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 microsonda termómetro (World Precision 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> Mango de bisturí y la cuchilla # 11, iris pinzas, fórceps Graefe, retractores bulldog pinza, destornillador, jeringa de 10 ml con aguja de calibre 26 biselado (World Precision Instruments, Inc., Sarasota, FL, EE.UU.) </li><li> Micromotor taladro y soporte estereotáxico, Quintessential inyección estereotáxica (Stoelting, Wood Dale, IL, EE.UU.) </li><li> 1,0 mm fresa fresa redonda, 1,0 mm cono invertido taladro fresa (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EE.UU.) </li></ol></li><li> Suministros Quirúrgicos <ol class="lalpha"><li> Tornillos de montaje 0-80 x 3/32 de 2,4 mm de longitud del eje, 21-calibre cánula guía [4 mm de largo por debajo del pedestal] y maniquí cánula (un plástico, Roanoke, VA, EE.UU.) </li><li> Jet acrílico de la dentadura y líquidos (Lang Dental Manufacturing Co., Inc., Chicago, IL, EE.UU.) </li><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> Máquinas de cortar el cabello (eléctricas Oster, Providence, Rhode Island, EE.UU.) </li></ol></li><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><li> Buprenorfina HCl (Hospira Inc., de Lake Forest, IL, 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> Iluminador de fibra óptica (TechniQuip Corp., Livermore, CA, EE.UU.) </li></ol></li><li> Flujometría láser Doppler del sistema (ADInstruments, Inc., Colorado Springs, CO, EE.UU.) <ol class="lalpha"><li> StPencil Probe Andard </li><li> Probe titular </li><li> Sangre FlowMeter </li><li> Powerlab 4/30 con LabChart 7 </li></ol></li><li> Medición del volumen del infarto <ol class="lalpha"><li> Rata cerebro matriz (Zivic-Miller Lab., Inc., Allison Park, PA, EE.UU.) </li><li> 2,3,5-trifeniltetrazolio (Sigma-Aldrich, St Louis, MO, EE.UU.) diluido al 0,05% en PBS </li><li> Escáner plano (Epson Perfection V30 de Epson America, Inc., Long Beach, CA, EE.UU.) </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 el 100% de mezcla gas O 2 en una cámara de inducción hasta que la rata ya no retiradas a pinch pata trasera. </li><li> Latécnica séptica se debe mantener durante este procedimiento, incluyendo el uso de guantes estériles y estériles instrumentos quirúrgicos y un campo estéril quirúrgico 11. </li><li> La corona de la cabeza rapada con cortar el pelo eléctricas. </li><li> La rata se coloca en la posición de decúbito prono sobre una almohadilla absorbente tendido en una superficie de operación de temperatura controlada (manta térmica). </li><li> 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> Ungüento oftálmico 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 mantiene firmemente en la estereotáxicodispositivo, el área quirúrgica se limpia con alternancia de clorhexidina al 2% y solución salina tres veces. </li></ol> 2. Pasos quirúrgicos <ol><li> Con un escalpelo, una incisión central se hace en la piel que cubre la bóveda craneal de los aspectos más caudales de los ojos (nasión) a entre las orejas (línea nucal superior). </li><li> La piel se retrae lateralmente con 3 pinzas bulldog. </li><li> El tejido conectivo está retirado del cráneo utilizando hisopos de algodón seco para que múltiples estructuras se pueden ver claramente. Estos incluyen bregma, la sutura coronal, y la cresta lateral derecha del cráneo. Bastoncillos de algodón se utilizan para eliminar la sangre del campo quirúrgico. </li><li> El uso del microscopio quirúrgico, bregma se encuentra, y los manipuladores estereotáxica se ajustan hasta que la fresa 1,0 mm redondo taladro se pone a cero a bregma. </li><li> La fresa de perforación se mueve entonces a 1,6 mm anterior y 5,2 mm lateral al bregma. </li><li> Un agujero de fresa que penetra en el cráneo perforado por cannula colocación (Figura 1). El exceso de suciedad y la sangre están constantemente limpiado utilizando hisopos de algodón. </li></ol> En este punto, una cánula de guía puede ser insertado (paso 7) o una