Name: application of chronic stimulation to study contractile activity-induced rat skeletal muscle phenotypic adaptations
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Description: Watch this Scientific Journal Video about Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations at JoVE.com

Agradecemos a Liam Tyron su experta lectura del manuscrito. Este trabajo fue apoyado por fondos de las ciencias naturales e Ingeniería investigación Consejo de Canadá (NSERC) a D. A. campana. D. A. Hood también es titular de una Cátedra de investigación de Canadá en fisiología celular.

Todos los procedimientos relacionados con el animal fueron revisados y aprobados por el Comité de cuidado de Animal de Universidad de York. A su llegada en las instalaciones de animales en la Universidad de York, todas las ratas se les dio un mínimo de cinco días para aclimatarse a su entorno antes del procedimiento quirúrgico, con alimentos proporcionados ad libitum. Aunque este protocolo se ha aplicado previamente a otras especies de17,15,</su...

<ol>
	<li>Holloszy, J. O., Coyle, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptations+of+skeletal+muscle+to+endurance+exercise+and+their+metabolic+consequences.">Adaptations of skeletal muscle to endurance exercise and their metabolic consequences.</a> J Appl Physiol Respir Environ Exerc Physiol. 56 (4), 831-838 (1984).</li><li>Hood, 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=Invited+Review:+contractile+activity-induced+mitochondrial+biogenesis+in+skeletal+muscle.">Invited Review: contractile activity-induced mitochondrial biogenesis in skeletal muscle.</a> J Appl Physiol. 90 (3), 1137-1157 (2001).</li><li>Fernandes, 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=Exercise+training+restores+the+endothelial+progenitor+cells+number+and+function+in+hypertension:+implications+for+angiogenesis.">Exercise training restores the endothelial progenitor cells number and function in hypertension: implications for angiogenesis.</a> J Hypertens. 30 (11), 2133-2143 (2012).</li><li>Chabi, B., Adhihetty, P. J., O'Leary, M. F., Menzies, K. J., Hood, 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=Relationship+between+Sirt1+expression+and+mitochondrial+proteins+during+conditions+of+chronic+muscle+use+and+disuse.">Relationship between Sirt1 expression and mitochondrial proteins during conditions of chronic muscle use and disuse.</a> J Appl Physiol. 107 (6), 1730-1735 (2009).</li><li>Lessard, S. 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=Resistance+to+aerobic+exercise+training+causes+metabolic+dysfunction+and+reveals+novel+exercise-regulated+signaling+networks.">Resistance to aerobic exercise training causes metabolic dysfunction and reveals novel exercise-regulated signaling networks.</a> Diabetes. 62 (8), 2717-2727 (2013).</li><li>Irrcher, I., Adhihetty, P. J., Sheehan, T., Joseph, A. M., Hood, 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=PPARgamma+coactivator-1alpha+expression+during+thyroid+hormone-+and+contractile+activity-induced+mitochondrial+adaptations.">PPARgamma coactivator-1alpha expression during thyroid hormone- and contractile activity-induced mitochondrial adaptations.</a> Am J Physiol Cell Physiol. 284 (6), C1669-C1677 (2003).</li><li>Tamura, Y., 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=Postexercise+whole+body+heat+stress+additively+enhances+endurance+training-induced+mitochondrial+adaptations+in+mouse+skeletal+muscle.">Postexercise whole body heat stress additively enhances endurance training-induced mitochondrial adaptations in mouse skeletal muscle.</a> Am J Physiol Regul Integr Comp Physiol. 307 (7), R931-R943 (2014).</li><li>Mosole, 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=Long-term+high-level+exercise+promotes+muscle+reinnervation+with+age.">Long-term high-level exercise promotes muscle reinnervation with age.</a> J Neuropathol Exp Neurol. 73 (4), 284-294 (2014).</li><li>Irrcher, I., Walkinshaw, D. R., Sheehan, T. E., Hood, 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=Thyroid+hormone+(T3)+rapidly+activates+p38+and+AMPK+in+skeletal+muscle+in+vivo.">Thyroid hormone (T3) rapidly activates p38 and AMPK in skeletal muscle in vivo.</a> J Appl Physiol. 104 (1), 178-185 (2008).</li><li>Lesmana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+change+in+thyroid+hormone+signaling+by+altered+training+intensity+in+male+rat+skeletal+muscle.">The change in thyroid hormone signaling by altered training intensity in male rat skeletal muscle.</a> Endocr J. 63 (8), 727-738 (2016).</li><li>Hokama, J. Y., Streeper, R. S., Henriksen, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+exercise+training+enhances+glucose+transport+in+muscle+stimulated+by+insulin-like+growth+factor+I.">Voluntary exercise training enhances glucose transport in muscle stimulated by insulin-like growth factor I.</a> J Appl Physiol. 82 (2), 508-512 (1997).</li><li>Tyler, K. R., Wright, A. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+weight+portable+stimulators+for+stimulation+of+skeletal+muscles+at+different+frequencies+and+for+cardiac+pacing.">Light weight portable stimulators for stimulation of skeletal muscles at different frequencies and for cardiac pacing.</a> J Physiol Lond. 307, 6-7 (1980).</li><li>Takahashi, M., Rana, A., Hood, 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=Portable+electrical+stimulator+for+use+in+small+animals.">Portable electrical stimulator for use in small animals.