Damos las gracias a Annalisa Buffo sugerencias interesantes y comentarios, y Maurizio Grassano, Marina Boido y Ermira Pajaj para el rodaje. Este trabajo fue financiado por el FP7-MC-214003-2 (Marie Curie Initial Training Network AXREGEN) y la Compagnia di San Paolo, proyecto gliarep.

1. Pre-cirugía <ol><li> Pesar Rosa de Bengala en un tubo de 1,5 ml y se disuelven en solución salina estéril hasta alcanzar una concentración final de 15 mg / ml. Filtrar esterilizar a través de un filtro de 0,2 micras y almacenar en la oscuridad a temperatura ambiente hasta por dos meses. </li><li> Esterilizar todos los instrumentos quirúrgicos en autoclave. El área quirúrgica debe ser desinfectado menos de una hora antes de iniciar la cirugía. </li><li> Registre el peso corporal del ratón para ajustar la dosis de Rosa de Bengala a inyectar. Hemos inyectado 10 l / g de peso del animal en ratones hembras CD1 12 semanas de edad en el presente protocolo. La cantidad de Rosa de Bengala necesario para producir el tamaño de la lesión cortical deseada se puede determinar fácilmente en un conjunto separado de experimentos preliminares mediante pruebas de diferentes dosis (típicamente 2 l / g, 5 l / g o 10 l / g de peso corporal). Tenga en cuenta que la cantidad de Rosa de Bengala a inyectar es altamente dependiente de las condiciones del experimento, en particular del tipo de fuente de luzutilizado y la duración de la exposición a la luz. En el presente estudio, 10 l / g (150 mg / g) se encontró dosis a ser necesario para inducir photothrombosis después de la exposición a la luz durante 15 min, mientras que otros grupos informaron de que, o bien 50 mg / g 6,8 y 100 mg / g 10 inyección intraperitoneal fue suficiente para inducir una lesión photothrombotic. </li></ol> 2. Procedimiento Anestesia <ol><li> Anestesiar a los ratones con isoflurano en una cámara de inducción transparente (3,5-4% para la inducción, 1,5-2% para el mantenimiento) en 50% (v / v) oxygen/50% (v / v) mezcla de gas de monóxido de dinitrógeno. Anestesia gaseosa permite una llamada rápida de los animales y el nivel de gas anestésico se puede ajustar fácilmente. Alternativamente, los ratones también pueden ser anestesiados por una mezcla de ketamina-xilazina. </li><li> Cuando se alcanza la anestesia profunda, retire el animal anestesiado de la cámara de inducción, colóquelo en el marco estereotáxico y mantener la anestesia con una máscara facial. Ajuste el isoflurane dosis para lograr un nivel de anestesia adecuado. Monitorear la frecuencia respiratoria durante todo el procedimiento y asegúrese de que es constante (40-60 respiraciones por minuto). </li><li> Usa pellizcos del dedo del pie con el fin de garantizar que el animal es profundamente anestesiados. </li><li> Aplicar pomada para los ojos, con el fin de evitar que los ojos se sequen. </li><li> Insertar suavemente la sonda rectal para controlar la temperatura a lo largo de los procedimientos quirúrgicos. Ajuste la almohadilla de calefacción controlado por retroalimentación asociado para el mantenimiento de la temperatura del cuerpo del ratón a 37 ± 0,5 ° C. </li></ol> 3. Cirugía para la iluminación de la zona de destino <ol><li> Afeitar el cuero cabelludo ratón con una rasuradora eléctrica. </li><li> Asegure