Este trabajo fue financiado por una Universidad de Texas en la beca del programa de investigación interdisciplinario de Arlington.

Este protocolo sigue las directrices de la Junta de revisión institucional de la Universidad de Texas en Arlington y se ajusta a las normas establecidas por la última versión de la declaración de Helsinki. Por consiguiente, consentimiento de informado escrita era (y debe) obtenido previo al inicio de los procedimientos de investigación.
1. instrumentación
Nota: La siguiente descripción de la instrumentación se basa en el infrarrojo cercano (NIR) Espectrómetro y datos adquisición sistema utilizado en nuestro laboratorio (véase Tabla de materiales). Así, las instrucciones incluyen pasos que son necesarios para la función óptima de estos dispositivos. Estos pasos incluyen la calibración de la sonda NIR, utilizando los software y calibración fantasma y la aplicación de un paño oscuro para excluir la luz ambiente. En caso de que se utiliza diversos datos colección hardware o software, los investigadores deben consultar sus propios manuales específicos por razones de calibración y luz ambiente. La figura 1 ilustra el montaje experimental e instrumentación que se describe inmediatamente a continuación.
<ol><li>Indicar el tema mentir supino con las piernas dentro de una cámara de presión negativa (LBNP) de cuerpo inferior (figura 1A), por lo que su línea de la correa es aproximadamente incluso con la apertura de la caja LBNP. Para obtener instrucciones sobre cómo construir una cámara LBNP, ver referencias17.
</li>
	<li>Coloque tres electrodos de electrocardiograma en el sujeto: dos en una posición inferior, mediados de-clavicular y uno sobre el tema de lado izquierdo medial a la cresta ilíaca. Esta configuración ofrece los mejores resultados debido al acceso limitado a las extremidades inferiores, la instrumentación de los miembros superiores y el movimiento del brazo durante el ejercicio de apretón de mano.</li>
	<li>Lugar un módulo de monitor de presión arterial no invasiva en la muñeca dominante del sujeto. Colocar los manguitos de presión arterial del dedo en cada dedo y conectar con el módulo (figura 1B). Asegúrese de que los manguitos de presión arterial del dedo están calibrados correctamente según el manual del usuario que acompaña a su dispositivo.</li>
	<li>Indicar el tema de captar un dinamómetro de agarre de la mano (HGD) con su brazo no dominante en una posición ligeramente abducida. El brazo debe colocarse cómodamente en una mesa de noche. La distancia y el ángulo de la DGH deben ajustarse para permitir la fuerza de agarre óptimo con movimiento mínimo del brazo (figura 1).</li>
	<li>Asegure el Dag a una mesa de noche.</li>
	<li>Medir la contracción voluntaria máxima (MVC) del participante. Dígale a los participantes que, cuando se le solicite, debe exprimir el HGD tan duro como sea posible utilizando sólo los músculos de la mano y el antebrazo. Indicar al tema que deben abstenerse de reclutar su brazo, pecho, hombro o los músculos abdominales al realizar la máxima adherencia.</li>
	<li>Repita paso 1.6 tres veces, separadas por al menos 60 registro s. la fuerza máxima alcanzada (mejor de 3). Esta fuerza máxima se utilizará para calcular la intensidad del ejercicio para la capacidad oxidativa del músculo esqueléticas y neurovasculares de acoplamiento (abajo).</li>
	<li>Coloque un puño rápido inflación alrededor de la parte superior del brazo de la mano de ejercicio. Conecte la línea aérea desde el controlador de la inflación rápida a la banda.</li>
	<li>Identificar digitorum del flexor profundo. Use un marcador de piel para delimitar las fronteras del músculo palpable.</li>
	<li>Asegúrese de que el espectrómetro NIR es calibrado correctamente según el manual del usuario incluido con su dispositivo. Limpiar la piel sobre la cual se colocará la sonda NIR con un paño con alcohol prep.</li>
	<li>Coloque la sonda NIR sobre el centro del vientre del músculo (flexor digitorum profundus) y fijar firmemente en el antebrazo.</li>
	<li>Envolver la sonda y del antebrazo con el paño oscuro, reduciendo al mínimo la interferencia de la luz ambiental (figura 1, figura 1).</li>
	<li>Cuando esté listo para llevar a cabo la simpatolisis funcional parte del estudio, sello al tema en la cámara de LBNP.</li>
</ol>2. capacidad oxidativa del músculo esquelético
Nota: Un trazo de datos representativos que ilustran el procedimiento experimental para medir la capacidad oxidativa del músculo esquelético se representa en la figura 2. Este enfoque experimental ha sido previamente validado contra en vivo fósforo MRS18 y en situ muscular respirometria19y está ganando la aceptación extensa20.
<ol><li>Instrumento al tema como se indicó anteriormente (instrumentación).</li>
	<li>Indique el tema a mentir aún durante 2 minutos mientras monitoreo desoxihemoglobina (HHb) y oxihemoglobina (HbO2) a través de la sonda NIR. Nota: Este período de descanso permite al sujeto recuperar de cualquier artefacto de movimiento relacionada con el proceso de instrumentación y garantiza mediciones de línea base estable. Si después de 2 min que no hay fluctuaciones significativas han ocurrido, el tema puede considerarse en un estado estacionario, o reposo basal.</li>