</a> J Appl Physiol. 74 (2), 942-945 (1993).</li><li>Ljubicic, V., Adhihetty, P. J., Hood, 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=Application+of+animal+models:+chronic+electrical+stimulation-induced+contractile+activity.">Application of animal models: chronic electrical stimulation-induced contractile activity.</a> Can J Appl Physiol. 30 (5), 625-643 (2005).</li><li>Pette, D., Vrbova, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+chronic+electrical+stimulation+teach+us+about+muscle+plasticity?.">What does chronic electrical stimulation teach us about muscle plasticity?.</a> Muscle Nerve. 22 (6), 666-677 (1999).</li><li>Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Perspectives:+plasticity+of+mammalian+skeletal+muscle.">Historical Perspectives: plasticity of mammalian skeletal muscle.</a> J Appl Physiol. 90 (3), 1119-1124 (2001).</li><li>Pette, D., Vrbova, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Contribution+of+Neuromuscular+Stimulation+in+Elucidating+Muscle+Plasticity+Revisited.">The Contribution of Neuromuscular Stimulation in Elucidating Muscle Plasticity Revisited.</a> Eur J Transl Myol. 27 (1), 6368 (2017).</li><li>Skorjanc, D., Jaschinski, F., Heine, G., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+increases+in+capillarization+and+mitochondrial+enzymes+in+low-frequency-stimulated+rabbit+muscle.">Sequential increases in capillarization and mitochondrial enzymes in low-frequency-stimulated rabbit muscle.</a> Am J Physiol. 274 (3 Pt 1), C810-C818 (1998).</li><li>Kim, Y., Hood, 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=Regulation+of+the+autophagy+system+during+chronic+contractile+activity-induced+muscle+adaptations.">Regulation of the autophagy system during chronic contractile activity-induced muscle adaptations.</a> Physiol Rep. 5 (14), (2017).</li><li>Memme, J. M., Oliveira, A. N., Hood, 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=Chronology+of+UPR+activation+in+skeletal+muscle+adaptations+to+chronic+contractile+activity.">Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity.</a> Am J Physiol Cell Physiol. 310 (11), C1024-C1036 (2016).</li><li>Ljubicic, V., 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=Molecular+basis+for+an+attenuated+mitochondrial+adaptive+plasticity+in+aged+skeletal+muscle.">Molecular basis for an attenuated mitochondrial adaptive plasticity in aged skeletal muscle.</a> Aging (Albany NY). 1 (9), 818-830 (2009).</li><li>Schwarz, G., Leisner, E., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+telestimulation+systems+for+chronic+indirect+muscle+stimulation+in+caged+rabbits+and+mice.">Two telestimulation systems for chronic indirect muscle stimulation in caged rabbits and mice.</a> Pflugers Arch. 398 (2), 130-133 (1983).</li><li>Simoneau, J. A., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+effects+of+chronic+nerve+stimulation+upon+tibialis+anterior+muscle+in+mouse,+rat,+guinea+pig,+and+rabbit.">Species-specific effects of chronic nerve stimulation upon tibialis anterior muscle in mouse, rat, guinea pig, and rabbit.</a> Pflugers Arch. 412 (1-2), 86-92 (1988).</li><li>Ohlendieck, K., 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=Effects+of+chronic+low-frequency+stimulation+on+Ca2+-regulatory+membrane+proteins+in+rabbit+fast+muscle.">Effects of chronic low-frequency stimulation on Ca2+-regulatory membrane proteins in rabbit fast muscle.</a> Pflugers Arch. 438 (5), 700-708 (1999).</li><li>Brown, M. D., Cotter, M. A., Hudlicka, O., Vrbova, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+different+patterns+of+muscle+activity+on+capillary+density,+mechanical+properties+and+structure+of+slow+and+fast+rabbit+muscles.">The effects of different patterns of muscle activity on capillary density, mechanical properties and structure of slow and fast rabbit muscles.</a> Pflugers Arch. 361 (3), 241-250 (1976).</li><li>Skorjanc, D., Traub, I., Pette, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identical+responses+of+fast+muscle+to+sustained+activity+by+low-frequency+stimulation+in+young+and+aging+rats.">Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats.</a> J Appl Physiol. 85 (2), 437-441 (1998).</li><li>Kim, Y., Triolo, M., Hood, 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=Impact+of+Aging+and+Exercise+on+Mitochondrial+Quality+Control+in+Skeletal+Muscle.">Impact of Aging and Exercise on Mitochondrial Quality Control in Skeletal Muscle.</a> Oxid Med Cell Longev. 2017, 3165396 (2017).</li><li>Callewaert, L., Puers, B., Sansen, W., Jarvis, J. C., Salmons, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+implantable+device+for+investigating+the+adaptive+response+of+skeletal+muscle+to+chronic+electrical+stimulation.">Programmable implantable device for investigating the adaptive response of skeletal muscle to chronic electrical stimulation.</a> Med Biol Eng Comput. 29 (5), 548-553 (1991).</li><li>Kern, 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=Electrical+stimulation+counteracts+muscle+decline+in+seniors.">Electrical stimulation counteracts muscle decline in seniors.</a> Front Aging Neurosci. 6, 189 (2014).</li><li>Zampieri, 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=Physical+exercise+in+aging+human+skeletal+muscle+increases+mitochondrial+calcium+uniporter+expression+levels+and+affects+mitochondria+dynamics.">Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics.</a> Physiol Rep. 4 (24), (2016).</li></ol>