bien la cabeza e inserte las barras de oído en el meato externo. Tenga cuidado de no dañar los tímpanos. </li><li> Desinfectar la piel en la superficie con golpes de 70% de etanol y betadine usando hisopos de algodón alterna. </li><li> Utilice un bisturí para realizar una incisión a lo largo de la línea media del ojo leVel hasta el cuello. Aplicar retractores de la piel para mantener el cráneo expuesto. </li><li> Retraer con suavidad el periostio de los bordes del cráneo con un bisturí y dejar secar la superficie del cráneo con hisopos de algodón estériles. Identificar el bregma y lambda. Coloca una micropipeta de vidrio sobre el bregma como punto de referencia, a continuación, pasar a las coordenadas de la zona de interés. La región de interés elegido para este artículo es más o menos en forma de rotundamente, centrada aproximadamente 2 mm lateral a la bregma, y que cubre un área de aproximadamente 30 mm 2, que incluye una gran parte de la corteza sensitivomotora de acuerdo con el atlas de cerebro de ratón por Franklin y Paxinos 11. </li><li> Marque la posición de interés con los puntos de referencia y poner fibra óptica en estrecho contacto con la superficie del cráneo para evitar la dispersión de la luz pero preste atención a no ejercer presión sobre él. El área iluminada puede ser restringido mediante la aplicación en el cráneo de una máscara con una pequeña abertura o una tapa de la punta de la fibra óptica GUIde. </li></ol> 4. Rosa de Bengala inyección y activación <ol><li> Cargar la solución de Rosa de Bengala en una jeringa de 1 ml y calcular la cantidad a inyectar de acuerdo con una dosis de 10 l / g de peso corporal. </li><li> Proceder a una inyección intraperitoneal lento. </li><li> Deje que el tinte difuso y entrar en el torrente sanguíneo. Después de 5 minutos, encienda el iluminador de luz fría. Evite la iluminación del animal por cualquier otra fuente de luz. Utilizamos iluminador de fibra óptica de 150 W intensidad. </li><li> Después de 15 min de la iluminación, deje de exposición a la luz y suturar la herida. Cinco minutos de iluminación que ya producen de miocardio y 10 min es probablemente suficiente para obtener un efecto máximo 12. Con el fin de minimizar la variabilidad, se elige para iluminar durante 15 minutos. </li></ol> 5. Sutura <ol><li> Retire los retractores piel y aplicar una solución salina estéril para evitar la deshidratación. </li><li> Cierre de la herida utilizando needl corte inversae y de la seda o hilo de sutura de nylon. </li><li> Interrupción de suministro de anestesia, retire con cuidado el ratón del aparato estereotáxico y lo puso en una almohadilla caliente precalentada hasta que esté completamente despierto, y luego devolverlo a su jaula. La temperatura del cuerpo se debe monitorizar cuidadosamente a través de los procedimientos para limitar la variabilidad en la extensión del infarto. De acuerdo con la concentración y la vía de administración, Rosa de Bengala todavía puede ser detectada en el torrente sanguíneo durante varias horas después de la inyección 13. Prefiero la manta de calefacción para el calentamiento de la lámpara para evitar daños secundarios potenciales (Rosa de Bengala absorción de longitud de onda en el espectro de color verde). </li></ol>

