Los autores desean agradecer al Dr. Paul Witting y Genevieve Fong por su ayuda con la extracción de ARNm y PCR, y la Garnett Passe y Rodney Williams Memorial Fundación para el apoyo.

Declaración de Ética: Todos los procedimientos descritos a continuación fueron aprobados por la Universidad de Comité de Ética Animal Sydney. 1. Los animales NOTA: 1 y 8 - Un 9 meses de edad ratones (C57 / BL6) se obtuvieron del Centro de Recursos Animales (Perth, Australia). Los animales fueron alojados en el Fondo para Bosch de roedores en la Universidad de Sydney. <ol><li> Ratones Casa en jaulas estándar del ratón en un 12/12 horas de luz / oscuridad ciclo con el acceso a alimentos y agua ad libitum. </li><li> Divida a los ratones en cada grupo de edad en el infrarrojo cercano (NIR) tratados o tratamiento simulado (control) grupos de comparación. </li></ol> 2. Infrarrojo Cercano (NIR) Irradiación y Sham Tratamiento <ol><li> Afeitarse la piel en la región de la cabeza y el cuello del ratón con una afeitadora eléctrica tan estrechamente como sea posible para prevenir el rebrote rápida del cabello antes de la finalización del régimen de tratamiento. A fin de mantener el estado libre de patógenos de los ratones, utilice etanol al 70%para limpiar instrumentos entre los animales afeitado y desinfectante veterinaria F10 entre los animales de jaulas separadas. Haga esto 2-3 días antes del comienzo del tratamiento para que los animales no son excesivamente manipulado. </li><li> Restringir ratón manteniendo el extremo proximal de la cola y dejar que se relaje en la palma de una mano o de banco con el fin de minimizar el estrés que podría conducir a resultados falsos. </li><li> Sostenga el NIR (670 nm) LED (diodo emisor de luz) dispositivo 1-2 cm de distancia de la zona expuesta (rebajado) y encienda el dispositivo durante 90 segundos (Figura 1). NOTA: El cambio de temperatura como resultado de 90 segundos de exposición se midió como &lt;0,2 ° C en 100 ml de agua. </li><li> Repita los pasos 2.2 a 2.3 para el grupo de tratamiento simulado de los animales, pero deje el dispositivo apagado (Figura 1B). </li><li> Repita los pasos 2.2 a 2.3 para el grupo de tratamiento bloqueado-NIR de los animales, pero cubre el dispositivo con papel de aluminio. </li><li> Repita los pasos 2.2 a 2.5 días a las approximatintervalos hr e 24 durante 5 días consecutivos. </li><li> Extraer el epitelio vestibular sensorial (3 crestas y 1 mácula utricular) de ambos oídos en el post-tratamiento quinto día (véase la sección 3). </li></ol> 3. Extracción de tejido 20 <ol><li> Preparar 300 ml de un fluido cefalorraquídeo artificial (ACSF) basado en glicerol que consiste en (en mM): 26 NaHCO3, 11 glucosa, 250 glicerol, 2,5 KCl, 1,2 NaH 2 PO 4, 1,2 MgCl2, y 2.5 CaCl 2. Antes de la adición de CaCl2, gas la solución con carbógeno (95% de O2 y 5% de CO 2) para establecer un pH de 7,4 y evitar la precipitación de calcio (nubosidad). Enfriar la solución en un congelador a -80ºC durante 45 min de manera que se forma una suspensión de hielo. </li><li> Preparar el tampón de lisis aislamiento de ARN en microtubos de tapa de rosca marcados (se detiene el estallido de las tapas cuando los cambios de presión del tubo entre la congelación y descongelación en nitrógeno líquido) de acuerdoa las instrucciones del fabricante o las prácticas habituales de laboratorio. Tener nitrógeno líquido listo en un frasco Dewar de aluminio. NOTA: Utilice ropa protectora y eyeware al manejar nitrógeno líquido. Asegúrese de que se utiliza nitrógeno líquido en una habitación bien ventilada, ya que el volumen de su forma de gas es aproximadamente 700 veces mayor que la de su forma líquida y puede causar asfixia. </li><li> Profundamente anestesiar a los ratones con ketamina (400 mg / kg) a través de una inyección intra-peritoneal. Deje que el reflejo de las extremidades posteriores se desplome por completo como indicación de que los ratones están completamente anestesiados. </li><li> Decapitar a los ratones con tijeras de acero inoxidable afiladas y hacer una incisión a lo largo de la piel sagital del cráneo usando una cuchilla de afeitar (redondeado # 22). En este punto y los pasos a lo largo de 3.5 a 3.9, mantener la bóveda del cráneo, el cerebro y aparato vestibular subyacente lo más fresco posible por la aplicación regular de ACSF helada sobre el tejido. </li><li> Utilizando el brazo con punta de tijeras patrón estándar hacer una Small incisión en el cráneo en Lambda y cortar a lo largo de la sutura sagital. </li><li> Deslice suavemente un brazo de una gubia poco profundas debajo del hueso parietal de mantenimiento de la cuchilla lo más cerca posible a la superficie inferior del hueso sin arrastrar el cerebro. Seguros y alejarse del hueso parietal lateral y el hueso occipital posterior hasta que se vea el cerebro. </li><li> Use una pequeña espátula de acero inoxidable y levante el cerebro lejos de la anterior y la fosa craneal media para exponer el nervio vestíbulo coclear (CN VIII). Seccionar el nervio para evitar tensiones innecesarias en los axones aferentes primarias que inervan directamente las células ciliadas vestibulares. </li><li> Retire el cerebro en su totalidad después de la sección del CN VIII. </li><li> Observe el laberinto óseo contiene la cóclea y los órganos vestibulares periféricas en la fosa craneal media. Haga dos incisiones pequeñas al lado de cada laberinto óseo y extirpar toda la estructura sosteniendo el canal semicircular anterior y tirando despuésaliado. </li><li> Inmediatamente sumerja laberintos extirpados en un plato de la disección de la solución que contiene ACSF enfriado en hielo (como se describe en el paso 3.1), mientras continua perfusión con carbógeno. </li><li> Bajo un microscopio estereoscópico, mantenga pulsado el laberinto de la cóclea y fijarlo a la parte inferior del plato con fórceps. <ol><li> El uso de fórceps recto finas rayar una pequeña abertura en el hueso por encima del canal semicircular (SSC) ampolla anterior. </li><li> Ampliar suavemente esta apertura al llegar inmediatamente debajo del hueso y chasqueando hacia el exterior lejos de la ampolla. Tenga precaución para no empujar fórceps en la abertura y dañar el laberinto membranoso frágil continuación. Continuar de esta manera hasta el utrículo, anterior y lateral ampollas están expuestos. Si es posible eliminar la ampolla posterior también. </li></ol></li><li> Con unas pinzas finas, levante suavemente el utrículo y ampollas lejos del laberinto óseo hasta que estén completamente separados. Siempre que sea posible, mantenerlospor sus canales semicirculares asociados para evitar dañar el epitelio sensorial. En algunos casos, puede ser necesario cortar con tijeras iris para liberar las ampollas de la médula la parte proximal del conducto membranoso semicircular. </li><li> Agarrar firmemente los órganos vestibulares entre la punta del fórceps y el lugar en el tampón de lisis preparado anteriormente en el paso 3.2. Agite suavemente la pinza alrededor en el búfer para asegurar órganos vestibulares han desprendido de los fórceps. Compruebe este es el caso de llevar a los fórceps bajo el microscopio estereoscópico. <ol><li> Tornillo en la tapa de los microtúbulos y congelar la muestra en nitrógeno líquido inmediatamente. </li></ol></li></ol> 4. Extracción de ARN y RT-PCR <ol><li> Siga métodos estándar de ARN mensajero (ARNm) de extracción de acuerdo con las instrucciones del fabricante o protocolos de laboratorio preferidos. NOTA: Un kit comercial que utilizó ARN portador para ayudar a aislar pequeños rendimientos de ARNm se utilizó en esteprotocolo. Volúmenes de elución más bajas pueden ser usados ​​para aumentar las concentraciones finales de mRNA. NOTA: Negativo controles &quot;sin enzima&quot; (CNE) y controles &quot;sin plantilla de ADN&quot; (NTC) también deben completarse para asegurar la validez de los efectos observados. </li><li> Aplicar métodos estándar de la transcripción inversa de ARNm en ADN complementario (ADNc) y la amplificación de los genes diana 21-23. </li></ol>