El modelo de actividad contráctil crónica (CCA) de ejercicio muscular de baja frecuencia estimulación en vivo, es un excelente modelo para el estudio de adaptaciones fenotípicas del músculo para el ejercicio de13,24,25 , 26. como se indica en anteriores estudios20,27, CCA es una herramienta eficaz por el cual los investiga...

Músculo esquelético puede adaptarse para el ejercicio de formación a través de cambios en su bioenergética y estructura física1. Una de las principales alteraciones por entrenamiento de resistencia es la Biogénesis mitocondrial, que se puede evaluar por un aumento en la expresión de los componentes mitocondriales (p. ej., subunidades de c oxidasa [COX] citocromo), así como la expresión de el coactivator transcripcional, PGC-1α2. Un creciente número de estudios ha indicado que numerosos otros factores, como volumen mitocondrial y mitofagia, también son importantes para las adaptaciones musculares. Sin embargo, los mecanismos por los cual ejercicio agudo o crónico regulan estos procesos en músculo esquelético aún no están claros.
Para delinear los caminos que se regulan las adaptaciones del músculo inducida por el ejercicio, varios modelos de ejercicio se han utilizado comúnmente en estudios de roedores, incluyendo rueda de ardilla, corriente de la rueda y ejercicio de natación. Sin embargo, estos protocolos tienen algunas limitaciones en eso ~ 4-12 semanas se necesitan para observar estos cambios fenotípicos3,4,5. Como un método experimental alternativo, baja frecuencia inducida por estimulación crónica actividad contráctil (CCA) se ha utilizado eficazmente, como puede llevar a adaptaciones musculares en un período mucho más corto (es decir, hasta 7 días) y sus efectos ser comparables, o incluso superior a otros protocolos de ejercicio. Además, la presencia de hormonal6, temperatura7y efectos neurológicos8 puede hacer difícil entender las respuestas del músculo-específica al ejercicio crónico. Por ejemplo, la hormona de tiroides9,10 y factor de crecimiento insulínico (IGF) -1 se han identificado11 para mediar adaptaciones inducidas por el entrenamiento muscular, que pueden regular también otras vías de señalización en esqueleto muscular. En particular, los efectos inducidos por la CCA mínimamente son regulados por factores sistémicos, lo que permite centrarse en la respuesta directa del músculo esquelético a la actividad contráctil.
La unidad externa para el CCA fue introducida por Tyler y Wright12y ha sido desarrollada con modificaciones12. En Resumen, la unidad se compone de tres partes principales: un detector de infrarrojos que puede activar y desactivar por exposición a luz infrarroja, un generador de pulsos y un indicador del pulso (figura 1). El diseño detallado del circuito de la unidad de estimulador ha sido descrito previamente13. Las características detalladas y específicas de CCA se pueden encontrar en mayor profundidad en un número de revisión artículos14,15,16,17. En Resumen, el protocolo de estimulación está diseñado para activar el nervio peroneo común a baja frecuencia (es decir, 10 Hz), y los músculos inervados (tibialis anterior [TA] y Músculo extensor digitorum largo [EDL]) se ven obligados a contratar un longitud predeterminada de tiempo (por ejemplo, 3-6 h). Con el tiempo, esto cambia los músculos antes mencionados a un fenotipo más aeróbico, demostrado por un aumento en la densidad capilar18 y contenido mitocondrial19,20,21. Así, este método es un modelo probado para imitar algunas de las adaptaciones de entrenamiento de resistencia importante en músculo esquelético de ratas.
