Este trabajo es apoyado por la American Heart Association 14FTF20370058 (DMA) y NIH T32 HL007101-35 (DMA).

1. Preparativos conductancia catéter y calibración de presión <ol><li> Conecte el catéter de conductancia al módulo catéter hemodinámica. Calibrar electrónicamente mediciones de la presión y el volumen mediante el registro de la presión preestablecida y el volumen establecido en el módulo de sonda. Grabar un rastreo de 0 mm Hg y 25 mm Hg (Figura 1A) y asignar las tensiones a ambos trazados de presión (Figura 1B y 1C). Del mismo modo, grabar un trazado volumen de 5 RVU y 25 RVU (Figura 1D) y asignar las tensiones a ambos trazados volumen (1E y 1F). </li><li> Confirme la calibración de presión electrónico con una calibración de presión manual, utilizando un esfigmomanómetro de columna de mercurio. Coloque el puerto manguito del esfigmomanómetro con una llave de paso de 3 vías. Llene un sistema de válvula hemostática de 3 puertos, a menudo utilizado para la angioplastia coronaria, con agua RT utilizando el puerto de lado. <ol><li> Coloque la punta de un catéter de conductancia en la válvula hemostática lleno de fluido y seguro suavemente ingenioHout retorcimiento del catéter. Conecte la válvula hemostática para el esfigmomanómetro e inflar hasta 200 mmHg y el bloqueo de 3 vías llave de paso. Examinar si la presión medida se corresponde con la presión de inflado del esfigmomanómetro. </li></ol></li><li> Place catéter en solución salina calentada a 37 ° C que está en el nivel de la presión campo de operación y medir. Ajuste el control de presión hasta que las presiones registradas está en cero. </li></ol> 2. Anestesia / intubación <ol><li> Administrar ketamina / xilazina (80-100 / 10 kg mg -1) como una inyección intraperitoneal. Nota: agentes anestésicos alternativos pueden ser utilizados. Una extensa lista de anestésicos se proporcionan en las revisiones anteriores de esta técnica 11,12. Anestesia adecuada puede ser confirmado por pizca de cola suave. </li><li> Una vez anestesiado, afeitarse el cuello y el pecho con cortar el pelo y el lugar en una almohadilla caliente. Mantener la temperatura rectal del ratón en 36,5 a 37,5 ºC. Las bajas temperaturas del cuerpodará lugar a ritmos cardíacos deprimidos. Aplique ungüento para los ojos para evitar la sequedad </li><li> Haga una incisión en la línea media en el cuello y diseccionar los músculos traqueales para exponer la tráquea. Coloque un tubo endotraqueal a través de la boca, mientras que la visualización de la tráquea para asegurar la intubación, y conectar con el respirador. </li><li> Mantener ratón en el ventilador durante el procedimiento. Configuración del conjunto de ventilador basan en el peso de los animales a lo descrito previamente 11. Volumen corriente (ml) = 6,2 x (peso de los animales en kilogramos) 1.01 y la frecuencia respiratoria = 53,5 x (peso de los animales en kilogramos) -0.26. </li></ol> 3. Colocación de la conductancia catéter en la cámara LV <ol><li> Enfoque de la carótida <ol><li> Para asegurar la esterilidad, dos conjuntos de instrumentos quirúrgicos estériles son uno Used- para la incisión inicial en la piel y uno para operar en la toracotomía. Los instrumentos deben ser descontaminados utilizando un esterilizador seco entre los animales durante una sesión quirúrgica individuo unand autoclave al final de cada día quirúrgico. </li><li> Después de que la piel ha sido descontaminado con tres ciclos de un exfoliante de la piel Clorhexidina + alcohol (solución de alcohol / 70% de clorhexidina al 0,5%), realizar una incisión sobre la carótida derecha desde la mandíbula al esternón. Diseccionar el tejido circundante para exponer la carótida derecha, y cortar el nervio vago, que corre junto a la carótida. </li><li> Coloque una sutura de seda 6-0 estéril alrededor del extremo distal (lejos del pecho) de la arteria carótida, la corbata y seguro. Coloque dos suturas adicionales debajo de la arteria carótida proximal (más cerca en el pecho) a la primera sutura, sin apretar atar la sutura media. Tire suavemente la sutura proximal y asegurar sujetando a la piel. Asegúrese de que la arteria carótida se ha sujetado tanto proximal y distal antes de proceder. </li><li> Hacer una pequeña incisión en la arteria carótida R., proximal a la primera sutura, y se extienden longitudinalmente hacia el pecho. </li><li> Inserte