<ol>
	<li>Agrawal, Y., Carey, J. P., Della Santina, ., C, C., Schubert, M. C., Minor, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+balance+and+vestibular+function+in+US+adults:+data+from+the+National+Health+and+Nutrition+Examination+Survey,+2001-2004.">Disorders of balance and vestibular function in US adults: data from the National Health and Nutrition Examination Survey, 2001-2004.</a> Archives of internal medicine. 169, 938-944 (2009).</li><li>Bessho, 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=Effect+of+subthreshold+infrared+laser+treatment+for+drusen+regression+on+macular+autofluorescence+in+patients+with+age-related+macular+degeneration.">Effect of subthreshold infrared laser treatment for drusen regression on macular autofluorescence in patients with age-related macular degeneration.</a> Retina. 25, 981-988 (2005).</li><li>Olk, R. 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=Therapeutic+benefits+of+infrared+(810-nm)+diode+laser+macular+grid+photocoagulation+in+prophylactic+treatment+of+nonexudative+age-related+macular+degeneration:+two-year+results+of+a+randomized+pilot+study.">Therapeutic benefits of infrared (810-nm) diode laser macular grid photocoagulation in prophylactic treatment of nonexudative age-related macular degeneration: two-year results of a randomized pilot study.</a> Ophthalmology. 106, 2082-2090 (1999).</li><li>Rodanant, N., 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=Predictors+of+drusen+reduction+after+subthreshold+infrared+(810+nm)+diode+laser+macular+grid+photocoagulation+for+nonexudative+age-related+macular+degeneration.">Predictors of drusen reduction after subthreshold infrared (810 nm) diode laser macular grid photocoagulation for nonexudative age-related macular degeneration.</a> American journal of ophthalmology. 134, 577-585 (2002).</li><li>De Taboada, 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=Transcranial+laser+therapy+attenuates+amyloid-beta+peptide+neuropathology+in+amyloid-beta+protein+precursor+transgenic+mice.">Transcranial laser therapy attenuates amyloid-beta peptide neuropathology in amyloid-beta protein precursor transgenic mice.</a> Journal of Alzheimer's disease : JAD. 23, 521-535 (2011).</li><li>Grillo, S. L., Duggett, N. A., Ennaceur, A., Chazot, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+infra-red+therapy+(1072+nm)+reduces+beta-amyloid+protein+levels+in+the+brain+of+an+Alzheimer's+disease+mouse+model.">Non-invasive infra-red therapy (1072 nm) reduces beta-amyloid protein levels in the brain of an Alzheimer's disease mouse model.</a> TASTPM. Journal of photochemistry and photobiology. B, Biology. 123, 13-22 (2013).</li><li>Purushothuman, S., Johnstone, D. M., Nandasena, C., Mitrofanis, J., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+with+near+infrared+light+mitigates+Alzheimer's+disease-related+pathology+in+cerebral+cortex+-+evidence+from+two+transgenic+mouse+models.">Photobiomodulation with near infrared light mitigates Alzheimer's disease-related pathology in cerebral cortex - evidence from two transgenic mouse models.</a> Alzheimer's researc., &amp; therapy. 6, 2 (2014).</li><li>Sommer, A. P., 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=670+nm+laser+light+and+EGCG+complementarily+reduce+amyloid-beta+aggregates+in+human+neuroblastoma+cells:+basis+for+treatment+of+Alzheimer's+disease.">670 nm laser light and EGCG complementarily reduce amyloid-beta aggregates in human neuroblastoma cells: basis for treatment of Alzheimer's disease.</a> Photomedicine and laser surgery. 30, 54-60 (2012).</li><li>Moro, 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=Photobiomodulation+preserves+behaviour+and+midbrain+dopaminergic+cells+from+MPTP+toxicity:+evidence+from+two+mouse+strains.">Photobiomodulation preserves behaviour and midbrain dopaminergic cells from MPTP toxicity: evidence from two mouse strains.</a> BMC neuroscience. 14, 40 (2013).</li><li>Peoples, 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=Photobiomodulation+enhances+nigral+dopaminergic+cell+survival+in+a+chronic+MPTP+mouse+model+of+Parkinson's+disease.">Photobiomodulation enhances nigral dopaminergic cell survival in a chronic MPTP mouse model of Parkinson's disease.</a> Parkinsonis., &amp; related. 18, 469-476 (2012).</li><li>Shaw, V. 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=Neuroprotection+of+midbrain+dopaminergic+cells+in+MPTP-treated+mice+after+near-infrared+light+treatment.">Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment.</a> The Journal of comparative neurology. 518, 25-40 (2010).</li><li>Ying, R., Liang, H. L., Whelan, H. T., Eells, J. T., Wong-Riley, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+with+near-infrared+light+via+light-emitting+diode+provides+added+benefit+against+rotenone-+and+MPP+-induced+neurotoxicity.">Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.</a> Brain research. 1243, 167-173 (2008).</li><li>Rajguru, S. 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=Infrared+photostimulation+of+the+crista+ampullaris.">Infrared photostimulation of the crista ampullaris.</a> The Journal of physiology. 589, 1283-1294 (2011).</li><li>Chung, 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=The+nuts+and+bolts+of+low-level+laser+(light)+therapy.">The nuts and bolts of low-level laser (light) therapy.</a> Annals of biomedical engineering. 40, 516-533 (2012).</li><li>Desmet, K. 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=Clinical+and+experimental+applications+of+NIR-LED+photobiomodulation.">Clinical and experimental applications of NIR-LED photobiomodulation.