Sandeep Khurana está apoyado por el NIH (K08DKO81479). Vikrant Rachakonda es apoyado por (T32DK067872).

Los ejemplos que se muestran aquí son de experimentos aprobados por IACUC en Georgia Regents de la Universidad - Protocolo nº: # 2011-0408 1. Preparación de los reactivos <ol><li> Prepare la solución de la disección de almacén: Para 500 ml de solución madre de disección (5x), se disuelven 21,18 g de NaCl, 0,875 g de KCl, 0,739 g MgSO4, 1,049 g MOPS y 0,019 g de EDTA en 450 ml de agua Milli-Q. Ajustar el pH a 7.3 a 7.4 utilizando 1 N NaOH. Completar el volumen hasta 500 ml con agua Milli-Q. Solución de Stock se pueden almacenar hasta 7-10 días. Ver Tabla 1 para una lista de productos químicos y sus proveedores. Ver Tabla 2 para la concentración en mM. </li><li> Prepare la solución de la disección de trabajo: Preparar la solución de trabajo de disección fresco todos los días. Por 100 ml de solución de trabajo, se disuelven 0,091 g de glucosa, 0,016 g de NaH 2 PO 4 y 0,022 g de piruvato de sodio en 79,8 ml de agua Milli-Q. Añadir 0,2 ml de 1 M de CaCl2 y 20 ml de solución de disección de acciones a llevar volume a 100 ml. </li><li> Preparar una solución salina fisiológica (PSS): Para preparar 1000 ml PSS, disuelven 0,365 g de KCl, NaCl 6,545 g, 0,296 g MgSO4, 0,163 g de KH 2 PO 4, 2,072 g de glucosa, 2,184 g de NaHCO 3 y 2,383 g de HEPES en 950 ml de agua Milli-Q. Ajustar el pH a 7.3 a 7.4 utilizando 1 N NaOH. Completar el volumen a 1000 ml con agua Milli-Q. Retire 2 ml de solución y reemplazarla con 2 ml de 1 M de CaCl2. (Fresh PSS tiene que estar preparado al día) </li><li> Preparar calcio (Ca2 +) PSS libre: Para 100 ml PSS sin Ca 2+, se disuelven 0,036 g de KCl, NaCl 0,654 g, 0,029 g MgSO4, 0,016 g de KH 2 PO 4, 0,207 g de glucosa, 0,218 g de NaHCO3, 0.238 g HEPES, 0,015 g EGTA y 0,0026 g Sodio nitroprusiato (SNP) en 95 ml de agua Milli-Q. Ajustar el pH a 7.3 a 7.4 utilizando 1 N NaOH. Completar el volumen hasta 100 ml con agua Milli-Q. </li></ol> 2. Preparación de la Copa cánulas <ol><li> Tire de pipetas de vidriopara generar los 100 a 150 micras cánulas de punta utilizando un extractor de pipeta según las directrices del fabricante. </li><li> Bisel las puntas de la cánula de vidrio utilizando un beveller microelectrodos, fuego pulir ellos y doblar las puntas de la cánula de vidrio por ~ 45 ° utilizando una sonda de calor. </li><li> Cargue las cánulas en el soporte micropipeta y adjuntar el soporte micropipeta a la cámara de perfusión. </li></ol> 3. Preparación de cámara de perfusión <ol><li> Cámara de perfusión Enjuague con agua Milli-Q seguido de solución de disección durante 5 minutos cada uno. Cargar la cámara con 2 ml de solución de disección. </li><li> Solución de disección de succión a través de la cánula utilizando 10 ml jeringa y llenar cuidadosamente toda la cánula y el tubo conectado sin burbujas. Aplicar succión suavemente para evitar la generación de burbujas. </li><li> Preparar dos suturas con un medio nudo cada uso de fórceps romos. Desde suturas de nylon de monofilamento oftálmicas (10-0, 0,2 métrico) se utilizan para preparar los nudos que son sólo1-2 mm de diámetro, puede ser necesaria microscopio de disección. </li><li> La visualización al microscopio de disección, utilizar pinzas de disección para cargar ambas cánulas con nudos de sutura parcialmente cerrados un poco lejos de la punta. Más tarde estos nudos se deslizaron con cuidado sobre los extremos arteriales canulados