Este trabajo fue apoyado por becas de la Asociación Americana del Corazón Afiliado Mayor Sudeste (09GRNT2060421), la American Medical Association, y de la Universidad de Florida Clínica y Traslacional Science Institute. Adán Meca es un compañero NIH / NINDS, NRSA predoctoral (F30 NS-060335). Robert Regenhardt recibido apoyo beca predoctoral de la Universidad de Florida Programa de Entrenamiento Multidisciplinario de Hipertensión (T32 HL-083810).

Este protocolo fue aprobado por el Cuidado de Animales institucional y el empleo Comisión (IACUC) de la Universidad de Florida y está en conformidad con la &quot;Guía para el Cuidado y Uso de Animales de Laboratorio&quot; (octava edición de la Academia Nacional de Ciencias, 2011). Materiales <ol><li> Animales: Ocho semanas de edad, macho, Sprague Dawley (Charles River Farms, Wilmington, MA, EE.UU.) con un peso de 250-300 g en el momento de la cirugía. </li><li> Anestesia <ol class="lalpha"><li> Inhalación sistema de anestesia (VetEquip Inc., Pleasanton, CA, EE.UU.) </li><li> El isoflurano anestesia (Farmacia Baxter, Deerfield, IL, EE.UU.) </li></ol></li><li> Estereotáxica sistema (David Kopf Instruments, Tujunga, CA, EE.UU.) <ol class="lalpha"><li> Animal Pequeño sistema estereotáxico </li><li> Para no romper los oídos barras para ratas </li><li> Anestesia Gas cabeza titular para ratas </li></ol></li><li> Regulación de la temperatura <ol class="lalpha"><li> BAT-12 termómetro microsonda (WorldPrecision Instruments, Inc., Sarasota, FL, EE.UU.) </li><li> T / PUMP, TP600 manta térmica (Industrias Gaymar, Inc., Orchard Park, Nueva York, EE.UU.) </li></ol></li><li> Los instrumentos quirúrgicos <ol class="lalpha"><li> Metzenbaum tijera iris pinzas, separadores bulldog abrazadera, jeringa de 10 ml con aguja de calibre 26 biselado, Bovie cauterización kit (World Precision Instruments, Inc., Sarasota, FL, EE.UU.) </li><li> Micromotor de perforación (Stoelting, Wood Dale, IL, EE.UU.) </li><li> 0,8 mm redondo taladro fresa (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EE.UU.) </li><li> STORZ Bonn sutura Pinzas (Bausch and Lomb, Inc., Rochester, NY, EE.UU.) </li></ol></li><li> Suministros Quirúrgicos <ol class="lalpha"><li> 3,0 nylon sutura (Oasis, Mettawa, IL, EE.UU.) </li><li> Hisopos de algodón, pomada ocular Puralube (Fisher Científico, Pittsburgh, PA, EE.UU.) </li><li> Puntos quirúrgicos y tiras (Medtronic Xomed, Inc., Jacksonville, FL, EE.UU.) </li><li> Máquinas de cortar el cabello (eléctricas Oster, Providence, Rhode Island, EE.UU.) </li></ol></li&gt; <li> Químicos <ol class="lalpha"><li> La endotelina-1 (American Peptide, Sunnyvale, CA, EE.UU.) </li><li> La clorhexidina al 2% (Agrilabs, St. Joseph, MO, EE.UU.) </li></ol></li><li> Visualización de equipos <ol class="lalpha"><li> Microscopio quirúrgico (Seiler Instrument and Manufacturing, St. Louis, MO, EE.UU.) </li><li> Sony Handycam HDR-SR12 (Sony, Minato, Tokio, Japón) </li><li> Iluminador de fibra óptica (TechniQuip Corp., Livermore, CA, EE.UU.) </li></ol></li><li> Medición del diámetro de los vasos <ol class="lalpha"><li> VLC media player (París, Francia) </li><li> Image J software (software ImageJ 1.42q, EE.UU. Institutos Nacionales de Salud, Bethesda, MA, EE.UU.) </li></ol></li></ol> 1. Pre-quirúrgicos Pasos <ol><li> Antes de la cirugía, las ratas se alojaron en un 12:12 luz / oscuridad ciclo con acceso libre a comida y agua roedores. </li><li> La anestesia se indujo con 4% de isoflurano en 100% de mezcla de gas O 2 en una cámara de inducción. </li><li> El cuervo<html> n de la cabeza rapada con cortar el pelo eléctricas. </li><li> La rata se coloca en posición de decúbito prono sobre una almohadilla absorbente tendido en una superficie de operación de temperatura controlada (manta térmica) y la cabeza se coloca en el aparato estereotáctico de partida con la colocación de la máscara facial gas anestésico. </li><li> A continuación, las barras del oído se inserta y se aprieta. </li><li> Durante el procedimiento de anestesia se mantiene con 2% de isoflurano en 100% de mezcla de gas O 2. </li><li> Pomada oftálmica lubricante se aplica a ambos ojos y los párpados están cerrados para prevenir la desecación del ojo durante el procedimiento quirúrgico. </li><li> Una sonda de temperatura rectal es insertado para mantener una temperatura constante animales núcleo de 37 ± 0,5 ° C. </li><li> Con la cabeza de la rata anestesiada mantiene firmemente en el dispositivo estereotáxico el área quirúrgica se limpia con alternancia de clorhexidina y solución salina tres veces. </li></ol> 2. Pre-craneal Ventana Preparación ONTENIDO &quot;&gt; Antes de la creación de una ventana craneal, la rata debe estar preparado para cualquier experimento mediante la implantación de hardware requerida y debe recibir ningún procedimientos quirúrgicos requeridos. Para este protocolo, se han implantado previamente una cánula guía para la endotelina-1 (ET- 1) inducida por MCAO como se muestra en una publicación que titula &quot;La endotelina-1 inducida cerebral media oclusión arterial Modelo para el ictus isquémico con Orientación flujometría láser Doppler en la rata&quot;. 