directa ET-1 de inyección a través del orificio de fresa se puede realizar (vaya directamente al paso 16). <ol start="7"><li> Luego los agujeros, BUR 3 para los tornillos de montaje están perforados a través de espesor parcial del cráneo usando un cono invertido 1,0 mm taladro fresa (Figura 1). Un agujero es perforado en cada hueso frontal aproximadamente 1-2 mm por delante de la sutura coronal y 1-2 mm lateral a la sutura sagital. Un agujero es perforado en el hueso parietal de unos 2-3 mm posterior a la sutura coronal y 2-3 mm lateral a la sutura sagital ipsilateral al agujero de guía para cánula fresa. Tres 0-80 x 3/32 tornillos de montaje se colocan en los agujeros fresa y proporcionará apoyo para el cemento que en la cánula. Los tornillos deben ser avanzado 2 o 3 vueltas a fin de no dañar la duramadre. </li&gt; <li> La cánula de guía se coloca en el soporte de cánula estereotáxico y se encuentra bregma. </li><li> Los manipuladores estereotáxica se ajustan hasta que la cánula guía se pone a cero a bregma. La cánula se mueve en el orificio situado fresa de 1,6 mm anterior y 5,2 mm lateral al bregma. </li><li> Finalmente, la cánula guía se introduce en el agujero de la fresa con la posición de la punta final de 4,5 mm ventral a la bregma. </li><li> Laser Doppler sonda de colocación (opcional) <ol class="lalpha"><li> Con el fin de controlar el flujo sanguíneo cerebral usando LDF, un soporte de la sonda se puede colocar en posición antes de la colocación de la cánula guía con cemento dental. </li><li> La base de soporte de la sonda se recorta a ras con el pedestal a excepción de pestaña en forma de cuña pequeña. </li><li> El soporte de la sonda se coloca posterior a la cánula guía y justo medial al canto lateral del cráneo con la pestaña orientado medialmente (Figura 1). </li><li> El soporte de la sonda y guía cánula se fijan entre sí mediante cemento dental. </li></ol></li><li> Cemento dental se utiliza entonces para asegurar la cánula en su lugar. El cemento está en contacto con los tres tornillos y rodea toda la base de la cánula. </li><li> El cemento debe estar completamente seca antes de la retirada del soporte de cánula. Esto toma alrededor de 5 min. </li></ol> Después de estos pasos, la incisión quirúrgica se puede cerrar y el maniquí cánula se puede atornillar en la cánula guía. Alternativamente, ET-1 inducida por MCAO se puede realizar en la rata después de un período de recuperación de la cirugía de implante de la cánula. Para este método, el paso 19 se debe realizar próximo y pasos 14-18 se puede realizar en un momento posterior. Para llevar a cabo la implantación cánula guía y la inyección de ET-1 durante la misma cirugía, el paso 14 se debe realizar después. <ol start="14"><li> La jeringuilla de infusión se carga con ET-1 (diluida a 80 mM en PBS) y luego se monta en el inyector estereotáxica. </li><li> Lamanipuladores estereotáxicas se ajustan hasta que la punta de la aguja se pone a cero en el borde de la cánula guía. </li><li> La punta de la aguja se baja a través de la cánula guía hasta una posición de 17,2 mm ventral a la llanta de la cánula guía. Si una cánula guía no se utiliza, la punta de la aguja se pone a cero al bregma y bajado a través del agujero de la fresa para una posición de 8,7 mm ventral a la bregma. </li><li> 3 l de 80 mM ET1 se infunde a una velocidad de 1 l por min. </li><li> La jeringa se deja en su lugar durante 3 min después de la infusión es completa y luego se extrae lentamente. </li><li> La incisión se cierra con sutura de nylon 3,0 y el maniquí cánula se enrosca en la cánula. </li><li> Una dosis de analgésico apropiado (es decir, la buprenorfina en 0.05-0.1 mg / kg) se debe utilizar después de la cirugía para minimizar el dolor y la incomodidad durante el período de recuperación. </li><li> La rata se retira de la sala de operaciones y se coloca en un lugar caliente y seco recuperación para prevenir la hipotermia, con acceso libre y fácil a una alimentación suave y agua. </li&gt; </ol>