y cerraron por completo. </li></ol> 4. Recolección de la arteria mesentérica Arcade de ratas Sprague-Dawley <ol><li> Buscar la aprobación del Comité de Cuidado y Uso de Animales institucional local (IACUC) antes de realizar estos experimentos. Animales de la vivienda en las instalaciones de animales con temperatura controlada y la iluminación y permitir el libre acceso a agua y una comida para roedores comercial. </li><li> Anestesiar las ratas por inyección intraperitoneal de ketamina (80 mg / kg) y xilazina (10 mg / kg). Confirmar profunda anestesia por toe-pinch y si Administrar necesaria anestésicos adicionales. </li><li> Después de confirmar la anestesia quirúrgica, la eutanasia a los animales por decapitación. Siga las directrices AAALAC para utilizing métodos apropiados para la eutanasia de animales. </li><li> Use una tijera de disección y una pinza para realizar una laparotomía línea media de la pelvis al esternón. Esto se hace en dos pasos: primero, haga una incisión en la piel y segundo, haga una incisión de la capa muscular subyacente. Se debe tener cuidado de no lesionar los órganos intraabdominales. </li><li> Cortar el extremo proximal del intestino cerca del píloro y el extremo distal cerca de la unión íleo-cecal. Tie ambos extremos por separado para evitar la fuga de quimo y las heces, evitando así la contaminación de solución de baño extracelular. Incisión en el mesenterio en su base, cerca de la alimentación vasculatura es decir, la arteria mesentérica superior y la transferencia de todo el intestino cama mesentérica intestinal a un vaso de precipitados de 50 ml que contenía solución de disección enfriado con hielo. </li><li> Permita tejido recogido de alojarse en el hielo solución de disección fría durante 5 minutos y enjuague con solución disección fresca para deshacerse de la sangre. </li></ol> 5. El aislamiento y la canulación de 4º </sup&gt; Solicitar mesentérica arteria <ol><li> Pin hacia abajo el extremo proximal del intestino en el lado de la mano derecha en un plato Sylgard recubiertos. Extender el intestino restante en un camino en sentido antihorario, el fijar el segmento hacia abajo para difundir el mesenterio y la exposición de los vasos sanguíneos (Figura 1). Nota: Nos aislamos segmentos arteriales a temperatura ambiente. De lo contrario ponemos la arcada mesentérica contiene plato en hielo. Algunos laboratorios, incluidos los que en nuestra institución, utilizan unidades de refrigeración para diseccionar las arterias a 4 ° C. </li><li> Bajo el microscopio zoom estéreo diseccionar 3 ° y 4 ° de orden pequeñas arterias mesentéricas (~ 260 m) paralela al intestino delgado utilizando unas tijeras pequeñas. Primero diseccionar toda la grasa de cobertura. Entonces diseccionar la vena y aislar la arteria con punto de bifurcación en forma de V. Tenga cuidado de no perforar el segmento seleccionado. Iniciar la disección de la grasa cerca de una rama para 2º y encontrar la manera de 3º o 4º ordvasos er. <ol><li> Nota: Las arterias y las venas se pueden distinguir en función de su espesor de pared - pared arterial es más gruesa que la de la vena. Además, cuando contiguo tejido conectivo se tira suavemente perpendicular a los vasos, venas colapso fácilmente mientras que las arterias no. Desde arterias con diámetro de la luz &lt;400 micras son los principales sitios de la resistencia vascular sistémica, para este protocolo que utilizamos 4º arterias mesentéricas fin de rata (diámetro de la luz &lt;300 micras). </li></ol></li><li> Aislar una sección de 4-5 mm de la arteria paralelo al intestino delgado. Visualice todas las 5 ramas de orden ª incrustación en el intestino delgado y se cortan un poco lejos del origen de las ramas y preservar una parte. Estas porciones conservadas de ramas sirven como sitios de la celebración (con unas pinzas de disección) para transferir segmentos arteriales a una cámara de perfusión y, posteriormente guiar su canulación. </li><li> A continuación, corte los segmentos arteriales al hacer 2 incisiones distallas ramas de orden 5 th en cada lado de la arteria y transferirla a la cámara de perfusión (véase la Figura 1C y la leyenda). </li><li> Canular un extremo de los vasos en una de micropipeta de vidrio (diámetro: 100-150 micras) utilizando pinzas de disección mediante la celebración de las puntas del segmento arterial con unas pinzas de disección. Deslice la sutura parcialmente cerrada previamente cargado en el extremo canulado y asegurarla. Nota: El extremo proximal de la arteria puede ser una cánula en la cánula de vidrio que está conectado al dispositivo de regulación de presión servo-controlado para imitar entorno in situ. </li><li> Adjuntar una solución disección cargado 10 ml jeringa para la llave de paso conectado a esta cánula de tal manera que la solución disección en el tubo de conexión de la cánula y se fusiona con llave de paso que en la jeringa. Levantar suavemente la jeringa. La fuerza gravitatoria sobre la solución eliminará la sangre vascular intra desde el extremo abierto del recipiente. Después de retirar la sangre intra-arterial, Cierre la llave de paso. Nota: Como alternativa, coloque la llave de paso para el controlador de presión, encenderlo y aumentar suavemente la presión de 5-10 mm Hg para conseguir el mismo resultado. </li><li> Ate el extremo distal de los vasos sobre una segunda cánula de vidrio trayendo cuidadosamente la otra cánula tan cerca como sea posible del extremo desatado del segmento arterial. Deslice la sutura parcialmente cerrada previamente cargado en el extremo canulado y asegurarla. Se debe tener cuidado no tirar o tirar de los segmentos arteriales. Asegúrese de que las llaves de paso unidas a ambas cánulas están cerrados. </li><li> Traslado cámara de perfusión a la etapa de microscopio invertido equipado con grabación de vídeo en directo. </li><li> Conectar la llave de paso de la cánula atada al extremo proximal del segmento arterial a un dispositivo de regulación de presión servo-controlado y asegúrese de que la llave de paso unido a la otra cánula permanece cerrada para mantener la presión intraluminal estable. </li><li> A continuación, conecte el tubo de vacío al puerto de succión de unaencontrar la perfusión tubería al puerto de la perfusión de la cámara. Nota: el puerto de aguja biselada se utiliza para la aspiración y el puerto aguja roma para la perfusión. </li><li> Iniciar la perfusión del recipiente con PSS caliente a través del calentador solución en línea única (37 ° C, equilibrada con mezcla de gas: 5% de CO 2, 5% O 2 y 90% de N para mantener el pH neutro y una oxigenación adecuada 17) en 2 ml / min utilizando una bomba peristáltica. Gire el vacío también. Coloque un termistor en la cámara para controlar la temperatura continuamente. </li><li> Como la temperatura del PSS en la cámara se acerca a ~ 37 ° C (generalmente dentro de 5 min), aumentar lentamente la presión intraluminal