	<li>Antes de la oclusión del manguito, notificar al sujeto que se infla el brazalete. Inflar la parte superior del brazo brazalete por lo menos 30 mmHg sobre la presión arterial sistólica durante 5 minutos (es decir, suprasistólica). Instruir al sujeto a mantener el brazo quieto y relajado como sea posible, tanto durante el manguito inflado y desinflado del siguiente. Nota: Esta 5 min protocolo de oclusión de arteria braquial brazalete cerca refleja que el estándar clínico actualmente aceptado para oclusión vascular pruebas21,22,23,24,25.</li>
	<li>Registre el valor inicial/basal (antes de la oclusión del manguito) y el valor nadir de saturación de tejidos (StO2) durante la oclusión del manguito y determinar el punto medio entre estos dos valores. <img alt="Equation 1" src="/files/ftp_upload/57317/57317eq1.jpg">
</li>
	<li>Permita que el tema para recuperarse de la oclusión del manguito y volver a los valores basales reposo. Una vez que el sujeto ha mantenido una línea de base descanso durante al menos 1 minuto completo, continúe al siguiente paso.</li>
	<li>Instruir a sujetos a presión y agarre una mano isométricas al 50% de la MVC. Fomentar el tema para mantener su contracción isométrica hasta que elimina la saturación de los tejidos en un 50%. Al alcanzar este valor, Dile el sujeto relaje su mano y les informará que se necesita no más ejercicio o movimiento.</li>
	<li>Dentro de 3-5 s siguiente ejercicio dejar de fumar, administrar las siguientes series de oclusión del manguito rápido (una serie = 1 inflación + 1 deflación), como se ha establecido18: Serie #1 - 6: 5 a/5 s s off Serie #7 - 10: 7 en/10 s s off Serie #11-14:10 s a/15 s de Serie #15-18:10 s a/20 s de</li>
	<li>Después de completar la serie de inflación/deflación 18th , instruir el tema para el descanso, permitiendo la saturación de tejido volver a los valores de línea de base inicial. Después de que estos valores han permanecido constantes durante al menos 2 minutos, repita los pasos del 2.4 y 2.5.</li>
	<li>
Cálculo de la capacidad oxidativa del músculo esquelético
	<ol>
<li>Calcular la pendiente del cambio en el StO2para cada una de las oclusiones de 18 manguito individuales, formando los puntos de recuperación de monoexponential ilustrados en la figura 2.</li>
		<li>Ajuste los datos calculados del 2.7 al siguiente monoexponential curva18,19,26 y = End - Δ x e-kt Nota: 'y' es la tasa de consumo de oxígeno muscular relativa (mV̇O2) durante el inflado del manguito, 'Poner fin' representa el mV̇O2 inmediatamente después de la cesación del ejercicio; Delta ('Δ') significa el cambio de mV̇O2 de descanso al final del ejercicio; 'k' es la conexión constante de velocidad; ' t ' es el momento. Tau se calcula como 1/k.</li>
	</ol>
</li>
</ol>3. reactiva hiperemia
Nota: Un trazo de datos representativos que ilustran el procedimiento experimental para medir la hiperemia reactiva se representa en la figura 3.
<ol><li>Con el tema mentira supina y equipado como se describe anteriormente (instrumentación), instruir el tema mentir como todavía posible.</li>
	<li>Una vez que el sujeto ha logrado un consistente estado de reposo, continuar a grabar al menos 1 minuto de la base de datos y luego inflar rápidamente un esfigmomanómetro en el brazo a una presión suprasistólica (30 mmHg sobre la presión arterial sistólica).</li>
	<li>En la marca de 5 minutos, rápidamente desinfle el manguito mientras graba la respuesta hiperémica.</li>
	<li>Continuar la grabación de al menos 3 minutos para capturar la recuperación del sujeto.</li>
	<li>
Cálculo de hiperemia reactiva Nota: Los parámetros NIRS calculados se muestran en la figura 3.<ol>
<li>Calcular base StO2 como promedio StO2 durante 1 minuto completo previo al inicio de la oclusión arterial brazalete.</li>
		<li>Determinar la tasa metabólica de reposo músculo esquelético como el índice de desaturación (es decir, la pendiente media) durante el brazalete oclusión (definida como 1 pendiente)27,28.</li>
		<li>Calcular la hiperemia reactiva como sigue: a) la media ascendente tras liberación de puño (es decir, reperfusión tasa, definida como pendiente 2), calculados desde el momento de la liberación del manguito a través de la fase creciente linealmente de la traza de rebote; b) StO2 el valor máximo alcanzado después de lanzamiento del manguito (denotado como StO2máximo); c) la zona de hiperemia reactiva bajo la curva (AUC). calculado desde el momento de la liberación del manguito a 1, 2 y 3 min post oclusión del manguito (1min, AUC AUC 2 min y AUC 3-min, respectivamente); y d) el hyperemic reserva, calculada como el cambio en StO2 por encima de la línea de base y como un cambio de porcentaje (%). Este valor se calcula como la saturación más alto alcanzada durante la oclusiva posterior rebote menos la saturación promedio calculada en el paso 3.5.1 (véase arriba). Nota: Las diferencias grandes en base de datos afectará grandemente la interpretación de la reserva hiperémica.</li>
	</ol>
</li>
</ol>4. funcional simpatolisis
Nota: Un trazo de datos representativos que ilustran el procedimiento experimental para medir la simpatolisis funcional se representa en la figura 4.
<ol><li>Instrumento al tema como se indicó anteriormente (instrumentación).</li>