la punta del catéter de conductancia,empapado previamente en solución salina caliente durante 30 min, en el recipiente a través de la incisión y asegurar el catéter usando la sutura media. </li><li> Avanzar suavemente el catéter en el ventrículo izquierdo a través de la carótida, mientras ve el bucle presión-volumen de rastreo para asegurar la colocación correcta. Nota: La ubicación óptima del catéter debe producir bucles de presión-volumen que aparecen rectangular (Ver Figura 2). Si se encuentra cualquier resistencia al paso del catéter, tire suavemente hacia adelante y avanzar de nuevo con una suave presión. Rotación suave del catéter puede ayudar con la colocación en el ventrículo izquierdo. Forzar el catéter de conductancia puede llevar a complicaciones cardiovasculares graves o daños al catéter. </li><li> Record línea de base de presión-volumen bucles de ~ 10 min después de la colocación del catéter y el logro de un estado estacionario (Figura 2). </li></ol></li><li> Enfoque Apical <ol><li> En un ratón anestesiado y ventilado, hacer una incisión from el proceso xifoides y cortar a través de la pared torácica lateralmente hasta que el diafragma es visible. </li><li> Cortar aunque el diafragma y visualizar el vértice del corazón. </li><li> Insertar el catéter de conductancia en el ápex del ventrículo izquierdo a través de una herida de arma blanca aguja (usando una aguja G 25-30), hasta que el electrodo proximal está justo en el interior del ventrículo. </li><li> Record línea de base de presión-volumen bucles de ~ 10 min después de la colocación del catéter y el logro de un estado estacionario. </li></ol></li></ol> 4. Varying poscarga Usando transitoria oclusión aórtica <ol><li> Para realizar la oclusión aórtica transitoria, hacer una pequeña incisión horizontal en la parte superior del pecho y diseccionar el tejido circundante para exponer la aorta transversal. </li><li> Colocar una ligadura de seda 6-0 por debajo de la aorta transversal. Después de bucles de presión-volumen han regresado a la línea de base, junte ambos extremos de la sutura con una presilla de la aguja, suave y lentamente elevar la sutura durante 1-2 seg, y liberar lentamente la tensión. </li&gt; <li> Repita este procedimiento, hasta tres grabaciones óptimas separados están hechos del mismo animal. Nota: las grabaciones óptimas deben tener al menos 5 ciclos de bucle de volumen de presión y un aumento constante de la presión sistólica finales durante la aplicación de la tensión en la sutura (Figura 3A y 3C). </li></ol> 5. Variación de precarga Usando transitoria vena cava inferior Oclusión (IVC) <ol><li> Para llevar a cabo la oclusión transitoria vena cava inferior, hacer una incisión horizontal debajo de la apófisis xifoides, debajo del diafragma para exponer la IVC. </li><li> Coloque una ligadura de seda 6-0 por debajo de la IVC. Después de bucles de presión-volumen han regresado a la línea de base, junte ambos extremos de la sutura con una presilla de la aguja; suave y lentamente aumentar la sutura durante 1.2 segundos, y suelte lentamente la tensión. IVC oclusión también se puede realizar por la aplicación suave de presión usando un hisopo de algodón con punta. </li><li> Repita este procedimiento hasta tres grabaciones óptimas separados son made del mismo animal. Nota: las grabaciones óptimas deben tener al menos 5 ciclos de bucle de volumen de presión y una caída constante de la presión diastólica final del ventrículo izquierdo durante la aplicación de la tensión en la sutura (Figura 4A y 4B). </li></ol> 6. Saline Calibración <ol><li> En la conclusión del estudio, una conductancia en paralelo (V p) valor puede ser obtener mediante la inyección de un bolo de 10 l de solución salina hipertónica (15%) en el animal a través de la vena yugular (Figura 5A y 5B). Nota: Esta bolo causará un aumento aparente en volumen con ningún cambio en la presión. Esta aparente cambio de volumen es el resultado de un cambio en la conductancia de la acumulación de sangre en lugar de debido a un aumento real en volumen. Una disminución transitoria de la dP / dt max puede ser observado, como solución salina hipertónica tiene un efecto inotrópico negativo 13. El V p calculada se puede introducir en el volumen de la presión software de análisis de bucle a lo largocon los parámetros de calibración y cubeta para convertir