Este trabajo presenta un procedimiento detallado de la cirugía de implante de electrodo para inducir CCA para que los investigadores pueden aplicar este modelo en sus estudios de ejercicio. CCA es un excelente modelo para estudiar el curso del tiempo de las adaptaciones del músculo, proporcionando así una herramienta eficaz para la investigación de varios eventos moleculares y de señalización en ambos puntos del tiempo temprano y más tarde tras el inicio del entrenamiento.

<table>
	<tbody>
		<tr>
			<td>Sprague Dawley Rat</td>
			<td>Charles River</td>
			<td>Strain 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chronic contractile activity unit</td>
			<td>Home-made</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>CCA unit protective box (3.5 x 3.5 x 2.5 cm)</td>
			<td>Home-made</td>
			<td>n/a</td>
			<td>Box should be made of opaque material or covered in an opague tape</td>
		</tr>
		<tr>
			<td>Coin lithium ion batteries (3V)</td>
			<td>Panasonic</td>
			<td>CR2016</td>
			<td></td>
		</tr>
		<tr>
			<td>Medwire</td>
			<td>Leico Industries</td>
			<td>316SS7/44T</td>
			<td></td>
		</tr>
		<tr>
			<td>Solder pin (socket)</td>
			<td>Digi-Key</td>
			<td>ED6218-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Zonas porous tape</td>
			<td>Johnson &#38; Johnson</td>
			<td>5104</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture silk (Size 5)</td>
			<td>Ethicon</td>
			<td>640G</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture silk (Size 6)</td>
			<td>Ethicon</td>
			<td>706G</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved blunt scissor (11.5 cm Length)</td>
			<td>F.S.T.</td>
			<td>14075-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved blunt scissor (15 cm Length)</td>
			<td>F.S.T.</td>
			<td>14111-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate haemostatic forceps (16 cm Length)</td>
			<td>Lawton</td>
			<td>06-0230</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless-steel rod (30 cm; 7mm diameter)</td>
			<td>Home-made</td>
			<td>n/a</td>
			<td>Rod should have 5 mm slit in one end to hold the wire for tunneling under the skin</td>
		</tr>
		<tr>
			<td>Clip applying forceps</td>
			<td>KLS Martin</td>
			<td>20-916-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Staples (clips)</td>
			<td>Bbraun</td>
			<td>BN507R</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal hooks/retractor</td>
			<td>Home-made</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine (500 mL)</td>
			<td>Rougier</td>
			<td>#NPN00172944</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium</td>
			<td>Novopharm</td>
			<td>#DIN00872644</td>
			<td></td>
		</tr>
		<tr>
			<td>Metacam</td>
			<td>Boehringer</td>
			<td>#DIN02240463</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital multimeter (voltmeter)</td>
			<td>Soar Corporation</td>
			<td>ME-501</td>
			<td></td>
		</tr>
		<tr>
			<td>LED digital stroboscope</td>
			<td>Lutron Electronic Enterprise</td>
			<td>DT-2269</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of chronic stimulation to study contractile activity-induced rat skeletal muscle phenotypic adaptations