	<li>Asegurar un sellado hermético de la cámara LBNP.</li>
	<li>Con el sujeto acostado aún y en reposo, recoger 3 min de la base de datos.</li>
	<li>En la marca de 3 minutos, encienda la aspiradora. Ajuste el vacío para que la presión dentro de la cámara LBNP es entre-20 y -30 mmHg. Permita que la aspiradora a 2 min mientras la respuesta del sujeto.</li>
	<li>En la marca de 5 minutos, apagar el vacío y dejar que el tema reposar 3 minutos.</li>
	<li>En la marca de 8 minutos, iniciar el mensaje de voz guiando el tema a través del ejercicio de agarre de mano rítmica (20% MVC).</li>
	<li>Confirmar que el tema es mantener su apretón en la totalidad de cada fase de agarre y relajarse completamente durante entre cada repetición. Monitorear su fuerza de salida y confirmar que están alcanzando 20% MVC con cada apretón. Continuar el ejercicio hasta el minuto 11.</li>
	<li>En la marca de 11 minutos, encienda la aspiradora animando el tema para continuar su ejercicio rítmico. Permita que la aspiradora desde el 11-13 min., después los apaga.</li>
	<li>Que el tema continúe realizando ejercicio de agarre de mano rítmica al 20% de la MVC para un 2 minutos adicionales. Sobre cese de ejercicio, tienen el resto del tema silencio y mentira todavía.</li>
	<li>
Cálculo funcional simpatolisis
	<ol>
<li>Normalizar el cambio de oxihemoglobina con LBNP la señal inestable total (TLS), determinado durante la oclusión del manguito de 5 min: <img alt="Equation 2" src="/files/ftp_upload/57317/57317eq2.jpg"> <img alt="Equation 3" src="/files/ftp_upload/57317/57317eq3.jpg">
</li>
		<li>Calcular cada evento como el promedio final de 20 min de cada evento.</li>
		<li>Calcular la atenuación inducida por el ejercicio de la reducción de la oxihemoglobina: <img alt="Equation 4" src="/files/ftp_upload/57317/57317eq4.jpg">
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Huang, A. 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=Predictive+value+of+reactive+hyperemia+for+cardiovascular+events+in+patients+with+peripheral+arterial+disease+undergoing+vascular+surgery.">Predictive value of reactive hyperemia for cardiovascular events in patients with peripheral arterial disease undergoing vascular surgery.</a> Arterioscler Thromb Vasc Biol. 27 (10), 2113-2119 (2007).</li><li>Suryapranata, H., 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=Predictive+value+of+reactive+hyperemic+response+on+reperfusion+on+recovery+of+regional+myocardial+function+after+coronary+angioplasty+in+acute+myocardial+infarction.">Predictive value of reactive hyperemic response on reperfusion on recovery of regional myocardial function after coronary angioplasty in acute myocardial infarction.</a> Circulation. 89 (3), 1109-1117 (1994).</li><li>Richardson, R. 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=High+Muscle+Blood-Flow+in+Man+-+Is+Maximal+O2+Extraction+Compromised.">High Muscle Blood-Flow in Man - Is Maximal O2 Extraction Compromised.</a> J of Appl Physiol. 75 (4), 1911-1916 (1993).</li><li>Clifford, P. S., Hellsten, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasodilatory+mechanisms+in+contracting+skeletal+muscle.">Vasodilatory mechanisms in contracting skeletal muscle.</a> J Appl Physiol. 97 (1), 393-403 (2004).</li><li>Hansen, J., Thomas, G. D., Jacobsen, T. N., Victor, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+metaboreflex+triggers+parallel+sympathetic+activation+in+exercising+and+resting+human+skeletal+muscle.">Muscle metaboreflex triggers parallel sympathetic activation in exercising and resting human skeletal muscle.</a> Am J Physiol. 266 (6 Pt 2), H2508-H2514 (1994).</li><li>Thomas, G. D., Victor, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+mediates+contraction-induced+attenuation+of+sympathetic+vasoconstriction+in+rat+skeletal+muscle.">Nitric oxide mediates contraction-induced attenuation of sympathetic vasoconstriction in rat skeletal muscle.</a> J Physiol. 506 (Pt 3), 817-826 (1998).</li><li>Hansen, J., Thomas, G. D., Harris, S. A., Parsons, W. J., Victor, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+sympathetic+neural+control+of+oxygenation+in+resting+and+exercising+human+skeletal+muscle.">Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.</a> J Clin Invest. 98 (2), 584-596 (1996).</li><li>Rosenmeier, J. B., Fritzlar, S. J., Dinenno, F. A., Joyner, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+NO+administration+and+alpha-adrenergic+vasoconstriction+in+human+limbs.">Exogenous NO administration and alpha-adrenergic vasoconstriction in human limbs.</a> J Appl Physiol. 95 (6), 2370-2374 (2003).</li><li>Fadel, P. J., Keller, D. M., Watanabe, H., Raven, P. B., Thomas, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+assessment+of+sympathetic+vasoconstriction+in+human+and+rodent+skeletal+muscle+using+near-infrared+spectroscopy+and+Doppler+ultrasound.">Noninvasive assessment of sympathetic vasoconstriction in human and rodent skeletal muscle using near-infrared spectroscopy and Doppler ultrasound.</a> J Appl Physiol. 96 (4), 1323-1330 (2004).</li><li>Nelson, M. 