</a> Photomedicine and laser surgery. 24, 121-128 (2006).</li><li>Huang, Y. Y., Chen, A. C., Carroll, J. D., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+dose+response+in+low+level+light+therapy.">Biphasic dose response in low level light therapy.</a> Dose-response : a publication of International Hormesis Society. 7, 358-383 (2009).</li><li>Rojas, J. C., Gonzalez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+light+therapy+of+the+eye+and+brain.">Low-level light therapy of the eye and brain.</a> Eye and brain. 3, 49-67 (2011).</li><li>Vranceanu, F., 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=Striated+organelle,+a+cytoskeletal+structure+positioned+to+modulate+hair-cell+transduction.">Striated organelle, a cytoskeletal structure positioned to modulate hair-cell transduction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 4473-4478 (2012).</li><li>Johnson, K. 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=Separate+and+combined+effects+of+Sod1+and+Cdh23+mutations+on+age-related+hearing+loss+and+cochlear+pathology+in+C57BL/6J+mice.">Separate and combined effects of Sod1 and Cdh23 mutations on age-related hearing loss and cochlear pathology in C57BL/6J mice.</a> Hearing research. 268, 85-92 (2010).</li><li>Tung, V. W., Di Marco, S., Lim, R., Brichta, A. M., Camp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+semi-intact+preparation+of+the+mouse+vestibular+sensory+epithelium+for+electrophysiology+and+high-resolution+two-photon+microscopy.">An isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy.</a> Journal of visualized experiments : JoVE. , e50471 (2013).</li><li>Kirby, J., Menzies, F. M., Cookson, M. R., Bushby, K., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+in+a+cell+culture+model+of+SOD1-related+familial+motor+neurone+disease.">Differential gene expression in a cell culture model of SOD1-related familial motor neurone disease.</a> Human molecular genetics. 11, 2061-2075 (2002).</li><li>Parry, S. N., Ellis, N., Li, Z., Maitz, P., Witting, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+induces+oxidative+stress+and+decreases+endocytosis+and+monolayer+permissiveness+in+cultured+kidney+epithelial+cells+without+affecting+viability.">Myoglobin induces oxidative stress and decreases endocytosis and monolayer permissiveness in cultured kidney epithelial cells without affecting viability.</a> Kidney and Blood Pressure Research. 31, 16-28 (2008).</li><li>Saee-Rad, 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=Analysis+of+superoxide+dismutase+1,+dual-specificity+phosphatase+1,+and+transforming+growth+factor,+beta+1+genes+expression+in+keratoconic+and+non-keratoconic+corneas.">Analysis of superoxide dismutase 1, dual-specificity phosphatase 1, and transforming growth factor, beta 1 genes expression in keratoconic and non-keratoconic corneas.</a> Molecular vision. 19, 2501-2507 (2013).</li><li>Albert, E. 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=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> Journal of neurophysiology. 107, 3227-3234 (2012).</li><li>Moro, 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=Photobiomodulation+inside+the+brain:+a+novel+method+of+applying+near-infrared+light+intracranially+and+its+impact+on+dopaminergic+cell+survival+in+MPTP-treated+mice.">Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice.</a> Journal of neuroscience. 120, 670-683 (2014).</li><li>Moreno, L. 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=Infrared+neural+stimulation:+beam+path+in+the+guinea+pig+cochlea.">Infrared neural stimulation: beam path in the guinea pig cochlea.</a> Hearing research. 282, 289-302 (2011).</li><li>Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+red+thread+as+a+guide+in+the+vestibular+labyrinth.">A red thread as a guide in the vestibular labyrinth.</a> The Journal of physiology. 589, 1241-1241 (2011).</li><li>Chakrabarti, 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=Mitochondrial+Dysfunction+during+Brain+Aging:+Role+of+Oxidative+Stress+and+Modulation+by+Antioxidant+Supplementation.">Mitochondrial Dysfunction during Brain Aging: Role of Oxidative Stress and Modulation by Antioxidant Supplementation.</a> Aging and disease. 2, 242-256 (2011).</li><li>Petrosillo, G., De Benedictis, V., Ruggiero, F. M., Paradies, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decline+in+cytochrome+c+oxidase+activity+in+rat-brain+mitochondria+with+aging.+Role+of+peroxidized+cardiolipin+and+beneficial+effect+of+melatonin.">Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin.</a> Journal of bioenergetics and biomembranes. 45, 431-440 (2013).</li><li>Zhu, H., Sun, A., Zou, Y., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+metabolic+adaptation+promotes+mesenchymal+stem+cell+therapy+for+ischemia:+a+hypoxia-induced+and+glycogen-based+energy+prestorage+strategy.">Inducible metabolic adaptation promotes mesenchymal stem cell therapy for ischemia: a hypoxia-induced and glycogen-based energy prestorage strategy.</a> Arteriosclerosis, thrombosis, and vascular biology. 34, 870-876 (2014).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Los resultados representativos descritos aquí muestran que breve entrega transcraneal de la luz NIR (90 seg / día durante 5 días) es suficiente para elevar los niveles de expresión de antioxidantes en ratones de edad avanzada en comparación con los ratones tratados con placebo. Mientras que el calor emitido podría representar una fuente de mitocondrial y / o activación neuronal, según lo informado por vestibular rata aferentes 24 - nuestra medición del calor emitido por el NIR LED dispositivo fue &lt...

