón de copulador. Designar el día y la hora en la que el tapón se ve por primera vez como días post-coito 0 (dpc0). Recuerde la hora específica cuando se inicia la grabación de la presión. </li><li> Realizar cirugías en dpc8-13. El DPC exacta dependerá de la cepa de ratón. </li><li> Para determinar si un ratón está embarazada antes de la cirugía, peimágenes de ultrasonido rforma del abdomen con una sonda de matriz lineal 40 MHz acoplado a un sistema de formación de imágenes antes de administrar la anestesia. Esto permitirá la visualización de sacos gestacionales. Si un sistema de ultrasonidos no está disponible, utilice el peso del ratón. NOTA: Un aumento de peso de al menos 2 g entre dpc0 y dpc8 y distensión del abdomen es un buen indicador de embarazo. </li></ol> 2. Telemetría Cirugía Prep <ol><li> Utilice un autoclave para esterilizar los instrumentos, gasas, torundas de algodón, paños quirúrgicos, y puntas de pipeta dentro de 24 horas antes de la cirugía. </li><li> Superficie cirugía limpia con etanol al 70%. </li><li> Encienda el cojín eléctrico (especializado para roedores) y esterilizador de cuentas. </li></ol> 3. Telemetría Cirugía <ol><li> Para la anestesia, utilizar 4-5% de isoflurano con una tasa de flujo de oxígeno de 1-2 L / min. </li><li> Espere hasta que el ratón muestra signos de anestesia y luego inyectar por vía subcutánea con buprenorfina (analgésico: 0,1 ml por 20 g). </li><li> Afeitar el abdomen del ratón desde la parte inferior de las costillas hacia abajo a la vejiga. </li><li> Traiga el ratón a la mesa de cirugía en el cojín de calefacción pre-calentado, y cubrir los ojos del ratón con pomada oftálmica para evitar el secado. </li><li> Comience técnica estéril. Póngase los guantes estériles y mantener la envoltura como una superficie estéril en el que colocar los matorrales de yodo y etanol. </li><li> Retire el telémetro del paquete o solución salina y utilizar pinzas estériles para colocarlo sobre una gasa estéril. <ol><li> Utilice la jeringa (proporcionado por el proveedor telémetro) para llenar la punta del catéter con gel. Apriete suavemente el catéter crear suficiente presión negativa para extraer el gel en el catéter. </li></ol></li><li> Coloque una punta estéril sobre la pipeta. Pipetear 2 l de pegamento quirúrgico y establecer la pipeta sobre una superficie estéril. </li><li> Para limpiar el sitio quirúrgico, limpie el área con yodo y luego con etanol al 70%. </li><li> Cortar un agujero en la sábana quirúrgica lo suficientemente grandepara el área necesaria para la incisión y el lugar sobre el ratón. </li><li> Realice una pizca pie (la respuesta del pedal) para asegurar que el ratón está completamente anestesiado antes de hacer una incisión. </li><li> Hacer una pequeña incisión en la línea media vertical. A continuación, hacer una incisión similar a través de la pared muscular del cuerpo subyacente. </li><li> Tire suavemente del útero de la cavidad del cuerpo y localice el cuerno uterino que contiene el mayor número de crías que sean viables. <ol><li> Opcional - Para añadir un miRNA, inyecte vector viral en la capa muscular del útero antes de la inserción del telémetro. </li></ol></li><li> Hacer una pequeña incisión en la punta del cuerno uterino con 3 mm de corte tijeras de primavera de borde. </li><li> Pase el catéter, sin apretar, entre la pared uterina y sacos fetales. Inserte el catéter pasado varios sacos de reducir su eliminación accidental. Asegúrese de que los sacos fetales no se vean perturbadas por la inserción. </li><li> Pipetear el pegamento quirúrgico para el sitio de inserción telémetro para adherir el catéter a la ucuerno terine y evitar que el catéter se deslice fuera del cuerno uterino. Espere unos segundos para que el pegamento se vuelven rígidas y coloque cuidadosamente el útero hacia el interior de la cavidad corporal. </li><li> Coloque el telémetro en la cavidad del cuerpo en el lado opuesto