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=PDE5+inhibition+alleviates+functional+muscle+ischemia+in+boys+with+Duchenne+muscular+dystrophy.">PDE5 inhibition alleviates functional muscle ischemia in boys with Duchenne muscular dystrophy.</a> Neurology. 82 (23), 2085-2091 (2014).</li><li>Nelson, M. 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=Sodium+nitrate+alleviates+functional+muscle+ischaemia+in+patients+with+Becker+muscular+dystrophy.">Sodium nitrate alleviates functional muscle ischaemia in patients with Becker muscular dystrophy.</a> J Physiol. 593 (23), 5183-5200 (2015).</li><li>Remensnyder, J. P., Mitchell, J. H., Sarnoff, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+sympatholysis+during+muscular+activity.+Observations+on+influence+of+carotid+sinus+on+oxygen+uptake.">Functional sympatholysis during muscular activity. Observations on influence of carotid sinus on oxygen uptake.</a> Circ Res. 11, 370-380 (1962).</li><li>Kodama, 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=Cardiorespiratory+fitness+as+a+quantitative+predictor+of+all-cause+mortality+and+cardiovascular+events+in+healthy+men+and+women:+A+meta-analysis.">Cardiorespiratory fitness as a quantitative predictor of all-cause mortality and cardiovascular events in healthy men and women: A meta-analysis.</a> JAMA. 301 (19), 2024-2035 (2009).</li><li>Westerblad, H., Place, N., Yamada, T., Rassier, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Muscle Biophysics: From Molecules to Cells. , 279-296 (2010).</li><li>Tyni-Lenn&#233;, R., Gordon, A., Jansson, E., Bermann, G., Sylv&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+endurance+training+improves+peripheral+oxidative+capacity,+exercise+tolerance,+and+health-related+quality+of+life+in+women+with+chronic+congestive+heart+failure+secondary+to+either+ischemic+cardiomyopathy+or+idiopathic+dilated+cardiomyopathy.">Skeletal muscle endurance training improves peripheral oxidative capacity, exercise tolerance, and health-related quality of life in women with chronic congestive heart failure secondary to either ischemic cardiomyopathy or idiopathic dilated cardiomyopathy.</a> Am J of Cardiol. 80 (8), 1025-1029 (1997).</li><li>Cabalzar, A. 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=Muscle+function+and+quality+of+life+in+the+Crohn's+disease.">Muscle function and quality of life in the Crohn's disease.</a> Fisioter Mov. 30, 337-345 (2017).</li><li>Esch, B. T., Scott, J. M., Warburton, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+lower+body+negative+pressure+chamber.">Construction of a lower body negative pressure chamber.</a> Adv Physiol Educ. 31 (1), 76-81 (2007).</li><li>Ryan, T. E., Southern, W. M., Reynolds, M. A., McCully, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cross-validation+of+near-infrared+spectroscopy+measurements+of+skeletal+muscle+oxidative+capacity+with+phosphorus+magnetic+resonance+spectroscopy.">A cross-validation of near-infrared spectroscopy measurements of skeletal muscle oxidative capacity with phosphorus magnetic resonance spectroscopy.</a> J Appl Physiol. 115 (12), 1757-1766 (2013).</li><li>Ryan, T. E., Brophy, P., Lin, C. T., Hickner, R. C., Neufer, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+in+vivo+skeletal+muscle+mitochondrial+respiratory+capacity+in+humans+by+near-infrared+spectroscopy:+a+comparison+with+in+situ+measurements.">Assessment of in vivo skeletal muscle mitochondrial respiratory capacity in humans by near-infrared spectroscopy: a comparison with in situ measurements.</a> J Physiol. 592 (15), 3231-3241 (2014).</li><li>Adami, A., Rossiter, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles,+insights+and+potential+pitfalls+of+the+non-invasive+determination+of+muscle+oxidative+capacity+by+near-infrared+spectroscopy.">Principles, insights and potential pitfalls of the non-invasive determination of muscle oxidative capacity by near-infrared spectroscopy.</a> J Appl Physiol. , (2017).</li><li>Corretti, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+ultrasound+assessment+of+endothelial-dependent+flow-mediated+vasodilation+of+the+brachial+artery+-+A+report+of+the+International+Brachial+Artery+Reactivity+Task+Force.">Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery - A report of the International Brachial Artery Reactivity Task Force.</a> J Am Coll Cardiol. 39 (2), 257-265 (2002).</li><li>Thijssen, D. H., 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=Assessment+of+flow-mediated+dilation+in+humans:+a+methodological+and+physiological+guideline.">Assessment of flow-mediated dilation in humans: a methodological and physiological guideline.</a> Am J Physiol Heart Circ Physiol. 300 (1), H2-H12 (2011).</li><li>Green, D. J., Jones, H., Thijssen, D., Cable, N. T., Atkinson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-mediated+dilation+and+cardiovascular+event+prediction:+does+nitric+oxide+matter?.">Flow-mediated dilation and cardiovascular event prediction: does nitric oxide matter?.</a> Hypertension. 57 (3), 363-369 (2011).</li><li>Southern, W. M., Ryan, T. E., Reynolds, M. A., McCully, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+near-infrared+spectroscopy+measurements+of+oxidative+function+and+postexercise+recovery+kinetics+in+the+medial+gastrocnemius+muscle.">Reproducibility of near-infrared spectroscopy measurements of oxidative function and postexercise recovery kinetics in the medial gastrocnemius muscle.