de RVU en microlitros. </li></ol> 7. Cubeta de calibración <ol><li> Para realizar una calibración de cubeta, coloque una cubeta con pozos de diámetros conocidos proporcionadas por el fabricante en una almohadilla eléctrica o un baño de agua se calienta a 37 ºC. Rellene los primeros 4-5 agujeros con sangre heparinizada caliente fresca de los ratones sometidos a evaluaciones hemodinámicas. Nota: Una calibración cubeta permite una evaluación precisa del ventrículo izquierdo charco de sangre utilizando la sangre de ratón y permite la conversión de los datos de volumen de RVU a microlitros. </li><li> Insertar el catéter de conductancia en el primer pocillo, hasta que se sumergen todos los electrodos. Mueva suavemente el catéter en el pozo, lo que generará la variación UVR. </li><li> Registrar los cambios de conductancia en el canal de volumen en UVR. Seleccionar la más alta RVU para la calibración. Nota: El volumen de los pocillos o bien se puede calcular mediante 1) utilizando la ecuación para el volume de un cilindro, donde el radio es el de la bien cubeta y la longitud se basa en la longitud entre los dos electrodos de detección de interiores o 2) el control en las instrucciones del fabricante. La salida de la conductancia se puede correlacionar con los volúmenes conocidos para desarrollar una ecuación de calibración que convierte los datos de RVU en 11 microlitros. </li></ol> 8. Eutanasia <ol><li> A la conclusión del protocolo, los ratones se sacrificaron por dislocación cervical, mientras que bajo anestesia. </li><li> Con el fin de asegurar la muerte, los ratones que tienen dislocación cervical sufrido deben someterse a un método secundario de la eutanasia. Utilizamos desangrado bajo anestesia, con la recolección de tejido cardíaco para la experimentación, o toracotomía bilateral bajo anestesia. </li></ol> 9. Análisis de datos usando Volumen Presión Loop Software de Análisis <ol><li> Calcular V p de Saline calibración <ol><li> Seleccione la presión y el volume bucles obtenidos durante la expulsión de la solución salina hipertónica (Figura 5A y 5B) </li><li> Exportación bucles de software de análisis de volumen de presión. Elija la opción para la calibración Saline (Figura 5C) </li><li> Registre el valor V p calculado (Figura 5D). Nota: El valor de p V se calcula mediante la identificación de la intersección del volumen 1) End-diastólica vs. volumen sistólico final de la calibración de solución salina y 2) línea de volumen volumen = final sistólica diastólica final. La intersección de estas líneas proporciona la V p, que se calcula por el volumen de presión software de análisis de bucle. </li></ol></li><li> Introduzca la conductancia de volumen (RVU) en relación con opciones de canales de volumen (Figura 1F) </li><li> Mida relación bucle presión-volumen de línea de base <ol><li> Seleccionar 8-10 ciclos cardíacos de los canales de presión y volumen, una vez se ha alcanzado el estado de equilibrio (Figura 2A) y la exportación a análisis suaveWare. Identificar 5-6 bucles final de la expiración (Figura 2B) </li><li> Utilice el valor p V para corregir la conductancia en paralelo. Elija la opción &quot;Estado Estacionario&quot; y generar una tabla resumen hemodinámica (Figura 2C) </li></ol></li><li> Medida bucle relación presión-volumen durante la constricción de la aorta <ol><li> Seleccionar 8-10 ciclos cardíacos de los canales de presión y volumen que corresponden a la constricción de la aorta antes del aumento de las presiones telediastólica (Figura 3A) y la exportación de software de análisis. Identificar 5-6 bucles final de la expiración (Figura 3C) </li><li> Seleccione el análisis &quot;contractilidad&quot; (Figura 3B), que calculará el volumen de presión Relación de fin sistólica (ESPVR) </li></ol></li><li> Medida bucle relación presión-volumen durante IVC Constricción <ol><li> Seleccionar 8-10 ciclos cardíacos de los canales de presión y volumen que corresponden a IVC constricción (Figure 4A) y la exportación de software de análisis. Identificar 5-6 bucles final de la expiración (Figura 4B) </li><li> Seleccione el análisis &quot;contractilidad&quot; (Figura 3B), que calculará precarga reclutable Trabajo Carrera (PRSW) (Figura 4C), Máximo dP / dt vs EDV (Figura 4D), así como la ESPVR y la relación volumen de la presión diastólica final (Figura 4E) </li></ol></li></ol>