del sitio de inserción del catéter. </li><li> Suturar la cavidad del cuerpo se cerró con 6,0 suturas absorbibles. En primer lugar, hacer una puntada bolso y luego tres y cincuenta y siete puntadas individuales. </li><li> Suturar la piel mediante el uso de la misma técnica, pero con 6-0 sutura no absorbible. </li><li> Dibucaina Swab (analgésico tópico) ungüento sobre el lugar de la incisión. </li><li> Inyectar 0,3 a 0,4 ml de solución salina estéril por vía subcutánea para ayudar a rehidratar al animal. </li><li> Monitorear el animal después de la cirugía hasta que esté completamente despierto. No coloque el ratón de nuevo en su jaula hasta que sea capaz de sostener libremente su cabeza por encima de la mesa, como ciertos tipos de ropa de cama pueden sofocar un ratón muy anestesiado. Para un ratón que ha sido anestesiado durante 30-45 min, uno debería esperar ªe ratón para ser completamente despierto por 1 hora y 15 min. </li><li> Si la realización de cirugías adicionales, limpie instrumentos con etanol al 70% y el talón esterilizar antes de su reutilización. </li></ol> 4. Después de la cirugía de recuperación <ol><li> Supervisar diariamente el progreso después de la cirugía en un registro. Catálogo aumento de peso y la pérdida y el reloj para letargo, sangrado o cierre incompleto de la incisión quirúrgica. <ol><li> Alimente el ratón chow suavizado ratón (~ la consistencia de puré de manzana) o especializada alimento de la dieta de recuperación. </li><li> Examinar las suturas y asegurarse de que no se sacan, pero trate de no manejar el ratón en exceso. </li></ol></li></ol> 5. La telemetría de grabación <ol><li> En el día de interés (por lo menos de dos a tres días después el ratón se ha recuperado de la cirugía), colocar un imán cerca del ratón para encender el telémetro. Coloque la jaula del ratón en el receptor. La luz en el receptor debería encenderse si se activa el telémetro. </li><li> Calibrar nueva Teleme marca<html> tros con calibraciones específicas del fabricante siempre. Especificaciones de calibración telémetro reutilizados serán guardados en el software. </li><li> Dentro del software, iniciar la grabación haciendo clic derecho sobre &quot;animal&quot; y haga clic en &quot;iniciar el muestreo, continua&quot;, que permite el muestreo a una velocidad de hasta 500 Hz. Asegúrese de que el software está configurado para guardar y rastrear. NOTA: Cuando el icono de muestreo del ratón cambia a verde, se están grabando las presiones. Perturbar el ratón durante el parto puede interrumpir o alterar el trabajo, así que observar con cautela. </li><li> Después del parto o periodo de tiempo de interés, detenga la grabación haciendo clic derecho sobre &quot;animal&quot; y seleccionando &quot;detener las grabaciones, todo&quot;. Utilice un imán para apagar telémetro para salvar la vida de la batería, ya que la batería telémetro normalmente sólo tendrá una duración de 1,5 meses de grabación continua. </li><li> La eutanasia el ratón por CO 2 sobredosis y recuperar el telémetro. Los cachorros son sacrificados por decapitación. </li></ol>tle &quot;&gt; 6. telémetro Esterilización <ol><li> Coloque el telémetro en una solución de 1% de enzima detergente durante la noche en agua desionizada a temperatura ambiente. </li><li> Enjuague el telémetro en agua desionizada y eliminar todo el tejido y el pegamento, teniendo cuidado de no apretar el catéter. </li><li> Incubar el telémetro en glutaraldehído al 2% durante 24 horas a temperatura ambiente para esterilizar. </li><li> Enjuague el telémetro con solución salina estéril y almacenarlo en una solución salina estéril hasta la próxima implantación o para ser enviado de nuevo a fabricante a ser reformado. Telémetros deben ser reformadas si el tejido se encuentra en el interior del catéter después de la esterilización. Normalmente telémetros se pueden utilizar 4-5 veces antes de necesitar ser renovado. </li></ol>