Disminución en los resultados equilibrio y caídas posteriores son comunes, y por desgracia a menudo características definitorias de envejecimiento natural 1. El impacto de esta disminución puede ser tanto físico y social, y reduce significativamente la calidad de vida de las personas mayores. En respuesta, las terapias físicas y de rehabilitación han sido el foco de la investigación de cataratas, pero no se han asociado con la reducción constante en la prevalencia de caídas repetidas. Al mismo tiempo, el trabajo de investigación de los cambios en el sistema vestibular periférico o central (el sistema responsable de mantener el equilibrio) es escasa, y posibles estrategias terapéuticas dirigidas a estos sistemas y las causas subyacentes del desequilibrio limitado. Trabajos recientes sobre los trastornos neurodegenerativos asociados con la edad, incluyendo la degeneración macular relacionada con la edad 2-4, modelos de enfermedad de Alzheimer 5-8, y la enfermedad de Parkinson 9-12 han demostrado efectos neuroprotectores de simple aplicación no invasiva de infrarrojo cercano (NIR) la luz. Además, en el sistema vestibular, NIR se ha utilizado para aumentar la actividad de las neuronas aferentes primarias vestibulares in vitro 13. Aunque no se entiende bien el mecanismo de la luz NIR, la mayoría de los estudios que utilizan NIR han sugerido que NIR estimula las mitocondrias complejo IV (citocromo c oxidasa) 14-17 para facilitar el metabolismo celular. En el epitelio sensorial vestibular la placa subcuticular de células que el cabello de tipo es denso en las mitocondrias de 18 años y, como tal, puede representar un punto de acción para el tratamiento terapéutico NIR. Aquí, una breve régimen, el tratamiento no invasivo de transcraneal aplica NIR que se puede utilizar para medir el metabolismo celular (y por implicación, la función mitocondrial) en el ratón epitelio sensorial vestibular se describe. También se discutió es una preparación del epitelio sensorial vestibular y se muestra que NIR aumenta la expresión de un ubiquitonos anti-oxidante (superóxido dismutasa 1) en el epitelio sensorial - previamente demostrado ser importante para la supervivencia de las células ciliadas cóclea 19.