<ol>
	<li>Clark, J. E., Marber, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+in+pressure-volume+catheter+technology+-+stress+remodelling+after+infarction.">Advancements in pressure-volume catheter technology - stress remodelling after infarction.</a> Exp Physiol. 98 (3), 614-621 (2013).</li><li>Sunagawa, K., Maughan, W. L., Burkhoff, D., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+interaction+with+arterial+load+studied+in+isolated+canine+ventricle.">Left ventricular interaction with arterial load studied in isolated canine ventricle.</a> Am J Physiol. 245 (Pt 1), H773-H780 (1983).</li><li>Suga, H., Sagawa, K., Demer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+instantaneous+pressure+in+canine+left+ventricle.+Time+and+volume+specification.">Determinants of instantaneous pressure in canine left ventricle. Time and volume specification.</a> Circ Res. 46 (2), 256-263 (1980).</li><li>Suga, H., Sagawa, K., Shoukas, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Load+independence+of+the+instantaneous+pressure-volume+ratio+of+the+canine+left+ventricle+and+effects+of+epinephrine+and+heart+rate+on+the+ratio.">Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio.</a> Circ Res. 32 (3), 314-322 (1973).</li><li>Kass, D. 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=Improved+left+ventricular+mechanics+from+acute+VDD+pacing+in+patients+with+dilated+cardiomyopathy+and+ventricular+conduction+delay.">Improved left ventricular mechanics from acute VDD pacing in patients with dilated cardiomyopathy and ventricular conduction delay.</a> Circulation. 99 (12), 1567-1573 (1999).</li><li>Kass, D. 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=Diastolic+Compliance+of+Hypertrophied+Ventricle+Is+Not+Acutely+Altered+by+Pharmacological+Agents+Influencing+Active+Processes.">Diastolic Compliance of Hypertrophied Ventricle Is Not Acutely Altered by Pharmacological Agents Influencing Active Processes.</a> Annals of Internal Medicine. 119 (6), 466-473 (1993).</li><li>Georgakopoulos, D., 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=In+vivo+murine+left+ventricular+pressure-volume+relations+by+miniaturized+conductance+micromanometry.">In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry.</a> Am J Physiol. 274 (4 pt 2), H1416-H1422 (1998).</li><li>Cingolani, O. H., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-volume+relation+analysis+of+mouse+ventricular+function.">Pressure-volume relation analysis of mouse ventricular function.</a> Am J Physiol Heart Circ Physiol. 301 (6), H2198-H2206 (2011).</li><li>Baan, J., 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=Continuous+Measurement+of+Left-Ventricular+Volume+in+Animals+and+Humans+by+Conductance+Catheter.">Continuous Measurement of Left-Ventricular Volume in Animals and Humans by Conductance Catheter.</a> Circulation. 70 (5), 812-823 (1984).</li><li>Pearce, J. A., Porterfield, J. E., Larson, E. R., Valvano, J. W., Feldman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+considerations+in+catheter+based+estimation+of+left+ventricular+volume.">Accuracy considerations in catheter based estimation of left ventricular volume.</a> Conf Proc IEEE Eng Med Biol Soc. 2010, 3556-3558 (2010).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nature Protocols. 3 (9), 1422-1434 (1038).</li><li>Hanusch, C., Hoeger, S., Beck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+of+small+rodents+during+magnetic+resonance+imaging.">Anaesthesia of small rodents during magnetic resonance imaging.</a> Methods. 43 (1), 68-78 (2007).</li><li>Georgakopoulos, D., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+parallel+conductance+by+dual-frequency+conductance+catheter+in+mice.">Estimation of parallel conductance by dual-frequency conductance catheter in mice.</a> Am J Physiol Heart Circ Physiol. 279 (1), H443-H450 (2000).