</a> Appl Physiol Nutr Metab. 39 (5), 521-529 (2014).</li><li>Ryan, T. E., Erickson, M. L., Brizendine, J. T., Young, H. J., McCully, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+evaluation+of+skeletal+muscle+mitochondrial+capacity+with+near-infrared+spectroscopy:+correcting+for+blood+volume+changes.">Noninvasive evaluation of skeletal muscle mitochondrial capacity with near-infrared spectroscopy: correcting for blood volume changes.</a> J Appl Physiol. 113 (2), 175-183 (2012).</li><li>Ryan, T. 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=Skeletal+muscle+oxidative+capacity+in+amyotrophic+lateral+sclerosis.">Skeletal muscle oxidative capacity in amyotrophic lateral sclerosis.</a> Muscle Nerve. 50 (5), 767-774 (2014).</li><li>Mayeur, C., Campard, S., Richard, C., Teboul, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+different+vascular+occlusion+tests+for+assessing+reactive+hyperemia+using+near-infrared+spectroscopy.">Comparison of four different vascular occlusion tests for assessing reactive hyperemia using near-infrared spectroscopy.</a> Crit Care Med. 39 (4), 695-701 (2011).</li><li>McLay, K. M., 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=Vascular+responsiveness+determined+by+near-infrared+spectroscopy+measures+of+oxygen+saturation.">Vascular responsiveness determined by near-infrared spectroscopy measures of oxygen saturation.</a> Exp Physiol. 101 (1), 34-40 (2016).</li><li>McLay, K. M., Nederveen, J. P., Pogliaghi, S., Paterson, D. H., Murias, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatability+of+vascular+responsiveness+measures+derived+from+near-infrared+spectroscopy.">Repeatability of vascular responsiveness measures derived from near-infrared spectroscopy.</a> Physiol Rep. 4 (9), (2016).</li><li>Ryan, T. E., Southern, W. M., Brizendine, J. T., McCully, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+changes+in+skeletal+muscle+metabolism+measured+with+optical+spectroscopy.">Activity-induced changes in skeletal muscle metabolism measured with optical spectroscopy.</a> Med Sci Sports Exerc. 45 (12), 2346-2352 (2013).</li><li>Southern, W. M., 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=Reduced+skeletal+muscle+oxidative+capacity+and+impaired+training+adaptations+in+heart+failure.">Reduced skeletal muscle oxidative capacity and impaired training adaptations in heart failure.</a> Physiol Rep. 3 (4), (2015).</li><li>Ryan, T. E., Brizendine, J. T., McCully, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+exercise+type+and+intensity+on+the+noninvasive+assessment+of+skeletal+muscle+mitochondrial+function+using+near-infrared+spectroscopy.">A comparison of exercise type and intensity on the noninvasive assessment of skeletal muscle mitochondrial function using near-infrared spectroscopy.</a> J Appl Physiol. 114 (2), 230-237 (2013).</li><li>Adami, A., Cao, R., Porszasz, J., Casaburi, R., Rossiter, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+NIRS+assessment+of+muscle+oxidative+capacity+in+smokers+with+and+without+COPD.">Reproducibility of NIRS assessment of muscle oxidative capacity in smokers with and without COPD.</a> Respir Physiol Neurobiol. 235, 18-26 (2017).</li><li>Lacroix, 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=Reproducibility+of+near-infrared+spectroscopy+parameters+measured+during+brachial+artery+occlusion+and+reactive+hyperemia+in+healthy+men.">Reproducibility of near-infrared spectroscopy parameters measured during brachial artery occlusion and reactive hyperemia in healthy men.</a> J Biomed Opt. 17 (7), 077010 (2012).</li><li>Bopp, C. M., Townsend, D. K., Warren, S., Barstow, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+brachial+artery+blood+flow+and+total+[hemoglobin+myoglobin]+during+post-occlusive+reactive+hyperemia.">Relationship between brachial artery blood flow and total [hemoglobin+myoglobin] during post-occlusive reactive hyperemia.</a> Microvasc Res. 91, 37-43 (2014).</li><li>Willingham, T. B., Southern, W. M., McCully, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+reactive+hyperemia+in+the+lower+limb+using+near-infrared+spectroscopy.">Measuring reactive hyperemia in the lower limb using near-infrared spectroscopy.</a> J Biomed Opt. 21 (9), 091302 (2016).</li><li>Kragelj, R., Jarm, T., Erjavec, T., Presern-Strukelj, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+postocclusive+reactive+hyperemia+measured+by+near+infrared+spectroscopy+in+patients+with+peripheral+vascular+disease+and+in+healthy+volunteers.">Parameters of postocclusive reactive hyperemia measured by near infrared spectroscopy in patients with peripheral vascular disease and in healthy volunteers.</a> Ann Biomed Eng. 29 (4), 311-320 (2001).</li><li>Gurley, K., Shang, Y., Yu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+quantification+of+absolute+blood+flow,+blood+oxygenation,+and+oxygen+consumption+rate+in+exercising+skeletal+muscle.">Noninvasive optical quantification of absolute blood flow, blood oxygenation, and oxygen consumption rate in exercising skeletal muscle.</a> J Biomed Opt. 17 (7), 075010 (2012).</li></ol>