Músculo esquelético es un tejido altamente adaptable, como sus propiedades bioquímicas y fisiológicas se alteran considerablemente en respuesta al ejercicio crónico. Para investigar los mecanismos subyacentes que producen diversas adaptaciones del músculo, un número de protocolos de ejercicio como la caminadora, rueda funcionamiento y ejercicio de natación se han utilizado en los estudios con animales. Sin embargo, estos ejercen modelos requieren un largo período de tiempo para lograr adaptaciones musculares, que pueden ser también reguladas por factores humorales o neurológicos, lo que limita sus aplicaciones en el estudio de las adaptaciones inducidas por la contracción músculo-específica. Estimulación indirecta baja frecuencia (10 Hz) para inducir la actividad contráctil crónica (CCA) se ha utilizado como un modelo alternativo de entrenamiento, con éxito puede llevar a adaptaciones mitocondriales musculares dentro de 7 días, independientes de factores sistémicos. Este documento detalla las técnicas quirúrgicas necesarias para aplicar el tratamiento de CCA en el músculo esquelético de ratas, para estudios de aplicación generalizada en el futuro.

Hemos demostrado que la actividad contráctil crónica (CCA) es una herramienta eficaz para inducir adaptaciones favorables mitocondriales en músculo esquelético. Ratas sometidas a 7 días de la CCA (6 h al día) pantalla mejorada la Biogénesis mitocondrial en el músculo estimulado en comparación con el miembro posterior contralateral sin estimular (control). Este aumento de la Biogénesis mitocondrial se indica por la expresión creciente de la proteína de PGC-1α (<strong class="x...

Watch this Scientific Journal Video about Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations at JoVE.com

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Este protocolo describe el uso del modelo de actividad contráctil crónica de ejercicio observar adaptaciones del músculo esquelético inducida por el estímulo en la trasera de la rata.

Aplicación de la estimulación crónica para estudiar adaptaciones fenotípicas de músculo esquelético de rata inducida por actividad contráctil