3. Creación de la ventana craneal Después de la colocación de una cánula guía o el equipo requerido para la experimentación, una ventana craneal se crea para visualizar directamente las porciones proximales de la arteria cerebral media durante un procedimiento de accidente cerebrovascular. <ol><li> En primer lugar, las tijeras se utilizan para incisión en la piel que recubre el músculo temporal medial de partida y trabajar lateralmente. </li><li> El músculo temporal es atravesada con electrocauterio y luego se retractó utilizando 3,0 sutura de nylon para visua<html> lizar la porción escamosa del hueso temporal. </li><li> Un cuadrado aproximadamente 3-4 mm se dibuja sobre la porción escamosa del hueso temporal caudal a la órbita y superior a la base del proceso cigomático como se refleja fuera del hueso temporal. </li><li> Un taladro se utiliza para cortar gradualmente la pieza resumidas de hueso libre del hueso temporal. Se debe tener cuidado para evitar la aplicación de demasiada presión a la broca, ya que es posible dañar la dura o corteza cerebral. </li><li> Enjuagues frecuentes con solución salina estéril se realizan para mejorar la visualización del campo quirúrgico y evitar el sobrecalentamiento del cráneo. </li><li> A partir de una esquina suelta, el trozo de hueso temoporal se retira cuidadosamente con finos dientes de rata pinzas asegurándose de no romper ningún buque asociados con la duramadre. </li><li> La duramadre se deja intacto y escombros se enjuaga con solución salina estéril. </li></ol> 4. Grabación de constricción de las arterias cerebral tienda de campaña &quot;&gt; Para demostrar cómo capturar imágenes en tiempo real, una rata que está pasando por ET-1 inducida por MCAO se utiliza para este protocolo. <ol><li> Antes de la aplicación de compuestos vasoactivos, un vídeo de línea de base debe ser registrada por lo menos durante 1 min. Para ET-1 inducida por MCAO, la grabación de línea de base se realiza una vez que la aguja ha sido bajada en el parénquima cerebral, pero antes de la inyección de ET-1. </li><li> La bomba de jeringa se inicia por inyección y grabado durante 1 hora o hasta que el punto final deseado. La aguja se deja en su lugar durante la grabación de vídeo con el fin de evitar perturbaciones en el plano focal. </li><li> La rata debe ser profundamente anestesiados y sacrificados de acuerdo con un protocolo aprobado siguiendo este procedimiento. </li></ol> 5. Análisis de Imagen El diámetro del vaso se puede determinar para cualquier parte de la visualizaron MCA. Como ejemplo, vamos a utilizar una rama de la MCA para medir el diámetro del vaso en los puntos de tiempo antes y después de la inyección de ET-1. Stimarcos ll del video se capturan a intervalos de 1 min utilizando VLC media player (VideoLAN). <ol><li> VLC jugador está instalado y se abrió. </li><li> Mediante la selección de herramientas y luego preferencias, el ajuste cambia a &quot;All&quot; en &quot;Mostrar valores&quot;. </li><li> El &quot;menú de vídeo&quot; se expande en la barra lateral izquierda y &quot;módulos de salida&quot; se expande entonces. &quot;Los filtros de Escena&quot; está seleccionado para que aparezca el menú de vídeo escena filtro. </li><li> &quot;Escena&quot; se escribe en el cuadro de nombre de archivo para el prefijo. </li><li> Un prefijo de ruta del directorio está definido. Aquí es donde se guardan imágenes fijas. </li><li> Una relación de grabación se selecciona basándose en la velocidad deseada de captura de imagen fija. Si un vídeo se capturó a 29 fotogramas por segundo, a continuación, &quot;1749&quot; (29 cuadros / seg x 60 seg / min) se consignarán en la casilla para guardar una imagen fija cada 1 min. Todos los cambios se guardarán. </li><li> El vídeo deseado se abrió en el reproductor VLC para guardar automáticamente imágenes fijas una vez cada 1 min. </li><li> El diámetro del vaso se mide entonces usando estosimágenes. En primer lugar, el software ImageJ (NIH) se abre. </li><li> A continuación, los cuadros fijos que se va a medir se abren en ImageJ. </li><li> Seleccionando la opción &quot;analizar&quot; menú &quot;, establecer medidas&quot; se abrió y todas las casillas estén marcadas. </li><li> A continuación, la herramienta de la línea recta se selecciona. </li><li> La combinación de teclas &quot;Ctrl +&quot; se utiliza para hacer un zoom según sea necesario y se coloca una línea perpendicular a la trayectoria recipiente para medir el diámetro del vaso. </li><li> Por último, la eslora del buque se obtiene seleccionando la opción &quot;analizar&quot; y haga &quot;medida&quot; (Ctrl + M) para obtener la eslora del buque. </li><li> Este proceso se repitió al menos tres veces y se promediaron para cada recipiente de medida se mide en cada trama todavía. </li><li> El diámetro del vaso en cada punto de tiempo se normaliza al diámetro del vaso de referencia de modo que se pueden hacer comparaciones múltiples utilizando ratas. Para ello, utilice la fórmula actual Diámetro / diámetro basal x 100% para calcular el diámetro basal% de cada buque. </li></ol>

