McCauley marca está respaldada por un mentor NIH-formación conceder 5T32HL066991-07 Xander Wehrens es apoyado por el NIH / NHLBI subvenciones 1R01HL091947-01A209 y 3R01HL089598 03S109-

1. Preparación de telémetro de implantación quirúrgica <ol><li> Antes de introducir un telémetro ECG en el ratón, es importante asegurarse de que el telémetro ECG es estéril y en buen estado de funcionamiento. Los nuevos dispositivos de telemetría se proporcionan típicamente en condiciones estériles por el fabricante. Telémetros ECG puede ser reutilizado siempre que el dispositivo se limpia con solución Tergazyme 1% durante al menos 4 horas. Usted puede enjuagar el telémetro con agua estéril después de la limpieza con Tergazyme. Además, el uso Wavicide-01 durante la noche desinfectante para esterilizar el telémetro ECG. Asegúrese de lavar desinfectante con agua estéril 48 horas antes de la implantación en ratones. Guardar en un recipiente estéril. </li><li> Compruebe el telémetro conduce a la integridad tanto de los hilos conductores y de la vaina aislante. Encienda el transmisor de telemetría mediante un imán saludó con la mano dentro de los 5 cm del telémetro, y prueba de la señal con una frecuencia de radio AM 530,. La señal debe ser fuerte y claro y que varían en intensidad y el tono basado en la manipulación de los cables. Anote el número de modelo, el número de serie telémetro, y el valor de calibración del ECG. Los fabricantes suelen recomendar que el telémetro debe ser por 24 horas antes de la implantación, consulte el manual de instrucciones de su telémetro específicos para más detalles. En este ejemplo, vamos a utilizar una Sciences Data International (DSI) telémetro para su implantación. </li><li> Preparación telémetro de plomo puede ser logrado mediante la reducción de los efectos negativos (blanco) dan lugar a aproximadamente 3,5 cm y el positivo (rojo) a 2,5 cm. Estas son las longitudes óptimas para la implantación del cable negativo en el lado derecho del pecho, y el cable positivo en el abdomen a la izquierda (ver figura 2). A continuación, retire la envoltura de aislamiento para exponer 7 mm de alambre. Pintar el extremo del cable de metal con pegamento estériles, como Vet-Bond, y luego coloque las tapas llevan a las puntas de metal. Estas tapas se evitará la erosión de la piel debido a la colocación del electrodo. Aproximadamente 2.3 mm de alambre de metal deben ser expuestos para la detección eléctrica del ritmo del corazón nativo. </li></ol> 2. La implantación quirúrgica del telémetro <ol><li> Para utilizar una técnica estéril en la cirugía de murino, tendrá que guantes estériles, un campo estéril, y las suturas de Prolene 6-0. Usted también tendrá que dos pares de pinzas esterilizadas, un par de extremos romos tijeras, un bisturí, y un conductor de la aguja. Los instrumentos quirúrgicos se pueden esterilizar en un esterilizador de cuentas de vidrio se calienta a 250 grados Celsius. Asegúrese de esterilizar y lavar el telémetro como se describió previamente antes de su uso. </li><li> Prepare el ratón para la cirugía por primera anestesiar el ratón en una caja de inducción con 3% de isoflurano en 0,5 L / min O2 al 100%. Use una almohadilla eléctrica, tales como T-bomba que hace circular el agua caliente, para mantener la temperatura del ratón anestesiado es. La temperatura puede ser controlada por la sonda rectal, si lo desea. Cuando el ratón está adecuadamente sedado, completamente afeitado el abdomen y el pecho del ratón con una maquinilla eléctrica. Volver a anestesiar a los ratones con isoflurano antes de transferir el ratón a un tubo de flujo constante, en decúbito supino, con la cabeza de espaldas y la cola hacia usted. Pegue las patas de los ratones a la mesa de operaciones con calefacción. Limpie el abdomen con un algodón con alcohol para eliminar el vello rasurado y la limpieza del campo operatorio. Aplicar tres capas diferentes de betadine con hisopos para desinfectar el abdomen y el pecho. Verificar el nivel correcto de la anestesia mediante la aplicación de presión sobre el lecho de la uña del ratón. Después de que el ratón se ha preparado para la cirugía, el uso de guantes estériles y aplicar el campo estéril en el campo quirúrgico. </li><li> Telémetro implantación intraperitoneal ofrece la ventaja de la experimentación de fisiología del ejercicio. Para la inserción intraperitoneal, comenzar la cirugía mediante el uso de un bisturí para crear una incisión vertical en la línea media de la piel que recubre el abdomen alrededor de 2,5 cm de longitud. Con cuidado, separa el pellejo de los tejidos conectivos subyacentes con extremos romos tijeras. A continuación, cree una incisión vertical en la línea media la línea alba que cubre el peritoneo, aproximadamente 1,5 cm de longitud. Además, crear un pequeño agujero en el peritoneo por encima (craneal) de la incisión peritoneal, el cual servirá como una salida por vía subcutánea conduce. Mientras se realiza la cirugía, mantener la humedad del lugar de la cirugía de vez en cuando gotas de solución salina estéril en el campo operatorio. </li><li> Inserte el telémetro en la cavidad peritoneal derecha. Inserte pinzas en el agujero superior al telémetro y tire de ambos lleva a través del orificio principal por lo que están saliendo de la cavidad peritoneal. Uso continuo 6-0 suturas de Prolene para cerrar la incisión de 1,5 cm peritoneal línea media vertical. </li></ol> 3. El plomo implantación y el cierre abdominal <ol><li> Los cables de ECG se colocan a la cabeza de configuración II. A la cabeza con la funda blanca / transparente es negativo, y se coloca en el abdomen superior izquierdo. En primer lugar, crear una incisión de 0,5 cm de la piel en el pecho superior derecho del ratón.A continuación, utilice