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

The chronic contractile activity model of exercise is useful to study skeletal muscle adaptations to exercise training stimuli in vivo. In particular, this model elicits the phenotypic adaptations of a six week training protocol within seven days. This method can help answer key questions in the muscle physiology field like mitochondrial biogenesis, autophagy, and many other exercise-associated cellular adaptations.The main advantage of this technique is that it allows for the observation of muscle specific adaptations over a short time course of seven days as compared to other exercise training protocols that require several months. The implications of this technique extend toward therapy of muscle atrophy ages because chronic muscle stimulation has been shown to have an effect on ameliorating aging-related muscle weakness. Generally individuals new to this method will struggle because of difficulty landmarking the common peroneal nerve and attaching electrical coils at either end as these require practice and precision.Begin by anesthetizing the rat under one to 3%isoflurane inhalation with oxygen and confirm full sedation by checking the response to a hind limb toe pinch and observing respiratory depth and rate. Apply ocular lubricant on eyes to avoid dryness and place the rat on a heating pad at a low to medium setting. Gently shave the left hind limb as well as a strip around the torso from the back of the neck around behind the forelimbs and across the anterior thorax.Gently wipe shaved areas with iodine and ethyl alcohol to disinfect. With the animal laying on its stomach use a sterile scalpel with a number 10 blade to make a small incision of around 0.5 centimeters on the back of the neck in the center of the shaved region. Then roll the animal onto its right side and make a two to three centimeter cut in the skin of the left hind limb between the dimple of the knee joint and close to the origin of the tail.Using blunt tipped curved surgical scissors dissect the subcutaneous area to around 3.5 to four centimeters separating the skin from the underlying muscle to make a pocket between the open skin and underlying muscle. Next, use surgical scissors to make a small incision of less than 0.5 centimeters on the biceps femoris muscle ensuring that the tips of the scissors are cutting directly down through the muscle. Then gently open the cut area until the internal muscle groups and the common peroneal nerve are visible.Use extreme caution to avoid cutting or damaging the nerve. Next prepare 50 to 60 centimeters of PTFE coated fine stainless steel wire and fold it in half. Hook the folded part of the wire into the slit of a 30 centimeter stainless steel rod.Use the rod to pass the wire through the open pocket of the hind limb up the leg and along the center of the back in an L shape pattern to reach the small incision area at the back of the neck. Fix the window by pulling it open with metal retractors until the size of the window is approximately 1.5 squared centimeters with the peroneal nerve lying in the center of the window. Using a scalpel, carefully strip the ends of the wire by 1.5 centimeters.Wrap the stripped wire ends around a blunted 21 gauge needle five times to make a coil. Once the coils are properly made release the needle from them. Make a knot at the very end of the coil and suture it on the left hand side of the nerve ensuring that the coil is at 1.5 to 2.5 millimeters from the nerve.To secure the coil apply two or three additional sutures along the coil. This step requires considerable practice and patience in order to avoid damaging the nerve and underlying muscle and to achieve all consistent stimulation condition across all animals. It's best to have one skilled researcher to complete all surgical procedures during a study.Apply two or three drops of antibiotic solution and then carefully suture the window using size 5-0 silk. Loosely wind the remaining slack of wire and push into the subcutaneous pocket above the sutured incision of the biceps femoris muscle above the hip. Apply two to three drops of antibiotic solution and close the open skin by stapling.Next, move the animal to the sternal position and cut the wire loop coming out of the incision at the top of the neck to create two wire ends. Using a scalpel, strip of the ends of the wires by 0.5 centimeters. Cut off any frayed wires.Slowly push the stripped parts of the wires into the hole of the pin sockets and using a soldering iron, solder the wires on the pin sockets. Pass the pin connected wire ends through a 4.4 centimeter sterile gauze pad. Then pass the wires through the hole in the base of the stimulator box.Insert the pins into the connection sockets on the CCA unit. Gently put the CCA unit into the chamber using a sticky tack to secure the CCA unit at the bottom of the chamber. Use athletic tape or porous surgical tape to fix the chamber around the shaved torso.Close the top of the chamber with three layers of taping. And finish by wrapping tape around the sides of the stimulator box to secure the box. Check if the CCA is working by exposing the unit to a single pulse of infrared light emitted by a portable infrared strobe light.If the CCA is properly working the hind limb muscles will contract in response to the infrared light. Weigh the animals shortly before beginning the CCA procedure to record a baseline measurement to help identify any severe stress or adverse effects by change of body weight after the CCA procedure. On the day of CCA stimulation turn on the CCA by exposing the stimulator unit to a single pulse of infrared light by a portable infrared strobe light and apply three or six hours of 10 hertz CCA stimulation.Check the stimulation and animal every 30 to 60 minutes. Following the desired CCA period turn off the CCA unit via infrared light exposure. Rats subjected to seven days of CCA for six hours per day display enhanced mitochondrial biogenesis in the stimulated muscle as compared to the unstimulated contralateral hind limb.This increase in mitochondrial biogenesis is indicated by increased protein expression of PGC-1 alpha. Examination of permeabilized muscle fibers to measure mitochondrial respiratory capacity reveals that CCA resulted in an increase in the maximal respiratory capacity of muscle relative to control muscle. Both subsarcolemmal and intermyofibrillar mitochondrial populations increased following seven days of CCA compared to control unstimulated muscle.Adaptations to the autophagy and lysosomal systems can also be brought about by CCA. An increase in the protein abundance of Transcription factor EB the principal regulator of lysosomal biogenesis is observed after CCA at all time points. Following this procedure other methods like general subcutaneous or intraperitoneal injections can be performed in order to answer additional questions such as estimation of autophagy flux with colchicine treatment.After watching this video you should have a good understanding of how to apply the current chronic contractile activity model in order to examine muscle phenotypic changes following endurance training.

Research

JoVE Journal

Developmental Biology

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Investigación

Biología del Desarrollo

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En la generación de Vitro del Somite derivados humanos inducida por células madre pluripotentes

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

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The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

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Realización de Xenoinjertos musculares esqueléticos humanos en ratones inmunodeficientes

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...