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Mathers, C. D., Boerma, T., Ma Fat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+causes+of+death.">Global and regional causes of death.</a> Br. Med. Bull. 92, 7-32 (2009).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circ. Res. 40, 320-328 (1977).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Ann. Neurol. 17, 497-504 (1985).</li><li>Bergeron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+photochemical+cortical+lesions+in+rat+brain.">Inducing photochemical cortical lesions in rat brain.</a> Curr. Protoc. Neurosci. Chapter 9, Unit 9 16 (2003).</li><li>Lee, J. 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=Photochemically+induced+cerebral+ischemia+in+a+mouse+model.">Photochemically induced cerebral ischemia in a mouse model.</a> Surg. Neurol. 67, 620-625 (2007).</li><li>Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D., Bethea, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+infarction.+I.+Early+microvascular+alterations.">Photochemically induced cerebral infarction. I. Early microvascular alterations.</a> Acta Neuropathol. 72, 315-325 (1987).</li><li>Schroeter, M., Jander, S., Stoll, G. <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+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J. Neurosci. Methods. 117, 43-49 (2002).</li><li>Kitagawa, 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=Cerebral+ischemia+after+bilateral+carotid+artery+occlusion+and+intraluminal+suture+occlusion+in+mice:+evaluation+of+the+patency+of+the+posterior+communicating+artery.">Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery.</a> J. Cereb. Blood Flow Metab. 18, 570-579 (1998).</li><li>Sigler, A., Goroshkov, A., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hardware+and+methodology+for+targeting+single+brain+arterioles+for+photothrombotic+stroke+on+an+upright+microscope.">Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope.</a> J. Neurosci. Methods. 170, 35-44 (2008).</li><li>Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Piao, M. S., Lee, J. K., Jang, J. W., Kim, S. H., Kim, H. 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+mouse+model+of+photochemically+induced+spinal+cord+injury.">A mouse model of photochemically induced spinal cord injury.</a> J. Korean Neurosurg. Soc. 46, 479-483 (2009).</li><li>Silva, V. M., Corson, N., Elder, A., Oberdorster, 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+rat+ear+vein+model+for+investigating+in+vivo+thrombogenicity+of+ultrafine+particles+(UFP).">The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP).</a> Toxicol. Sci. 85, 983-989 (2005).</li><li>Watson, B. D., Prado, R., Dietrich, W. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+spinal+cord+injury+in+the+rat.">Photochemically induced spinal cord injury in the rat.</a> Brain Res. 367, 296-300 (1986).</li><li>Van Reempts, J., Van Deuren, B., Van de Ven, M., Cornelissen, F., Borgers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flunarizine+reduces+cerebral+infarct+size+after+photochemically+induced+thrombosis+in+spontaneously+hypertensive+rats.">Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats.</a> Stroke. 18, 1113-1119 (1987).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2, 396-409 (2005).</li><li>Kleinschnitz, 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=Blocking+of+platelets+or+intrinsic+coagulation+pathway-driven+thrombosis+does+not+prevent+cerebral+infarctions+induced+by+photothrombosis.">Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis.</a> Stroke. 39, 1262-1268 (2008).</li><li>Porritt, M. 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=Photothrombosis-induced+infarction+of+the+mouse+cerebral+cortex+is+not+affected+by+the+Nrf2-activator+sulforaphane.">Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane.</a> PLoS One. 7, e41090 (2012).</li><li>Baskin, Y. K., Dietrich, W. D., Green, 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=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J. Neurosci Methods. 129, 87-93 (2003).</li><li>Markgraf, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+histopathologic+consequences+of+photothrombotic+occlusion+of+the+distal+middle+cerebral+artery+in+Sprague-Dawley+and+Wistar+rats.">Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.</a> Stroke. 24, 286-292 (1993).</li><li>Wester, P., Watson, B. D., Prado, R., Dietrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photothrombotic+'ring'+model+of+rat+stroke-in-evolution+displaying+putative+penumbral+inversion.">A photothrombotic 'ring' model of rat stroke-in-evolution displaying putative penumbral inversion.</a> Stroke. 26, 444-450 (1995).</li><li>Hu, X., Wester, P., Brannstrom, T., Watson, B. D., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+and+reproducible+focal+cortical+ischemia+with+or+without+late+spontaneous+reperfusion+generated+by+a+ring-shaped,+laser-driven+photothrombotic+lesion+in+rats.">Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.</a> Brain Res. Brain Res. Protoc. 7, 76-85 (2001).</li><li>Maxwell, K. A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+focal+ischemic+lesions+in+neonatal+mice+by+photothrombosis.">Induction of reproducible focal ischemic lesions in neonatal mice by photothrombosis.</a> Dev. Neurosci. 27, 121-126 (2005).</li><li>Kuroiwa, 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=Development+of+a+rat+model+of+photothrombotic+ischemia+and+infarction+within+the+caudoputamen.">Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen.</a> Stroke. 40, 248-253 (2009).</li></ol>