Los autores declaran que no tienen intereses financieros que compiten.

Los métodos descritos permiten una evaluación no invasiva, clínica de hiperemia reactiva, acoplamiento neurovascular y capacidad oxidativa del músculo esquelético en una sola visita de clínica o de laboratorio.
Consideraciones críticas
Aunque NIRS es relativamente robusto y fácil de usar, la recogida de estos datos requieren cuidadosa colocación de la optodes directamente sobre el vientre muscular, firmemente asegurado en su lugar ...

La respuesta hiperémica a un breve período de isquemia del tejido se ha convertido en una clave medida no invasiva de la función vascular (micro). Durante la oclusión de una arteria del conducto, las arteriolas descendentes dilatan en un esfuerzo por contrarrestar el insulto isquémico. Sobre el lanzamiento de la oclusión, la disminución de la resistencia vascular produce hiperemia, la magnitud de la que es dictada por la capacidad de dilatar la microvascularización aguas abajo. Mientras que la hiperemia reactiva es un fuerte predictor independiente de eventos cardiovasculares1,2 y, por tanto, un criterio de valoración clínicamente significativa, su significación funcional para ejercer la tolerancia y la calidad de vida es menos clara.
De hecho, el ejercicio dinámico representa un mayor estrés cardiovascular que exige una respuesta neurovascular altamente coordinada para coincidir con la entrega de oxígeno a demanda metabólica. Por ejemplo, el flujo sanguíneo músculo esquelético puede aumentar casi 100-fold durante musculares aisladas contracciones3, que pasarían a avasallar a la capacidad de bombeo del corazón si se extrapolaron una respuesta hemodinámica al ejercicio por todo el cuerpo. Por consiguiente, para evitar hipotensión severa, simpática (es decir, vasoconstrictor) aumenta la actividad nerviosa para redistribuir el gasto cardíaco de tejidos inactivos y viscerales y músculo esquelético activo4. Salida comprensiva está dirigida también al ejercicio músculo esquelético5; sin embargo, señalización metabólica local atenúa la respuesta vasoconstrictora para asegurar adecuado tejido oxígeno entrega6,7,8,9,10, 11. en conjunto, este proceso se denomina simpatolisis funcional12y es imprescindible para la normal regulación de flujo de sangre del músculo esquelético durante el ejercicio. Puesto que el flujo sanguíneo del músculo esquelético es un determinante clave de la capacidad aeróbica, un predictor independiente de la calidad de vida y de la morbilidad y mortalidad de enfermedad cardiovascular13— comprensión del control del oxígeno tejido y flujo sanguíneo del músculo esquelético entrega durante el ejercicio es de gran importancia clínica.
Entrega de oxígeno es sólo la mitad de la ecuación de Fick, sin embargo, con satisfacción de la otra mitad de la ecuación de la utilización del oxígeno. Entre los principales determina de utilización del oxígeno, la fosforilación oxidativa mitocondrial juega un papel esencial en el suministro de energía adecuada para los procesos celulares tanto en reposo como durante el ejercicio. De hecho, deficiencias en la capacidad oxidativa muscular pueden limitar funcional capacidad y calidad de vida14,15,16. Diversas medidas se utilizan para proporcionar un índice de la capacidad oxidativa del músculo, incluyendo las biopsias del músculo invasivo y costoso y desperdiciador de tiempo de resonancia magnética espectroscopia (MRS) técnicas.
Aquí, proponemos un enfoque novedoso, no invasivo, utilizando espectroscopia de infrarrojo cercano (NIRS) para evaluar cada uno de estos tres principales medidas de resultado clínicas (hiperemia reactiva simpatolisis y capacidad oxidativa muscular) en una sola visita de clínica o de laboratorio. Las principales ventajas de este enfoque son tres: en primer lugar, esta técnica es fácilmente portable, relativamente bajo costo y fáciles de realizar. Enfoques actuales de la ecografía Doppler para medir la hiperemia reactiva son altamente operador dependiente, que requieren gran habilidad y entrenamiento — y requiere software hardware y procesamiento posterior de adquisición de datos sofisticados, de alto costo,. Por otra parte, esto podría concebiblemente introducirse en la clínica y/o ensayos clínicos grandes para cabecera de monitoreo o prueba de eficacia terapéutica. En segundo lugar, en virtud de la metodología, esta técnica se centra específicamente en la microvascularización de músculo esquelético, aumentando la especificidad global de la técnica. Enfoques alternativos mediante ecografía Doppler centran totalmente en los vasos del conducto ascendente e infieren cambios aguas abajo, que pueden atenuar la señal. En tercer lugar, esta técnica es completamente no invasivo. Capacidad oxidativa del músculo esquelético se evalúa tradicionalmente con el invasor y las biopsias musculares dolorosos y funcionales simpatolisis pueden evaluarse con la inyección intrarterial de simpaticomiméticos y droga. Este enfoque evita estos requisitos todos juntos.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Dual-channel OxiplexTS Near-infrared spectroscopy machine</td>
			<td style="width:223px;">Iss Medical</td>
			<td align="right" style="width:119px;">101</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">NIRS muscle sensor</td>
			<td style="width:223px;">Iss Medical</td>
			<td align="right" style="width:119px;">201.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">E20 Rapid cuff inflation system</td>
			<td style="width:223px;">Hokanson</td>
			<td style="width:119px;">E20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">AG101 Air Source</td>
			<td style="width:223px;">Hokanson</td>
			<td style="width:119px;">AG101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Smedley Handgrip dynometer (recording)</td>
			<td style="width:223px;">Stolting</td>
			<td align="right" style="width:119px;">56380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Powerlab 16/35, 16 Channel Recorder</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">PL3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human NIBP Set</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">ML282-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Bio Amp</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">FE132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Quad Bridge Amp</td>
			<td style="width:223px;">ADInstruments</td>
			<td style="width:119px;">FE224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Connex Spot Monitor</td>
			<td style="width:223px;">Welch Allyn</td>
			<td style="width:119px;">71WX-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Origin(Pro) graphing software</td>
			<td style="width:223px;">OrignPro</td>
			<td style="width:119px;">Pro</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Lower body negative pressure chamber</td>
			<td style="width:223px;">Physiology Research Instruments</td>
			<td style="width:119px;">standard unit</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal muscle neurovascular coupling, oxidative capacity, and microvascular function with 'one stop shop' near-infrared spectroscopy