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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Las estrategias para atenuar la disminución en la función del equilibrio con la edad son predominantemente centrada en terapias físicas que incluyen tareas de equilibrio y ejercicios. Sin embargo, estos enfoques no abordan las causas subyacentes del declive de equilibrio. Utilizando ratones, se evaluó el impacto de la luz del infrarrojo cercano (NIR) en el metabolismo de las células en el epitelio sensorial vestibular. Los datos recogidos muestran que esta intervención simple y seguro puede proteger estas células vulnerables de los efectos perjudiciales del envejecimiento natural. ARNm se extrajo a partir del epitelio sensorial vestibular periférica aislada (ampullaris crista y la mácula del utrículo) y posteriormente se transcribe en una biblioteca de ADNc. Después, esta biblioteca se probaron para la expresión de antioxidante ubicua (SOD-1). Entonces Antioxidante expresión génica se utilizó para cuantificar el metabolismo celular. Utilizando la entrega transcraneal de Nir en jóvenes (4 semanas) y mayores (8-9 meses) los ratones, y un régimen de tratamiento breve (90 seg / día durante 5 díasays), este trabajo sugiere Nir solo puede ser suficiente para mejorar la función mitocondrial en el epitelio sensorial vestibular. Dado que actualmente no hay métodos disponibles, asequibles, no invasivos de la terapia para mejorar la función de las células ciliadas vestibulares, la aplicación de radiación NIR externa proporciona una posible estrategia para contrarrestar el impacto del envejecimiento en el metabolismo celular enel epitelio sensorial vestibular.