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Las modificaciones y sustituciones Debido a su pico de absorción a 562 nm, un láser de luz verde de una lámpara de arco de xenón filtrada fue elegido originalmente para irradiar el fotosensible Rosa de Bengala. Aunque excitación láser mediada todavía se usaba recently5, puede ser reemplazado por la lámpara de luz fría que también aseguran excitación colorante 10,15. Cold fibras ópticas de luz son más fáciles de manipular y menos caro que las fuentes de láser. Sin embar...

A principios del siglo 21, el ictus isquémico es una enfermedad devastadora que representa la segunda causa de discapacidad a largo plazo 1 y la segunda causa de mortalidad en todo el mundo, en los que accidentes cerebrovasculares ocuparon alrededor de 5,7 millones de muertes en 2004 2. A pesar de los muchos esfuerzos que se pusieron en, todavía no existe un tratamiento eficaz para mejorar la recuperación funcional después del accidente cerebrovascular. Los modelos animales de accidente cerebrovascular son ampliamente utilizados en el campo de la investigación del accidente cerebrovascular, ya que permiten el modelado de la fisiopatología del daño isquémico y poner a prueba la eficacia de diferentes estrategias neuroprotectoras in vivo. La mayoría de estos modelos tienen por objeto inducir infartos extensos mediante la interrupción (temporal o permanentemente) el flujo de sangre dentro de la arteria cerebral media, mientras que se han desarrollado otros modelos para estudiar las lesiones de pequeño tamaño en áreas específicas, por lo general el motor y la corteza somatosensorial. Sin embargo, varios factores pueden contribuir a generar corriente alternaErtain grado de variabilidad en los estudios de accidente cerebrovascular experimental, incluyendo la cepa de ratón utilizado, la edad y el sexo de los animales incluidos en el estudio y, sobre todo, la técnica adoptó para inducir el daño isquémico. Con respecto a este último punto, la duración y la intensidad de la cirugía (es decir, la necesidad de una craneotomía), así como la habilidad quirúrgica requerida para el operador para inducir fiable una lesión isquémica son determinantes críticos para el éxito de e imparcial en el estudio in vivo accidente cerebrovascular . El concepto de photothrombosis inicialmente fue propuesto por Rosenblum y El-Sabban en 1977 3 y se convirtió en famoso por su aplicación en cerebro de rata por Watson et al en 1985 4 en el que la técnica se mejoró en gran medida y establece la base de la corriente de modelo 3. - 6. El enfoque photothrombotic pretende inducir un infarto cortical a través de la foto-activación de un tinte sensible a la luz previamente entregada al sistema sanguíneo, quich da lugar a trombosis de los vasos locales en las áreas expuestas a la luz. Cuando el colorante de circulación se ilumina en la longitud de onda apropiada por una fuente de luz fría, se libera la energía a las moléculas de oxígeno, que a su vez generan una gran cantidad de productos de oxígeno singlete altamente reactivos. Estos intermedios de oxígeno inducen la peroxidación de la membrana celular endotelial, que conduce a la adhesión y agregación de plaquetas, y, finalmente, a la formación de trombos que determinan la interrupción de flujo local cerebral 7. Photothrombosis es un modelo isquémico no canónica que no ocluir o romper una sola arteria como suele ocurrir en el accidente cerebrovascular humana, pero induce lesiones en los vasos más superficiales, lo que resulta en la interrupción selectiva del flujo sanguíneo en las áreas expuestas a la luz. Por esta razón, este enfoque puede ser adecuado para estudios celulares y moleculares de la plasticidad cortical. La ventaja principal de esta técnica reside en su simplicidad de ejecución.Por otra parte, photothrombosis se puede realizar fácilmente en aproximadamente cuarenta minutos por animal, incluida la espera de veinte minutos (3 min para la anestesia; 1 min a afeitar el cuero cabelludo; 3 a 5 min a colocar el animal en el aparato estereotáxico; 2 min para fregar la cuero cabelludo con una solución antiséptica, haga una incisión y limpiar el cráneo y de 2 a 4 minutos para colocar la fibra de luz fría, 1 min para inyectar la solución de rosa de Bengala, 5 min, espera para la difusión intraperitoneal, 15 min de la iluminación, y 5 min a limpiar la herida y suturar el animal). Además, no se necesita experiencia quirúrgica para llevar a cabo esta técnica ya que la lesión es inducida a través de simples iluminación del cráneo intacto. A diferencia de la oclusión arterial clásica, este método determina oclusiones selectivos de microvasos pial y intraparenquimatosa dentro de la zona irradiada y reduce la variabilidad entre las lesiones ya que ningún vaso colateral se deja para suministrar oxígeno en el área de orientación. A pesar de su naturaleza particular, laacciones daños photothrombotic mecanismos esenciales que ocurre en el ictus cerebral. De manera similar a oclusión de la arteria en el accidente cerebrovascular humana, la agregación plaquetaria y la formación de coágulos determinan interrupción del flujo sanguíneo en el área irradiada 7. Del mismo modo, este modelo también comparte las respuestas inflamatorias esenciales como en la oclusión de la arteria cerebral media 8. Sin embargo, debido a los límites bien delimitada, la zona de penumbra, que corresponde a un área de metabolismo parcialmente conservado, es muy reducida o inexistente después de una lesión photothrombotic. Esta frontera clara puede facilitar el estudio de las respuestas celulares en el área cortical isquémica o intactos. Modelo de ratón Photothrombosis es particularmente adecuada para los estudios de accidente cerebrovascular en una variedad de animales transgénicos. De hecho los modelos clásicos no pueden caber a todas las cepas y los estudios de largo período en ratones C57BL / 6 cepa de ratón informó una alta relación de mortalidad que puede causar sesgo 9.