de 20 a 100 mmHg y comprobar si hay fugas de los vasos. Esto se hace usando el ajuste automático de la presión del regulador de presión. Deseche los vasos con fugas y reemplazar con otro segmento. Los vasos con fugas no será capaz de mantener la presión. </li><li> Evaluar el segmento arterial para curvas mientras se mantiene la presión a 100 mmHg.Utilizando el tornillo de la palanca, mueva la cánula para enderezar el segmento arterial. No más de estirar los segmentos arteriales; el objetivo es imitar en la longitud del segmento arterial vivo. </li><li> Reducir la presión a 70 mmHg (para imitar la presión en vivo en la arcada mesentérica 18) y permitir que el segmento arterial para estabilizar y desarrollar tono miogénico. Las arterias pueden estar presurizados de forma variable (40-70 mmHg) de acuerdo con la estrategia experimental y lecho vascular. Una revisión publicada anteriormente proporciona una excelente revisión de la variabilidad en MT en segmentos arteriales de diferentes lechos vasculares 8. </li></ol> 6. Medición de diámetro arterial <ol><li> Ver arterias en objetivo 10x en un microscopio equipado con una cámara de dispositivo de carga acoplada de vídeo monocromo. Mida el diámetro luminal mediante capturadora de video y sistema de detección de bordes en tiempo real. Una lista de los equipos utilizados se proporciona en la Tabla 3. </li><li> Monitorear y buque registro diameter continuamente. </li><li> Observar para el desarrollo de la MT. Nota: Hemos observado que en las arterias mesentéricas de resistencia de ratas, a los 70 mmHg, desarrollo de MT se caracteriza por ~ 20% de disminución en el diámetro. MT varía según la especie de cama y animales vasculares. </li><li> Confirmar la viabilidad vascular mediante la evaluación de las respuestas vasoconstrictoras y vasodilatadoras a 1 M fenilefrina (Phe) y 1 M de acetilcolina (ACh). </li><li> Al final de cada experimento, determinar el diámetro pasiva (PD) mediante la incubación de las arterias en Ca 2 + libre de PSS por 20 min. </li></ol> 7. Respuesta Miogénica <ol><li> Reducir la presión a 20 mm Hg y permitir que el diámetro se estabilice. Aumentar la presión intraluminal en pasos incrementales (20, 40, 60, 80 y 100) y en cada etapa de presión permiten arterias para lograr un diámetro estable (normalmente dentro de 5 min). </li><li> Reducir la presión intraluminal a 20 mmHg e incubar el segmento arterial en Ca 2 + libre PSS contiene 0,39 mMSNP mM EGTA y 0,1. Permitir que el diámetro arterial para estabilizar (por lo general 15 min). </li><li> Repetir la respuesta de la presión a paso en Ca 2 + libre PSS contiene 0,39 mM EGTA y 0,1 mM SNP. </li></ol> 8. Interpretación de los resultados y cálculo de los datos <ol><li> Calcular la MT como la diferencia porcentual de diámetro observado para el Ca 2 + que contienen frente a Ca 2 + libre PSS en cada presión según la siguiente cálculo: <img alt="Ecuación 1" fo:src="/files/ftp_upload/50997/50997eq1highres.jpg" src="/files/ftp_upload/50997/50997eq1.jpg" /></li><li> Para arterias sometidos vasomoción el diámetro puede calcularse promediando la fase de meseta durante 1 min. Expresar los datos recogidos como por ciento de relajación máxima (% PD) de acuerdo con la relación:% de Pd = 100 x [Dd / PD]; Delta d es la diferencia entre el diámetro antes y después de la adición de cualquier compuesto en investigación (por ejemplo, Phe); PD es el diámetro pasiva (también eldiámetro máximo). </li></ol>