</li><li>Esposito, G., 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=Increased+myocardial+contractility+and+enhanced+exercise+function+in+transgenic+mice+overexpressing+either+adenylyl+cyclase+5+or+8.">Increased myocardial contractility and enhanced exercise function in transgenic mice overexpressing either adenylyl cyclase 5 or 8.</a> Basic Res Cardiol. 103 (1), 22-30 (2008).</li><li>Kohout, T. 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=Augmentation+of+cardiac+contractility+mediated+by+the+human+beta(3)-adrenergic+receptor+overexpressed+in+the+hearts+of+transgenic+mice.">Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.</a> Circulation. 104 (20), 2485-2491 (2001).</li><li>Kim, K. S., 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=beta-Arrestin-biased+AT1R+stimulation+promotes+cell+survival+during+acute+cardiac+injury.">beta-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury.</a> Am J Physiol Heart Circ Physiol. 303 (8), H1001-H1010 (2012).</li><li>Suga, H., Sagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+Interrelationship+between+Instantaneous+Ventricular+Pressure-Volume+Ratio+and+Myocardial+Force-Velocity+Relation.">Mathematical Interrelationship between Instantaneous Ventricular Pressure-Volume Ratio and Myocardial Force-Velocity Relation.</a> Annals of Biomedical Engineering. 1 (2), 160-181 (1972).</li><li>Suga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+energetics.">Ventricular energetics.</a> Physiol Rev. 70 (2), 247-277 (1990).</li><li>Kass, D. 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=Influence+of+contractile+state+on+curvilinearity+of+in+situ+end-systolic+pressure-volume+relations.">Influence of contractile state on curvilinearity of in situ end-systolic pressure-volume relations.</a> Circulation. 79 (1), 167-178 (1989).</li><li>Little, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+left+ventricular+dP/dtmax-end-diastolic+volume+relation+in+closed-chest+dogs.">The left ventricular dP/dtmax-end-diastolic volume relation in closed-chest dogs.</a> Circ Res. 56 (6), 808-815 (1985).</li><li>Glower, D. D., 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=Linearity+of+the+Frank-Starling+relationship+in+the+intact+heart:+the+concept+of+preload+recruitable+stroke+work.">Linearity of the Frank-Starling relationship in the intact heart: the concept of preload recruitable stroke work.</a> Circulation. 71 (5), 994-1009 (1985).</li><li>Sharir, T., 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=Ventricular+systolic+assessment+in+patients+with+dilated+cardiomyopathy+by+preload-adjusted+maximal+power.+Validation+and+noninvasive+application.">Ventricular systolic assessment in patients with dilated cardiomyopathy by preload-adjusted maximal power. Validation and noninvasive application.</a> Circulation. 89 (5), 2045-2053 (1994).</li><li>Baan, J., Van der Velde, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+left+ventricular+end-systolic+pressure-volume+relation+to+type+of+loading+intervention+in+dogs.">Sensitivity of left ventricular end-systolic pressure-volume relation to type of loading intervention in dogs.</a> Circ Res. 62 (6), 1247-1258 (1988).</li><li>Wei, C. L., 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=Volume+catheter+parallel+conductance+varies+between+end-systole+and+end-diastole.">Volume catheter parallel conductance varies between end-systole and end-diastole.</a> IEEE Trans Biomed Eng. 54 (8), 1480-1489 (2007).</li><li>Porterfield, J. E., 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=Dynamic+correction+for+parallel+conductance,+GP,+and+gain+factor,+alpha,+in+invasive+murine+left+ventricular+volume+measurements.">Dynamic correction for parallel conductance, GP, and gain factor, alpha, in invasive murine left ventricular volume measurements.</a> J Appl Physiol (1985). 107 (6), 1693-1703 (2009).</li></ol>