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 <td></td>
 <td></td>
 <td>Solutions and chemicals</td>
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 <td>Rose Bengal </td>
 <td>Sigma, Italy </td>
 <td>330000 </td>
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 <td>Isoflurane Vet </td>
 <td>Merial </td>
 <td> 103120022 </td>
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 <td>Paraformaldehyde </td>
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 <td> 158127 </td>
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 <td>Fluosorber Filter </td>
 <td>Havard apparatus </td>
 <td> 340415 </td>
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 <td>150W fiber optic illuminator</td>
 <td>Photonic</td>
 <td>PL3000</td>
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 <td>Temperature Controller for Plate TCAT-2DF </td>
 <td>Havard apparatus</td>
 <td>727561 </td>
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 <td>Stereotaxic Instrument </td>
 <td>Stoelting</td>
 <td> 51950 </td>
 <td></td>
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 <td>Operating microscope </td>
 <td>Takagi </td>
 <td> OM8 </td>
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 <td>Heating pad </td>
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 <td>Oxygen and nitrogen gas</td>
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 <td>Surgery Tools</td>
 <td>World precision instrument </td>
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 <td>Optic fiber taps and mask are custom-made </td>
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photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies

El modelo de carrera photothrombotic pretende inducir un daño isquémico dentro de un área cortical determinado a través de la foto-activación de un tinte sensible a la luz inyectada previamente. Después de la iluminación, el colorante se activa y produce oxígeno singlete que los componentes de daños de las membranas celulares endoteliales, con la agregación de plaquetas y la posterior formación de trombos, lo que finalmente determina la interrupción del flujo sanguíneo local. Este enfoque, propuesto inicialmente por Rosenblum y El-Sabban en 1977, fue mejorado más adelante por Watson en 1985 en cerebro de rata y establece la base del modelo actual. Además, el aumento de la disponibilidad de líneas de ratones transgénicos contribuyó a aumentar el interés en el modelo photothrombosis. En pocas palabras, un colorante fotosensible (Rosa de Bengala) se inyecta por vía intraperitoneal y entra en el torrente sanguíneo. Cuando está iluminado por una fuente de luz fría, el colorante se convierte en activa e induce daño endotelial con la activación plaquetaria y la trombosis, lo que resulta en localesla interrupción del flujo sanguíneo. La fuente de luz se puede aplicar en el cráneo intacto sin necesidad de craneotomía, que permite la orientación de cualquier área cortical de interés de una manera reproducible y no invasiva. El ratón se sutura y se deja despertar. La evaluación del daño isquémico se puede lograr rápidamente por cloruro de trifenil-tetrazolio o tinción con violeta de cresilo. Esta técnica produce infarto de pequeño tamaño y los límites bien delimitados, lo cual es muy ventajoso para la caracterización precisa celular o estudios funcionales. Además, es particularmente adecuado para el estudio de las respuestas celulares y moleculares que subyacen a la plasticidad cerebral en ratones transgénicos.