<ol>
	<li>Levasseur, J. E., Wei, E. P., Raper, A. J., Kontos, A. A., Patterson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+description+of+a+cranial+window+technique+for+acute+and+chronic+experiments.">Detailed description of a cranial window technique for acute and chronic experiments.</a> Stroke. 6, 308-317 (1975).</li><li>Baumbach, G. L., Dobrin, P. B., Hart, M. N., Heistad, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+cerebral+arterioles+in+hypertensive+rats.">Mechanics of cerebral arterioles in hypertensive rats.</a> Circ. Res. 62, 56-64 (1988).</li><li>Regrigny, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+melatonin+on+rat+pial+arteriolar+diameter+in+vivo.">Effects of melatonin on rat pial arteriolar diameter in vivo.</a> Br. J. Pharmacol. 127, 1666-1670 (1999).</li><li>Mecca, A. P., O'Connor, T. E., Katovich, M. J., Sumners, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candesartan+pretreatment+is+cerebroprotective+in+a+rat+model+of+endothelin-1-induced+middle+cerebral+artery+occlusion.">Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion.</a> Exp. Physiol. 94, 937-946 (2009).</li><li>Mecca, 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=Cerebroprotection+by+angiotensin-(1-7)+in+endothelin-1-induced+ischaemic+stroke.">Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke.</a> Exp. Physiol. 96, 1084-1096 (2011).</li></ol>

No hay conflictos de interés declarado.