la tijera sin punta para crear un túnel de vuelta a la incisión abdominal. Saque el cable a través del túnel y el uso de una sutura de prolene 6-0 para fijar la cabeza en el músculo pectoral. Asegúrese de que la sutura en la parte superior de la parte expuesta de la cabeza, y crea un buen contacto entre el plomo y el músculo subyacente. Use una segunda sutura proximal a la mencionada una, para inmovilizar la cabeza con el músculo. Cierre la incisión en la piel con una sutura de Prolene 6-0. </li><li> El cable positivo (vaina roja) se coloca en el abdomen izquierdo debajo del diafragma izquierdo y debajo del corazón. La iniciativa se basa en el tejido peritoneal subyacente por sutura de Prolene 6-0 y debe tener un buen contacto con el tejido peritoneal. Se hace una incisión adicional no es necesario en este paso porque el sitio de implante del electrodo está cerca de la incisión de la línea media vertical, por lo tanto se puede acercar con el campo quirúrgico ya existente. Tenga en cuenta que el tejido peritoneal debe ser levantada para evitar la perforación del intestino subyacente. </li><li> Cerrar la fascia abdominal y la piel de forma secuencial con Prolene 6-0 en capas. </li></ol> 4. Cuidados post-operatorios <ol><li> Dar el ratón 0,1 ml de [1 mg / ml] buprenorfina para la analgesia en el postoperatorio inmediato. Deje que el ratón para recuperarse de una cirugía en un cojín de calefacción. Si el ratón ha tenido una cirugía prolongada&gt; 30 min, o si el ratón se vea seca, se puede inyectar la solución salina estéril al 0.2 hasta 0.3 mL en el peritoneo para la rehidratación. Para la continuación de la analgesia postoperatoria es una práctica estándar para proporcionar ratones con buprenorfina dos veces al día durante tres días, y luego, según sea necesario cada ocho horas. </li><li> Limpie el betadine con una gasa con alcohol para evitar la irritación post-quirúrgica. Recuerde que debe registrar el peso post-quirúrgico para la determinación posterior de la post-quirúrgica de la salud. </li><li> 8-24 horas después de la cirugía, darle al ratón analgesia con buprenorfina cuando sea necesario, ya que esto reducirá el dolor y reducir el fracaso en la principal de ratón de arañazos en el sitio de post-quirúrgica. </li></ol> 5. Resultados representante Cuando se realiza correctamente, el ratón se han cerrado con incisiones abdominales subcutánea lleva debajo de la piel. Por lo general, el ratón lleva 10-30 minutos para recuperarse de la anestesia. Se tarda unos 70-10 días antes de que los ratones se han recuperado completamente de la cirugía, como lo demuestra la recuperación de la pérdida de peso post-operatorio y la normalización de la movilidad. La complicación más frecuente de la cirugía es la erosión de las derivaciones de las extremidades a través de la piel en los primeros 10 días después de la cirugía. Esta complicación común se puede evitar firmemente adherido subcutánea lleva a los tejidos subcutáneos o el peritoneo. Esta técnica evita lleva floja, que crean la presión y la fricción en la piel cuando el ratón deambula. Aunque es menos común, la infección y la sepsis puede ocurrir por errores en la técnica estéril, o de la limpieza incompleta de los telémetros utilizado. Salida de ECG puede ser registrada por una matriz de receptor junto con el software de adquisición de datos, tales como Data Sciences International (DSI). Resultados de forma de onda debe incluir una claramente definida onda P, que indica la despolarización auricular, y también una onda QRS que representa la despolarización ventricular, como se muestra en la Figura 3. Una buena calidad del trazado del ECG debe tener claramente definida una onda P que precede a una ola de QRS claramente definidos en una proporción de 1:1. No debe ser la señal de fondo bajo y los intervalos RR debe ser regular en ratones de tipo salvaje. Una mala calidad del trazado del ECG pueden tener indistinta o las ondas P QRS, o la señal de fondo, lo que puede hacer el análisis, al siguiente equipo difícil. Un pobre el trazado del ECG pueden ser fijados por lleva telémetro re-implantación, por lo general uno a la vez. Para volver a implantar una ventaja de telémetro, coloque el ratón bajo anestesia, y el uso de la técnica quirúrgica, hacer una incisión en la piel que recubre directamente a la cabeza en cuestión. Cuando la cabeza se accede, corte el hilo de sutura de anclaje a la cabeza, y mover la cabeza a la posición deseada antes de anclar la ventaja de nuevo. Finalmente, cerca de la zona quirúrgica con Prolene 6-0. <img src="/files/ftp_upload/1739/1739fig1.jpg" alt="Figura 1" /> Figura 1: Un ejemplo de un telémetro de animales pequeños con cables positivo y negativo. En general, el cable rojo es el positivo y el cable blanco es el cable negativo. Cada cable debe tener una cubierta aislante, de 5-7 mm de cable expuesto, y una punta de plástico tapado, lo que evita la erosión de la iniciativa a través de la piel. <img src="/files/ftp_upload/1739/1739fig2.jpg" alt="Figura 2" /> Figura 2: dibujos animados que muestran los lugares de implantación adecuada telémetro de un ratón. El blanco (negativo) se implanta en el pecho superior derecho del ratón, y el rojo (positivo) se coloca en el abdomen izquierdo, de tal manera que los sentidos telémetro la &quot;ventaja de dos&quot; de configuración. &lt;clase p = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1739/1739fig3.jpg" alt="Figura 3" /> Figura 3: Representante de forma de onda de un ratón en ritmo sinusal normal. La onda P representa la despolarización auricular y el complejo QRS representa la despolarización ventricular. Una forma de onda de alta calidad debe tener una onda P diferente que precede a cada complejo QRS en una proporción de 1:1.