Los autores no tienen nada que revelar.

Se describe un método para el análisis perfoming bucle presión-volumen con un catéter de conductancia en ratones, para derivar el análisis exhaustivo de tanto la contractilidad cardíaca y la relajación. Suga, Sagawa y sus colegas utilizaron presión-volumen bucles para definir medidas de la contractilidad cardíaca, específicamente la pendiente de la ESPVR, o la elastancia telesistólico (s E), y E máx. Elastancia, definido por la relación de la presión a volumen (P / V), varía a lo lar...

Análisis de bucle de volumen de la presión cardíaca proporciona información detallada de la función cardíaca y son el estándar de oro para la evaluación funcional 1. Mientras que las técnicas de imagen como la ecocardiografía o resonancia magnética cardiaca proporcionan medidas funcionales, estas medidas son altamente dependientes de las condiciones de carga. Medidas independiente de la carga de la contractilidad cardíaca y la relajación requieren mediciones dinámicas de la presión ventricular y la relación volumen en un intervalo de precarga y la poscarga. Esta comprensión de la relación presión-volumen surge del trabajo pionero de Sagawa y colegas 2,3. Demostraron en ex vivo corazones perfundidos canina que las medidas derivadas de la contractilidad del bucle presión-volumen fueron independientes de las condiciones de carga 4. En la aplicación in vivo de estos análisis se hizo posible con el desarrollo de catéteres de conductancia en los años 1980. Este avance técnico permitido Kass y sus colaboradores para llevar a cabo el análisis de bucle presión-volumen en humanos 5,6. La miniaturización de los catéteres de conductancia y mejoras en las técnicas quirúrgicas en el finales de 1990 de 7 hizo el análisis de la función cardiaca roedor viable, lo que permite estudios genéticos y farmacológicos a realizar. Este avance tiene ya lugar a la utilización generalizada de análisis de bucle presión-volumen y ha generado una gran cantidad de información sobre la fisiología cardiaca de los mamíferos. Un concepto clave en el uso de catéteres de conductancia y la interpretación de los datos obtenidos a partir de que es la relación entre el volumen y la conductancia. La conductancia es inversamente proporcional a la tensión, que se mide usando un catéter con electrodos colocados de manera proximal, por lo general colocados debajo de la válvula aórtica, y distalmente, en el ápice LV 8. Los cambios en la tensión o de la conductancia se mide por cambios en la corriente que fluye desde proximal a distal del electrodo. Aunque la piscina de sangre contribuyens de manera significativa a la conductancia, la contribución de la pared ventricular, denominado conductancia en paralelo (V p), a la conductancia medida se debe restar de obtener mediciones de volumen LV absolutos. Los métodos para llevar a cabo esta corrección, llamada una calibración de solución salina, se discuten en el protocolo a continuación. La relación matemática entre la conductancia y el volumen, descrito por Baan y sus colegas, es que el volumen = 1 / α; (ρ L 2) (GG p), donde α = factor de corrección de campo uniforme, ρ = resistividad de la sangre, L = distancia entre los electrodos, G = conductancia y G p = conductancia no sangre 9. Es de destacar que el factor de corrección de campo uniforme en ratones se aproxima a 1,0 debido a los volúmenes de cámara pequeñas 10. Junto con transductores de presión, el catéter de conductancia proporciona datos de presión y volumen simultánea en tiempo real. Pressu Cardiacare-volumen de análisis presenta ventajas particulares sobre otras medidas de la función cardiaca, ya que permiten la medición de la función ventricular independiente de las condiciones de carga y de la frecuencia cardíaca. De carga independiente índices cardíacos específicos de la contractilidad incluyen: relación del volumen final de la presión sistólica (ESPVR), d P / d t max relación volumen--end diastólica, elastancia máxima (Emax) y precarga trabajo sistólico reclutable (PRSW). Una medida independiente de la carga de la función diastólica es la relación volumen de la presión diastólica final (EDPVR) 11. El siguiente protocolo describe la realización del análisis de bucle de volumen de presión cardiaca, utilizando tanto la carótida y un enfoque apical. Si bien la metodología para llevar a cabo estos estudios se han descrito en detalle previamente 8,11, vamos a revisar los pasos clave para obtener mediciones de presión-volumen precisos, incluyendo tanto la solución salina y la calibración cubeta de corrección, y proporcionar una demostración visual de thesprocedimientos e. La investigación con animales llevados a cabo para este estudio fue manejado de acuerdo a los protocolos aprobados y normas de bienestar animal de Comité de Cuidado y Uso de Animales Institucional de la Universidad de Duke Medical Center.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AnaSed (xylazine)&#160;</td>
			<td>Lloyd Laboratories</td>
			<td>NADA no. 139-236</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Ketaset (ketamine)</td>
			<td>Pfizer</td>
			<td>440842</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>VIP3000</td>
			<td>Matrx Medical Inc.</td>
			<td></td>
			<td>Anesthesia machine</td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>Model 683</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Tubing kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1049</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket&#160;</td>
			<td>Kaz Inc.</td>
			<td>5628</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Carl Zeiss Optical Inc.</td>
			<td>Stemi 2000</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Cole&#8211;Parmer</td>
			<td>41720</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Dumont no. 55 Dumostar Forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11295-51</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Graefe forceps, curved&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11052-10</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Moria MC31 forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11370-31</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Mayo scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14512-15</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Iris scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14041-10</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Halsey needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12501-13</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Olsen&#8211;Hegar needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12002-12</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>spring scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>15610-08</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>disposable underpads</td>
			<td>Kendall/Tyco Healthcare</td>
			<td>1038</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Sterile gauze sponges, sterile&#160;</td>
			<td>Dukal</td>
			<td>62208</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Cotton-tipped applicators, sterile&#160;</td>
			<td>Solon</td>
			<td>368</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Surgical suture,&#160; silk, 6-0&#160;</td>
			<td>DemeTECH</td>
			<td>FT-639-1</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>1 cc Insulin syringes&#160;</td>
			<td>Becton Dickenson</td>
			<td>329412</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Access-9&#8482; Hemostasis Valve</td>
			<td>Merit Medical&#160;</td>
			<td>MAP111</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Sphygmomanometer</td>
			<td>Baumanometer</td>
			<td>320</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Millar PV system MPVS-300/400 or MPVS Ultra (includes calibration cuvette)</td>
			<td>ADInstruments Inc</td>
			<td></td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>1.4F conductance catheter&#160;</td>
			<td>ADInstruments Inc</td>
			<td>SPR-839</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>PowerLab 4/30 with Chart Pro</td>
			<td>ADInstruments Inc.</td>
			<td>ML866/P</td>
			<td>Hemodynamic software</td>
		</tr>
		<tr>
			<td>animal clipper</td>
			<td>Wahl</td>
			<td>8787-450A</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-10 (Becton Dickinson, cat. no. )</td>
			<td>Becton Dickenson</td>
			<td>427401</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
			<td>Becton Dickenson</td>
			<td>427411</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>0.9% (wt/vol) sodium chloride injection, USP , cat. no. )</td>
			<td>Hospira</td>
			<td>NDC no. 0409-4888-50</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
	</tbody>
</table>