<ol>
	<li>Slattery, M. M., Morrison, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+delivery.">Preterm delivery.</a> Lancet. 360, 1489-1497 (2002).</li><li>Goldenberg, R. L., Culhane, J. F., Iams, J. D., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+causes+of+preterm+birth.">Epidemiology and causes of preterm birth.</a> Lancet. 371, 75-84 (2008).</li><li>Keelan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+inflammatory+pathways+for+the+prevention+of+preterm+birth.">Pharmacological inhibition of inflammatory pathways for the prevention of preterm birth.</a> Journal Of Reproductive Immunology. 88, 176-184 (2011).</li><li>Voltolini, 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=Understanding+spontaneous+preterm+birth:+from+underlying+mechanisms+to+predictive+and+preventive+interventions.">Understanding spontaneous preterm birth: from underlying mechanisms to predictive and preventive interventions.</a> Reproductive Sciences. 20, 1274-1292 (2013).</li><li>Ratajczak, C. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+parturition+biology+from+genetically+altered+mice.">Insights into parturition biology from genetically altered mice.</a> Pediatric research. 64, 581-589 (2008).</li><li>Rajakumar, A., 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=Placental+HIF-1+alpha,+HIF-2+alpha,+membrane+and+soluble+VEGF+receptor-1+proteins+are+not+increased+in+normotensive+pregnancies+complicated+by+late-onset+intrauterine+growth+restriction.">Placental HIF-1 alpha, HIF-2 alpha, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 293, (2007).</li><li>Whitesall, S. E., Hoff, J. B., Vollmer, A. P., D'Alecy, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.+American+journal+of+physiology.">Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. American journal of physiology.</a> Heart And Circulatory Physiology. 286, H2408-H2415 (2004).</li><li>Mackay, L. B., Shi, L. B., Maul, H. .., Maner, W. L., Garfield, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bilateral+pelvic+neurectomy+on+cervical+ripening+in+pregnant+rats.">The effect of bilateral pelvic neurectomy on cervical ripening in pregnant rats.</a> Journal Of Perinatal Medicine. 37, 263-269 (2009).</li><li>Pierce, S. L., Kutschke, W., Cabeza, R., England, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+intrauterine+pressure+by+telemetry:+a+new+approach+for+studying+parturition+in+mouse+models.">In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models.</a> Physiol Genomics. 42, 310-316 (2010).</li><li>Imamura, T., Luedke, C. E., Vogt, S. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+modulates+the+onset+of+murine+parturition+by+competing+ovarian+and+uterine+effects.">Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 279, R1061-R1067 (2000).</li><li>McCloskey, 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=The+inwardly+rectifying+K++channel+KIR7.1+controls+uterine+excitability+throughout+pregnancy.">The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.</a> EMBO Mol Med. 6 (9), 1161-1174 (2014).</li></ol>