<ol>
	<li>Cerrone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+ventricular+tachycardia+and+fibrillation+elicited+in+a+knock-in+mouse+model+carrier+of+a+mutation+in+the+cardiac+ryanodine+receptor.">Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor.</a> Circ Res. 96 (10), e77-e77 (2005).</li><li>Chelu, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+kinase+II-mediated+sarcoplasmic+reticulum+Ca2++leak+promotes+atrial+fibrillation+in+mice.">Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.</a> J Clin Invest. 119 (7), 1940-1940 (1940).</li><li>Sood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+leak+due+to+FKBP12.6+deficiency+in+mice+facilitates+the+inducibility+of+atrial+fibrillation.">Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.</a> Heart Rhythm. 5 (7), 1047-1047 (2008).</li><li>Swoap, S. J., Gutilla, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+changes+during+daily+torpor+in+the+laboratory+mouse.">Cardiovascular changes during daily torpor in the laboratory mouse.</a> Am J Physiol Regul Integr Comp Physiol. 297 (3), R769-R769 (2009).</li><li>Costa-Goncalves, d. a., C, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+multidomain+protein+spinophilin+in+blood+pressure+and+cardiac+function+regulation.">Role of the multidomain protein spinophilin in blood pressure and cardiac function regulation.</a> Hypertension. 52 (4), 702-702 (2008).</li><li>Wehrens, X. H., Kirchhoff, S., Doevendans, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+electrocardiography:+an+interval+of+thirty+years.">Mouse electrocardiography: an interval of thirty years.</a> Cardiovasc Res. 45 (1), 231-231 (2000).</li></ol>