Ejercicio representa un estrés hemodinámico importante que exige una respuesta neurovascular altamente coordinada para coincidir con la entrega de oxígeno a demanda metabólica. Hiperemia reactiva (en respuesta a un breve período de isquemia del tejido) es un predictor independiente de eventos cardiovasculares y proporciona la penetración importante en la salud cardiovascular y la capacidad de vasodilatador. Capacidad oxidativa del músculo esquelético es igualmente importante en la salud y la enfermedad, como lo determina el suministro de energía para los procesos de myocellular. Aquí, describimos un enfoque simple y no invasor usando la espectroscopia de infrarrojo cercano para evaluar cada uno de estos puntos finales clínicos principales (hiperemia reactiva, acoplamiento neurovascular y capacidad oxidativa muscular) durante una sola visita de clínica o de laboratorio. A diferencia de la ecografía Doppler, imágenes/espectroscopia de resonancia magnética, o las mediciones de flujo basadas en catéter invasivo o biopsias del músculo, nuestro enfoque es menos operador dependiente, bajo costo y completamente no invasivo. Datos representativos de nuestro laboratorio junto con los datos Resumen de la literatura previamente publicada ilustran la utilidad de cada uno de estos terminales. Una vez que se domina esta técnica, aplicación a poblaciones clínicas proporcionará importante visión mecanicista de intolerancia del ejercicio y disfunción cardiovascular.