Este protocolo va a producir una lesión cortical que ya es visible en la disección de la corteza a simple vista (Figuras 1A-1C). La lesión photothrombotic desarrolla en superficiales y profundas capas corticales en el que el tejido es suficientemente translúcido para permitir la foto-activación de la Rosa de Bengala. Medición de la extensión del infarto cerebral se puede realizar rápidamente mediante la tinción histológica con cloruro de trifenil-tetrazolio (TTC) en tejido fresco o por cresil ...

Watch this Scientific Journal Video about Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies at JoVE.com

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Photothrombosis es una técnica rápida, mínimamente invasivo para la inducción de infarto pequeño y bien delimitado en áreas de interés de una manera altamente reproducible. Es especialmente adecuado para el estudio de las respuestas celulares y moleculares que subyacen a la plasticidad cerebral en ratones transgénicos.

Isquemia photothrombotic: Un modelo de lesión cortical fotoquímico mínimamente invasiva y reproducible de Estudios Carrera Ratón

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

photothrombotic-ischemia-minimally-invasive-reproducible

Research

JoVE Journal

Medicine

34.4K vistas.  University of Turin. Photothrombosis es una técnica rápida, mínimamente invasivo para la inducción de infarto pequeño y bien delimitado en áreas de interés de una manera altamente reproducible. Es especialmente adecuado para el estudio de las respuestas celulares y moleculares que subyacen a la plasticidad cerebral en ratones transgénicos.

Video: Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modelo murino de hemorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Investigación

Medicina

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lesión Explorer: Un video-guiada, Protocolo normalizado para precisa y fiable volumétrica derivados de la RM en la Enfermedad de Alzheimer y normal Ancianos

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve patient outcomes. We present our experience with a minimally invasive pterional approach for anterior circulation aneurysms performed in a major tertiary cerebrovascular center and compare the results with an aged matched dataset from the Nationwide Inpatient Sample (NIS). From August 2008 to December 2012, 22 elective aneurysm clippings on patients &#8804;55 years of age were performed by the same dual fellowship-trained cerebrovascular/endovascular neurosurgeon. One patient (4.5%) experienced transient post-operative complications. 18 of 22 patients returned for follow-up imaging and there were no recurrences through an average duration of 22 months. A search in the NIS database from 2008 to 2010, also for patients aged &#8804;55 years of age, yielded 1,341 hospitalizations for surgical clip ligation of unruptured cerebral aneurysms. Inpatient length of stay and hospital charges at our institution using the minimally invasive thumb-sized pterional technique were nearly half that of NIS (length of stay: 3.2 vs 5.7 days; hospital charges: $52,779 vs. $101,882). The minimally invasive thumb-sized pterional craniotomy allows good exposure of unruptured small and medium-sized supraclinoid anterior circulation aneurysms. Cerebrospinal fluid drainage from key subarachnoid cisterns and constant bimanual microsurgical techniques avoid the need for retractors which can cause contusions, localized venous infarctions, and post-operative cerebral edema at the retractor sites. Utilizing this set of techniques has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimally invasive thumb-sized pterional craniotomy for aneurysm clipping has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Mínimamente Invasiva del pulgar de tamaño pterional craneotomía para la ligadura quirúrgica videoclip de aneurismas de la circulación anterior no roto

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Un accidente cerebrovascular trombótico Basado En transitoria cerebral por hipoxia-isquemia Modelo

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

El déficit motor reproducible después de la oclusión aórtica en un modelo de rata de la isquemia de la médula espinal

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Un modelo mínimamente invasivo para analizar la fractura endocondral en ratones en condiciones biomecánicas estandarizadas

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

Localización de nódulos transtorácicos de navegación electromagnética para cirugía torácica mínimamente invasiva

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Un modelo preclínico de rata para el estudio de la lesión por isquemia-reperfusión en microcirugía reconstructiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Evaluación de la compatibilidad y precisión de la línea de muestreo de capnografía cuando se utiliza con un monitor de capnografía portátil

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

Un método no invasivo para generar el modelo de lesión de cartílago intraarticular inducida por carga cíclica de la rodilla de rata

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...