The authors have nothing to disclose.

Las investigaciones de la contractilidad del miometrio se han basado en las mediciones in vivo ex de la tensión muscular. Esta metodología puede ser útil para realizar pruebas en etapa temprana de fármacos uterotónicos recientemente desarrollados que no pueden ser administrados a los animales vivos. Sin embargo, los enfoques in vivo son necesarios para comprender longitudinalmente la progresión de la mano de obra. Las mediciones in vivo sirven para varios propósitos. En primer lugar, pe...

El nacimiento prematuro es la principal causa de morbilidad y mortalidad perinatal en los países desarrollados; que es responsable del 50% de la morbilidad perinatal y el 75% de los 1,2 mortalidad perinatal. El parto prematuro es multifacética y puede ser idiopática. Aunque muchas investigaciones han surgido en las vías moleculares y eléctricas que transforman el miometrio de un tejido quiescente en un contráctil uno, la fisiopatología exacta del trabajo de parto prematuro sigue siendo difícil de alcanzar. Endocrino, inflamatoria, y la regulación de genes han sido relacionados con el parto prematuro 3,4. Sin embargo, las preocupaciones éticas limitan la capacidad para llevar a cabo la investigación sobre los mecanismos de trabajo de parto prematuro en los seres humanos. Dadas estas limitaciones, muchos investigadores han recurrido a los ratones como sistema modelo con el que el estudio de la fisiología del parto. Los ratones tienen gestaciones corta duración aproximadamente tres semanas y pueden ser fácilmente manipulable genéticamente. Además, varios modelos genéticos de ratón tienenha desarrollado para determinar las vías de señalización que son esenciales para el trabajo 5. A pesar de las diferencias clave entre el ratón y el parto humano, los ratones y los humanos comparten muchos de los mismos mecanismos que son esenciales para el trabajo, incluyendo la inflamación y la infección intrauterina 6. De este modo, los ratones sirven como una valiosa herramienta para examinar la actividad uterina. Hasta la fecha, el estándar de oro para medir la contractilidad uterina en ratones ha sido ex vivo grabaciones tensión isométrica; sin embargo, esto está limitado a un punto de tiempo gestacional por experimento y requiere la extirpación del útero a partir de su entorno fisiológico. Otro factor importante es el gran número de animales necesarios para estas investigaciones. Por último, esta metodología no permite estudios longitudinales que examinan la inducción del trabajo de parto y parto prematuro fisiopatología. Los recientes avances realizados en los dispositivos de telemetría radiométricas utilizadas para estudiar la presión arterial cambia 7 en ratones y intpresión rauterine en ratas 8 plantea la cuestión de si la misma tecnología podría usarse para estudiar los cambios en la presión intrauterina en ratones durante el embarazo. Después de la resolución de problemas inicial, se desarrolló un método con éxito para medir la inducción del parto y la progresión en un ratón. Este enfoque in vivo puede medir la transición desde el estado de baja presión del útero de reposo al estado de alta presión indicativa de fuertes contracciones del parto. Este método también ha sido capaz de detectar diferencias de presión significativas entre los ratones transgénicos con el parto comprometido y ratones de tipo salvaje, lo que demuestra el impacto fisiológico de la expresión génica 9. Este protocolo proporciona un tiempo real, en el método in vivo para estudiar la presión uterina ratón durante el embarazo.

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intrauterine telemetry to measure mouse contractile pressure in vivo

Se necesita una integración compleja de señales moleculares y eléctricos para transformar un útero en reposo en un órgano contráctil al final del embarazo. A pesar del descubrimiento de los principales reguladores de la contractilidad uterina, este proceso todavía no se comprende totalmente. Los ratones transgénicos proporcionan un modelo ideal en el que estudiar el parto. Anteriormente, el único método para estudiar la contractilidad uterina en el ratón era ex vivo grabaciones de tensión isométrica, que son subóptima por varias razones. El útero debe ser retirado de su entorno fisiológico, un curso de tiempo limitado de investigación es posible, y los ratones debe ser sacrificada. El reciente desarrollo de telemetría radiométrica ha permitido, mediciones en tiempo real longitudinales de la presión intrauterina in vivo en ratones. Aquí, la implantación de un telémetro intrauterino para medir los cambios de presión en el útero de ratón a partir de la mitad del embarazo hasta que se describe entrega. Mediante la comparación de las diferencias en la prensaUres entre tipo salvaje y ratones transgénicos, el impacto fisiológico de un gen de interés puede ser dilucidado. Esta técnica debería acelerar el desarrollo de terapias que se utilizan para tratar los trastornos del miometrio durante el embarazo, incluyendo el parto prematuro.

En tiempo real de las presiones intrauterinas in vivo se puede grabar mediante el uso de un sistema de adquisición de telemetría. Muestreo de presión junto con la grabación de vídeo simultánea del ratón se utiliza para capturar el momento exacto de la entrega de cada cachorro y correlacionar el tiempo de entrega a la presión intrauterina de la madre. Todos los puntos de datos pueden entonces ser trazados a lo largo de la grabación para generar una parcela (Figura 1A) de presi?...

Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetría intrauterina para medir la presión del ratón contráctil<em&gt; En Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

intrauterine-telemetry-to-measure-mouse-contractile-pressure-in-vivo

Research

JoVE Journal

Medicine

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

9.6K vistas.  Washington University in St. Louis. This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy. 

Video: Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

La presión de la ventilación controlada para inducir la lesión pulmonar aguda en ratones

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Investigación

Medicina

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Medición in vivo de la capa de ratón pulmonar superficie endotelial

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Ecocardiografía Fetal y Ecografía Doppler de onda pulsada en un modelo de conejo de Restricción del crecimiento intrauterino

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo MRI de 19F para el seguimiento de la célula

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Precisión en Medicina Dental, Una nueva forma de medir veracidad y precisión

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medición de la curva de presión-volumen en mouse Pulmones

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

Trabecular Respuesta malla de elevación de la presión en el Living Ojo humano

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cateterismo cardíaco en ratones para medir la relación de volumen Presión: Investigar el efecto Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

A largo plazo de medida de la presión arterial en ratones con libertad de movimientos mediante telemetría

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...