Para comparar el impacto del tratamiento en Nir joven (4 semanas) y mayores (8-9 meses) los ratones que mide la expresión de antioxidante superóxido dismutasa 1 (SOD-1) en jóvenes (n = 16) y mayores (n = 20) Los ratones que fueron NIR-tratada, tratada simulada, o bloqueado-NIR. La Figura 2 muestra un aumento significativo en normalizado SOD-1 la expresión de β-actina de más de 2 veces en los animales jóvenes tratados con NIR jóvenes en comparación con los animales tratados con placebo (p &lt;0,...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

La disfunción mitocondrial es una característica de la senescencia celular. En este trabajo se utiliza el tratamiento no invasivo de infrarrojo cercano (NIR) para mejorar la función mitocondrial en el ratón epitelio sensorial vestibular envejecimiento.

Infrarrojo Cercano (NIR) Luz aumenta la expresión de un marcador de la función mitocondrial en el ratón vestibular sensorial Epitelio

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.9K vistas.  University of Sydney.  La disfunción mitocondrial es una característica de la senescencia celular. En este trabajo se utiliza el tratamiento no invasivo de infrarrojo cercano (NIR) para mejorar la función mitocondrial en el ratón epitelio sensorial vestibular envejecimiento. 

Video: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

La neuromodulación y mitocondrial de transporte: Imágenes en vivo en las neuronas del hipocampo durante largos períodos de tiempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Investigación

Neurociencias

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Una preparación semi-intacta aislada del ratón epitelio sensorial vestibular de Electrofisiología y de alta resolución de la microscopía de dos fotones

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

Imágenes in vivo de Óptica de Fibras Nerviosas Integridad de la RM con contraste en ratones

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo la detección de la mitofagia en células humanas, C. Elegansy los ratones

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicina

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Un protocolo de Transcranial Photobiomodulation terapia en ratones

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Imágenes en vivo del estado redox de glutatión mitocondrial en neuronas primarias utilizando un indicador ratiométrico

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Un rastreable<em&gt; En Vivo</em&gt; Ensayo para mitocondrial Moduladores Usando transgénico bioluminiscente<em&gt; Caenorhabditis elegans</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Biología

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Medición del potencial de membrana mitocondrial in vivo utilizando un indicador de voltaje codificado genéticamente

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

Obtención de imágenes de peróxido mitocondrial en células de levadura vivas mediante mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Autor destacado: Avance de la investigación mitocondrial - Biosensor mtHyper7 para análisis subcelular 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Obtención de imágenes de mtHyPer7, un biosensor radiométrico para el peróxido mitocondrial, en células vivas de levadura

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Infrarrojo Cercano (NIR) Luz aumenta la expresión de un marcador de la función mitocondrial en el ratón vestibular sensorial Epitelio

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Una preparación semi-intacta aislada del ratón epitelio sensorial vestibular de Electrofisiología y de alta resolución de la microscopía de dos fotones

Imágenes in vivo de Óptica de Fibras Nerviosas Integridad de la RM con contraste en ratones

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Un rastreable

Medición del potencial de membrana mitocondrial in vivo utilizando un indicador de voltaje codificado genéticamente

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Obtención de imágenes de mtHyPer7, un biosensor radiométrico para el peróxido mitocondrial, en células vivas de levadura