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Pasos crítico, solución de problemas y modificaciones En una preparación típica embarcación isobárica, la arteria se presuriza a 70 mmHg entre dos cánulas de vidrio perfundidos con agua tibia (37 ° C) PSS. Después de 30-45 min, arterias desarrollan MT, caracterizado por disminución espontánea de diámetro que se estabiliza en 20-30 min. Las arterias de resistencia de varios lechos vasculares desarrollan MT variable. Por ejemplo la resistencia de rata arterias mesentéricas desarrolla...

Arterias de resistencia pequeños son los principales determinantes de la SVR y desempeñan un papel importante en la patofisiología de muchas enfermedades 1,2. Condiciones como la diabetes 3, embarazo 4, isquemia-reperfusión 5, la obesidad y la hipertensión 6,7 se asocian frecuentemente con la función microvascular alterado. Miografía vascular no sólo puede proporcionar importantes conocimientos sobre los cambios en la función microvascular en diversas enfermedades, sino también ayudar a identificar dianas terapéuticas y evaluar la eficacia de los compuestos vasoactivos. La función vascular se ha estudiado el uso de pequeñas arterias aisladas en condiciones de buques isométricos o isobáricas 8. Descripción detallada de miografía isométrica se proporciona en otro lugar 9. Sin embargo, hay diferencias en los datos obtenidos a partir de isométrica frente a preparaciones isobáricas 10-12. Desde preparaciones arteriales presurizados permiten el estudio de la función microvascular en condiciones casi fisiológicas, lahallazgos obtenidos pueden correlacionarse mejor con el comportamiento in vivo de la cama vascular 8,13. En 1902 Bayliss descrita por primera vez el efecto de la presión transmural del diámetro vascular 14. Se observó en pequeñas arterias de resistencia de diversos lechos vasculares de los conejos, gatos y perros que una disminución en la presión fue seguido por la vasodilatación y un aumento en la presión fue seguido por la vasoconstricción. Este fenómeno se conoce como respuesta miogénica. Bayliss y posteriores investigadores observaron que, en condiciones isobáricas arterias pequeña resistencia desarrollan constricción sostenida conocida como MT 15,16. Tanto la respuesta miogénica y MT pueden ser evaluados utilizando miografía presión (PM) técnica. PM se utiliza principalmente para determinar vasoactividad de pequeñas arterias, venas y otros vasos. Además de evaluar el efecto de compuestos vasoactivos en diámetro vascular, PM - como su nombre indica - se utiliza para evaluar ch presión intravascular mediadaanges en el diámetro vascular. Durante las últimas décadas los avances en los programas informáticos, que mejoran la microscopía de vídeo y pipeta de vidrio tirando, han hecho PM más fácil de realizar. Sin embargo, la disección de los segmentos intactos viables de pequeños vasos sanguíneos sigue siendo tedioso ya veces difícil. Aquí describimos un protocolo detallado para estudiar la respuesta miogénica en pequeñas arterias mesentéricas de resistencia aislados de ratas.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Chemical</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma Aldrich</td>
			<td>A6625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G5767</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene diamine tetra acetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E9884</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3784</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M5162</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Sigma Aldrich</td>
			<td>P6126</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate (NaHCO3 )</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside</td>
			<td>Sigma Aldrich</td>
			<td>13451</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P8574</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1.</td>
		 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>
 <table border="1">
		<tbody>
		 <tr>
			<td>Physiological salt solution (1000 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.365 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>6.545 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.296 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.163 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>2.072 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>2.184 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>2.383 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>998 ml</td>
		 </tr>
		 <tr>
			<td>Ca2+ free physiological salt solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.036 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>0.645 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.029 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>0.207 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>0.218 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>0.238 g</td>
		 </tr>
		 <tr>
			<td>EGTA </td>
			<td>0.39</td>
			<td>0.015 g</td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside </td>
			<td>0.1</td>
			<td>0.0026 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>100 ml</td>
		 </tr>
		 <tr>
			<td>Dissection solution, stock (500 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>145</td>
			<td>21.18 g</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.7</td>
			<td>0.875 g</td>
		 </tr>
		 <tr>
			<td>MgSO4</td>
			<td>1.2</td>
			<td>0.739 g</td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>2</td>
			<td>1.049 g</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>0.02</td>
			<td>0.019 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>500 ml</td>
		 </tr>
		 <tr>
			<td>Working dissection solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection solution stock</td>
			<td>20 ml</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>1.2</td>
			<td>0.091 g</td>
		 </tr>
		 <tr>
			<td>NaH2PO4</td>
			<td>5</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>2</td>
			<td>0.022 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>0.2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>79.8 ml</td>
		 </tr>
	 </tbody>
</table>
 </td>
	 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>Table 2. Composition of Experimetnal solutions</td>
	 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>CCD Monochrome Camera</td>
			<td>The imaging Source</td>
			<td>DMK 21AU04</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Single inline solution heater</td>
			<td>Warner Instruments</td>
			<td>64-0102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thermistor</td>
			<td>Warner Instruments</td>
			<td>64-0108</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual automatic temperature controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instruments </td>
			<td>P-97</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fluorescence System Interface</td>
			<td>IonOptix</td>
			<td>model FSI-700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps and scissors </td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ion Wizard-Core and Analysis</td>
			<td>IonOptix</td>
			<td>Ion Wizard 6.0</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Laboratory tubing</td>
			<td>Silastic </td>
			<td>508-005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Male Sprague Dawley rat</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Master flex console drive</td>
			<td>Cole-parmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Milli-Q Plus Ultrapure Water System</td>
			<td>Millipore</td>
			<td>ZD5211584</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophthalmic monofilament nylon suture </td>
			<td>Ethicon </td>
			<td>9007G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photometry and Dimensioning Microscope</td>
			<td>Motic</td>
			<td>AE31</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pressure Servo Controller with peristaltic pump and pressure transducer</td>
			<td>Living Systems Instrumentation</td>
			<td>PS-200</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereomicroscope</td>
			<td>Nikon Instruments Inc</td>
			<td>SMZ660</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Vessel Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection dish</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-90-S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW120-6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microforge</td>
			<td>Stoelting</td>
			<td>51550</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3.</td>
		 </tr>
	 </tbody>
</table>