Capacidad oxidativa del músculo esquelético
La figura 2 ilustra una respuesta participante representante durante una evaluación de capacidad oxidativa del músculo esquelético derivado de NIRS. Panel A muestra la saturación de tejido perfil durante un minuto 5 arterial brazalete protocolo de obstrucción, el ejercicio de la empuñadura y la oclusión arterial intermite...

Watch this Scientific Journal Video about Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy at JoVE.com

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Músculo esquelético Neurovascular acoplamiento, capacidad oxidativa y la función Microvascular con espectroscopia del infrarrojo cercano "Una parada tienda"

Aquí, describimos un enfoque simple y no invasor usando la espectroscopia de infrarrojo cercano para evaluar hiperemia reactiva, acoplamiento neurovascular y capacidad oxidativa del músculo esquelético en una sola visita de clínica o de laboratorio.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

skeletal-muscle-neurovascular-coupling-oxidative-capacity

Research

JoVE Journal

Medicine

12.4K vistas.  University of Texas at Arlington. Aquí, describimos un enfoque simple y no invasor usando la espectroscopia de infrarrojo cercano para evaluar hiperemia reactiva, acoplamiento neurovascular y capacidad oxidativa del músculo esquelético en una sola visita de clínica o de laboratorio.

Video: Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mecánico Análisis de músculo esquelético aislado

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Investigación

Medicina

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

Ensayo in vivo del canino función muscular

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Músculo Esquelético dimorfismo de género de Proteómica

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

La evaluación no invasiva de la función endotelial microvascular y

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Imágenes por resonancia magnética cuantitativa de la enfermedad del músculo esquelético

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Imagen Fluorescente combinado del infrarrojo cercano y la tomografía computarizada-Micro para visualizar de manera directa tromboembolia cerebral

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

El fósforo-31 espectroscopía de resonancia magnética: una herramienta para medir<em&gt; En Vivo</em&gt; Capacidad de la fosforilación oxidativa mitocondrial en el músculo esquelético humano

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Evaluación de la función Microvascular del tejido adiposo humano mediante videomicroscopía

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Una mejor práctica para la validación de las inyecciones de vena de cola en rata con agentes cerca infrarrojo-marcado con

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

Cómo administrar espectroscopia de infrarrojo cercano en neonatos, bebés y niños gravemente enfermos

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...