cardiac pressure-volume loop analysis using conductance catheters in mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Análisis bucle presión-volumen se puede utilizar para medir la función cardiaca en ratones modificados genéticamente o ratones sometidos a 14,15 estudios fármaco 16. Bucles de volumen de presión representativos se proporcionan desde el trabajo publicado previamente 16 que investiga el efecto de ß-Arrestin sesgada ligando AT1R, TRV120023. Para probar si TRV120023 afecta a la función cardíaca in vivo, se realizó un análisis bucle presión-volumen en ratones de tipo salv...

Watch this Scientific Journal Video about Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice at JoVE.com

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiaca Presión-Volumen Análisis Loop Utilizando conductancia catéteres en ratones

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

19.3K vistas.  Duke University Medical Center. Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters. 

Video: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Investigación

Biología

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingeniería Humanos redes microvasculares en ratones inmunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Medición de conductancia de la hoja hidráulica y la conductancia estomática y sus respuestas a la irradiación y la deshidratación mediante el método de evaporación Flux (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital de la microcirculación en el músculo de ratón Cremaster para el análisis de la migración de la célula madre periféricas

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

La purificación de la fibrosis quística regulador de conductancia transmembrana de proteínas expresadas en<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Una nueva técnica de análisis cuantitativos de la pérdida del pelo en ratones Uso del análisis de escala de grises

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Relacionar estomática conductancia de la hoja Rasgos funcionales

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Contractilidad Mediciones en músculos papilares aislados para la Investigación de Cardíaca inotropía en ratones

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

La medición de volumen de bucles de presión en el ratón

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...