assessing myogenic response and vasoactivity in resistance mesenteric arteries using pressure myography

Arterias de resistencia pequeños constreñir y dilatar, respectivamente, en respuesta a un aumento o disminución de la presión intraluminal; este fenómeno se conoce como respuesta miogénica es un regulador clave del flujo sanguíneo local. En condiciones isobáricas arterias de resistencia pequeños desarrollan sostenidas constricción conocido como tono miogénico (MT), que es un importante factor determinante de la resistencia vascular sistémica (SVR). Por lo tanto, los preparativos ex vivo a presión de las arterias de resistencia pequeñas son las principales herramientas para el estudio de la función microvascular en estados-cerca fisiológica. Para lograr esto, un segmento intacto recién aisladas de una arteria resistencia pequeña (diámetro ~ 260 micras) se monta sobre dos cánulas pequeño de vidrio y presurizado. Estas preparaciones arteriales conservan la mayor parte en las características in vivo y evaluación permiso del tono vascular en tiempo real. Aquí le ofrecemos un protocolo detallado para evaluar vasoactividad en presurizadas pequeñas arterias mesentéricas de resistencia de ratas; estas arterias desarrollanvasoconstricción sostenida - aproximadamente 25% del diámetro máximo - cuando presuriza a 70 mmHg. Estas preparaciones arteriales se pueden utilizar para estudiar el efecto de compuestos en investigación sobre la relación entre la presión intra-arterial y vasoactividad y determinar cambios en la función microvascular en modelos animales de diversas enfermedades.

Representación esquemática de un myograph presión típica configuración se muestra en la Figura 1. Los dos extremos de la embarcación se canulan con una micropipeta de vidrio y se aseguran con suturas en ambos lados. Via tubería y una llave de paso abierto, una cánula está conectado a un regulador de presión servo-controlado; la otra cánula está conectado a una llave de paso cerrada. La cámara se perfundió con son observados por un microscopio invertido conectado a una cámara CCD PSS y di?...

Watch this Scientific Journal Video about Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography at JoVE.com

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Miografía de presión se utiliza para evaluar vasoactividad de las pequeñas arterias que se desarrollan constricción sostenida cuando se presuriza. Este manuscrito proporciona un protocolo detallado para evaluar en segmentos aislados de pequeñas arterias mesentéricas de ratas, vasoactividad y el efecto de la presión intraluminal del diámetro vascular.

Evaluar Miogénica Respuesta y Vasoactividad En arterias mesentéricas Resistencia Usando Miografía Presión

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

assessing-myogenic-response-vasoactivity-resistance-mesenteric

Research

JoVE Journal

Medicine

16.6K vistas.  Georgia Regents University.  Miografía de presión se utiliza para evaluar vasoactividad de las pequeñas arterias que se desarrollan constricción sostenida cuando se presuriza. Este manuscrito proporciona un protocolo detallado para evaluar en segmentos aislados de pequeñas arterias mesentéricas de ratas, vasoactividad y el efecto de la presión intraluminal del diámetro vascular. 

Video: Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Contracción de la arteria mesentérica y Estudios de relajación con miografía cable automático

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Investigación

Medicina

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Medición de la presión intracraneal epidural en ratas, utilizando un transductor de presión de fibra óptica

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

El aislamiento y uso de las Secciones de bovino mesentérica arteria y la vena como un bioensayo para la Prueba de Vasoactividad en el intestino delgado

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

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. ...

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 ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inducida por la forskolina Hinchazón en organoides intestinal: Una<em&gt; In Vitro</em&gt; Ensayo para evaluar la respuesta de drogas en pacientes con fibrosis quística

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

Un modelo de cultivo celular de las arterias de resistencia

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Evaluación de mecanismos de Control Vascular utilizando microscopia Video de arterias de resistencia aislada de ratas

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Cardiac Response to &amp;#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Respuesta cardíaca a la estimulación β-adrenérgica determinada por análisis de asa presión-volumen

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...