En resumen, esta técnica de ventana craneal preparación es muy versátil ya que puede modificarse para satisfacer las necesidades de muchos experimentos con modificaciones menores 4, 5. Por ejemplo, hemos logrado controlarse el flujo sanguíneo cerebral en determinadas ramas de MCA con la flujometría láser Doppler para centrarse directamente en una arteria cerebral visualizada a través de una ventana craneal (Meca AP 2009 y 2011). Además, una preparación similar con el incisa duramadre puede ser utiliz...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Inhalación sistema de anestesia </td><td> VetEquip Inc., Pleasanton, California, EE.UU. </td><td> 901806 </td><td></td></tr><tr><td> El isoflurano anestesia </td><td> Baxter Farmacia, Deerfield, IL, EE.UU. </td><td> 1001936060 </td><td></td></tr><tr><td> Animal Pequeño sistema estereotáxico </td><td> David Kopf Instruments, Tujunga, CA, EE.UU. </td><td> 900 </td><td></td></tr><tr><td> Los no ruptura bares oído, rata </td><td> David Kopf Instruments, Tujunga, CA, EE.UU. </td><td> 957 </td><td></td></tr><tr><td> Gas Rat anestesia cabeza titular </td><td> David Kopf Instruments, Tujunga, CA, EE.UU. </td><td> 1929 </td><td></td></tr><tr><td> BAT-12 microsonda termómetro </td><td> World Precision Instruments, Inc., Sarasota, FL, EE.UU. </td><td> BAT-12 </td><td&gt; </td></tr><tr><td> T / PUMP, manta térmica </td><td> Gaymar Industries, Inc., Orchard Park, Nueva York, EE.UU. </td><td> T / PUMP, TP600 </td><td></td></tr><tr><td> Metzenbaum Scissors </td><td> World Precision Instruments, Inc., Sarasota, FL, EE.UU. </td><td> 501254 </td><td></td></tr><tr><td> Iris fórceps </td><td> World Precision Instruments, Inc., Sarasota, FL, EE.UU. </td><td> 15915 </td><td></td></tr><tr><td> Bulldog abrazadera retractores </td><td> World Precision Instruments, Inc., Sarasota, FL, EE.UU. </td><td> 14119-G </td><td></td></tr><tr><td> 10 l jeringa 26-gaugue </td><td> World Precision Instruments, Inc., Sarasota, FL, EE.UU. </td><td> SGE010RNS </td><td></td></tr><tr><td> Bovie, alta temperatura kit cauterización </td><td> World Precision Instruments, Inc., Sarasota, FL, EE.UU. </td><td> 500392 </td><td></td></tr><tr><td> Diente de rata fórceps 0,12 </td><td> Stotz </td><td> E1811 </td><td></td></tr><tr><td> Micromotor taladro </td><td> Stoelting,Wood Dale, IL, EE.UU. </td><td> 51449 </td><td></td></tr><tr><td> 0,8 mm fresa fresa redonda </td><td> Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EE.UU. </td><td> RS-6280C-1 </td><td></td></tr><tr><td> STORZ Bonn sutura Pinzas </td><td> Bausch and Lomb, Inc., Rochester, NY, EE.UU. </td><td></td><td></td></tr><tr><td> Sutura Nylon, tamaño 3,0 </td><td> Oasis, Mettawa, IL, EE.UU. </td><td> MV-663 </td><td></td></tr><tr><td> Esponjas de algodón </td><td> Fisher Scientific, Pittsburg, PA, EE.UU. </td><td> 22-029-488 </td><td></td></tr><tr><td> Puralube pomada para los ojos </td><td> Fisher Scientific, Pittsburg, PA, EE.UU. </td><td> NC0138063 </td><td></td></tr><tr><td> Máquinas de cortar el pelo eléctricas </td><td> Oster, Providence, RI, EE.UU. </td><td> 78005-301 </td><td></td></tr><tr><td> ET-1 se diluye a 80 μ M de concentración en PBS </td><td> American Peptide, Sunnyvale, CA, EE.UU. </td><td> 88-1-10A </td><td></td></tr><tr><td> Clorhexidina, 2% </td&gt; <td> Agrilabs, St. Joseph, MO, EE.UU. </td><td> 1040, Rev. 6.6, NAC No.: 10580322 </td><td></td></tr><tr><td> Microscopio quirúrgico </td><td> Seiler Instrument and Manufacturing, St. Louis, MO, EE.UU. </td><td> Evolución XR6 </td><td></td></tr><tr><td> Sony Handycam </td><td> Sony, Minato, Tokio, Japón </td><td> HDR-SR12 </td><td></td></tr><tr><td> Iluminador de fibra óptica </td><td> TechniQuip Corp., Livermore, CA, EE.UU. </td><td> FO1-150 </td><td></td></tr><tr><td> VLC Media Player </td><td> (París, Francia) </td><td></td><td></td></tr><tr><td> Image J software </td><td> EE.UU. Institutos Nacionales de Salud, Bethesda, MA, EE.UU. </td><td></td><td></td></tr></tbody></table>

utilizing a cranial window to visualize the middle cerebral artery during endothelin-1 induced middle cerebral artery occlusion