Los pasos críticos de este procedimiento son la limpieza y la preparación correcta del ratón antes de la cirugía la supervivencia. Cuando un telémetro y el sitio del ratón operativo se limpian correctamente, hay un mucho mejor resultado post-quirúrgico. Además, mucha atención a la colocación del electrodo reduce post-quirúrgica erosión de la piel. Posibles modificaciones de la técnica incluyen configuraciones alternativas para evaluar la función del VI pared lateral (derivación I de configuración), o el ...

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<td>Steri 250 Bead Sterilizer</td>
<td>&nbsp;</td>
<td>Inotech</td>
<td>IS-250</td>
<td>&nbsp;</td>
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<td>Blunt-Ended Scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5980</td>
<td>&nbsp;</td>
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<td>Blunt-Ended Forceps (x2)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5138</td>
<td>&nbsp;</td>
<tr>
<td>Veterinary Anesthesia Vaporizer</td>
<td>&nbsp;</td>
<td>Surgivet</td>
<td>Isotec 4</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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ambulatory ecg recording in mice

Telemétricas registro del ECG en los ratones es esencial para entender los mecanismos detrás de las arritmias, trastornos de la conducción, y la muerte cardiaca repentina. Aunque el ECG de superficie se utiliza para mediciones de corta duración de los intervalos de la forma de onda, no es práctico para los estudios a largo plazo de la variabilidad del ritmo cardíaco o la captura de raros episodios de arritmias. Implantables telémetros ECG ofrecen las ventajas de la implantación quirúrgica sencilla, a largo plazo de registro de los electrogramas en ratones ambulatoria, y escalabilidad con registros simultáneos de varios animales. A continuación, presentamos una guía paso a paso para la implantación de telémetros para el registro del ECG ambulatorio en los ratones. La adhesión cuidadosa a la técnica aséptica se requiere para obtener resultados favorables de supervivencia con la posibilidad de la implantación y la grabación de semanas a meses. Por lo tanto, la telemetría ECG implantable es una herramienta valiosa para la detección de información crítica sobre la electrofisiología cardíaca en modelos animales ambulatorios como el ratón.

Watch this Scientific Journal Video about Ambulatory ECG Recording in Mice at JoVE.com

Ambulatory ECG Recording in Mice

ECG telemétrico se ha convertido en una herramienta esencial en la evaluación de modelos animales para las arritmias cardíacas y muerte súbita cardíaca. A continuación, presentamos una guía paso a paso para telemetría ECG para su aplicación a largo plazo de la monitorización ambulatoria de ECG en ratones.

Ambulatorio de grabación ECG en ratones

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Research

JoVE Journal

Biology

23.5K vistas.  Baylor College of Medicine (BCM). ECG telemétrico se ha convertido en una herramienta esencial en la evaluación de modelos animales para las arritmias cardíacas y muerte súbita cardíaca. A continuación, presentamos una guía paso a paso para telemetría ECG para su aplicación a largo plazo de la monitorización ambulatoria de ECG en ratones.

Video: Ambulatory ECG Recording in Mice

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

El trasplante cardíaco heterotópico en ratones

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Investigación

Biología

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Trasplante de intestino delgado en ratones

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

Medición de la presión arterial en ratones con registro del volumen de presión, un método de la cola del manguito

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Registro electrofisiológico en el Drosophila Embrión

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Constricción de la aorta transversa en ratones

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal celular conectado a Patch-clamp grabación

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Las inyecciones intravenosas en ratones recién nacidos

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Grabación de la presión intracavernosa para evaluar la función eréctil en roedores

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

Inducir la Periodontitis Apical en Ratones

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Establecimiento de un modelo de fractura de fémur diafisario en ratones

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...