Creación de una ventana craneal es un método que permite la visualización directa de las estructuras en la superficie cortical del cerebro 1-3. Esta técnica se puede realizar en muchas localizaciones que recubren el cerebro de rata, pero es más fácil de llevar a cabo mediante la creación de una craniectomía sobre los huesos de fácil acceso frontal o parietal. Con más frecuencia, se ha utilizado esta técnica en combinación con la endotelina-1 medio modelo oclusión de la arteria cerebral del accidente cerebrovascular isquémico para cuantificar los cambios en el diámetro medio vaso cerebral arteria que se producen con inyección de endotelina-1 en el parénquima cerebral adyacente a la MCA proximal 4, 5. Con el fin de visualizar la parte proximal de la MCA durante endotelina -1 inducida MCAO, se utiliza una técnica para crear una ventana craneal a través del hueso temporal en el aspecto lateral del cráneo de rata (Figura 1). Arterias cerebrales puede ser visualizado o bien con la duramadre intacta o con la duramadre y incisa RetraDirección Ejecutiva. Por lo general, dejamos la duramadre intacta durante la visualización desde la endotelina-1 inducida por MCAO implica la entrega del péptido vasoconstrictor en el parénquima cerebral. Esto evita la necesidad de una incisión en la duramadre directamente sobre los vasos visualizado para la administración de fármacos. Este protocolo se describe cómo crear una ventana craneal para visualizar las arterias cerebrales de una forma gradual, así como la manera de evitar muchos de los problemas potenciales relacionados con este método.

Las imágenes fijas tomadas de la serie de vídeo capturado que el cambio en el diámetro de la arteria cerebral después de la inyección de ET-1 se puede apreciar fácilmente utilizando esta técnica de ventana craneal (Figura 2). Pocos minutos después de la inyección de ET-1, el recipiente comienza a constreñir. Eventualmente los vasos será difícil de visualizar y el tejido cerebral se convertirá pálido. Después de unos 20 min de los efectos de ET-1 disminuye y los vasos comenzará a dilatars...

Watch this Scientific Journal Video about Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion at JoVE.com

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Este artículo describe un método para la visualización de las arterias cerebrales de rata a través de una ventana craneal mediante craniectomía temporal con el fin de ver las partes proximales de la arteria cerebral media (<strong&gt; Figura 1</strong&gt;). Este método versátil se puede combinar con diversas técnicas de administración de fármacos para medir la reactividad de la arteria cerebral<em&gt; In vivo</em&gt;.

Utilizando una ventana craneal para visualizar la arteria cerebral media Durante la endotelina-1 inducida por oclusión de la arteria cerebral media

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

utilizing-cranial-window-to-visualize-middle-cerebral-artery-during

Research

JoVE Journal

Medicine

14.8K vistas.  University of Florida. Este artículo describe un método para la visualización de las arterias cerebrales de rata a través de una ventana craneal mediante craniectomía temporal con el fin de ver las partes proximales de la arteria cerebral media ( Figura 1). Este método versátil se puede combinar con diversas técnicas de administración de fármacos para medir la reactividad de la arteria cerebral In vivo.

Video: Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modelo de ratón de la oclusión arteria cerebral media

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

La aplicación de la Permanente Ligadura arteria cerebral media en el ratón

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

La endotelina-1 inducida por la arteria cerebral media oclusión Modelo para el ictus isquémico con Orientación flujometría láser Doppler en la rata

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Sistema optimizado para la monitorización de la perfusión cerebral en el modelo de rata de Carrera intraluminal oclusión arteria cerebral media

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Arteria embólico cerebral media oclusión (MCAO) para el ictus isquémico con homólogas coágulos de sangre en las ratas

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Cuantificación de Protección neurovascular Siguiendo repetitivo hipóxico Precondicionamiento y transitoria cerebral media oclusión de la arteria en ratones

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Realización Permanente distal cerebral media carótida común con oclusión de la arteria en ratas de edad avanzada para el Estudio de la isquemia cortical con sostenida de la discapacidad

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Técnica quirúrgica para la oclusión de la arteria cerebral media y la reperfusión del movimiento inducidos en ratones

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transitoria de la arteria cerebral media oclusión Modelo de accidente cerebrovascular neonatal en ratas P10

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...