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No hay conflictos de interés declarado.

La ET-1 inducida por MCAO es un modelo establecido de accidente cerebrovascular isquémico experimental que se utiliza regularmente en múltiples cepas de ratas. Muchas variables, como la cepa de rata, la edad del animal, la temperatura corporal, el método de anestesia, y la experiencia del operador puede conducir a una mayor variabilidad en los volúmenes de infarto cuando se utiliza este modelo de 5, 14. Sin embargo, varios investigadores han demostrado que las ventajas de este modelo incluyen el enfoque ...

<table border="1"><tbody><tr><td></td><td></td><td></td><td><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 microsonda termómetro (World Precision 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> Mango de bisturí y la cuchilla # 11, iris pinzas, fórceps Graefe, retractores bulldog pinza, destornillador, jeringa de 10 ml con aguja de calibre 26 biselado (World Precision Instruments, Inc., Sarasota, FL, EE.UU.) </li><li> Micromotor taladro y soporte estereotáxico, Quintessential inyección estereotáxica (Stoelting, Wood Dale, IL, EE.UU.) </li><li> 1,0 mm fresa fresa redonda, 1,0 mm cono invertido taladro fresa (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EE.UU.) </li></ol></li><li> Suministros Quirúrgicos <ol class="lalpha"><li> Tornillos de montaje 0-80 x 3/32 de 2,4 mm de longitud del eje, 21-calibre cánula guía [4 mm de largo por debajo del pedestal] y maniquí cánula (un plástico, Roanoke, VA, USA) </li><li> Jet acrílico de la dentadura y líquidos (Lang Dental Manufacturing Co., Inc., Chicago, IL, EE.UU.) </li><li> 3,0 nylon sutura (Oasis, Mettawa, IL, EE.UU.) </li><li> Hisopos de algodón, pomada ocular Puralube (Fisher Scientific, Pittsburg, PA, EE.UU.) </li><li> Máquinas de cortar el cabello (eléctricas Oster, Providence, Rhode Island, EE.UU.) </li></ol></li><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><li> Buprenorfina HCl (Hospira Inc., de Lake Forest, IL, 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> Iluminador de fibra óptica (TechniQuip Corp., Livermore, CA, EE.UU.) </li></ol></li><li> Flujometría láser Doppler del sistema (ADInstruments, Inc., Colorado Springs, CO, EE.UU.) <ol class="lalpha"><li> Probe lápiz estándar </li><li> Probe titular </li><li> Sangre FlowMeter </li><li> Powerlab 4/30 con LabChart 7 </li></ol></li><li> Medición del volumen del infarto <ol class="lalpha"><li> De cerebro de ratamatriz (Zivic-Miller Lab., Inc., Allison Park, PA, EE.UU.) </li><li> 2,3,5-trifeniltetrazolio (Sigma-Aldrich, St Louis, MO, EE.UU.) diluido al 0,05% en PBS </li><li> Escáner plano (Epson Perfection V30 de Epson America, Inc., Long Beach, CA, EE.UU.) </li><li> Image J software (software ImageJ 1.42q, EE.UU. Institutos Nacionales de Salud, Bethesda, MA, EE.UU.) </li></ol></li></ol></td></tr></tbody></table>

endothelin-1 induced middle cerebral artery occlusion model for ischemic stroke with laser doppler flowmetry guidance in rat

El ictus es la primera causa de discapacidad y la tercera causa de muerte en el mundo, con un costo estimado de $ 70 mil millones en los Estados Unidos en 2009 1, 2. Varios modelos de isquemia cerebral se han desarrollado para imitar la condición humana de accidente cerebrovascular. Se ha sugerido que hasta el 80% de todos los accidentes cerebrovasculares resultado de daño isquémico en la arteria cerebral media (MCA) zona 3. En la década de 1990, la endotelina-1 (ET-1) 4 fue utilizado para inducir isquemia aplicándolo directamente adyacente a la superficie de la MCA después de la craneotomía. Más tarde, este modelo fue modificado 5 mediante el uso de una inyección estereotáxica de ET-1 adyacente a la MCA para producir isquemia cerebral focal. Las principales ventajas de este modelo incluyen la capacidad de realizar el procedimiento rápidamente, la capacidad de controlar la constricción de las arterias mediante la alteración de la dosis de ET-1 emitido, no hay necesidad de manipular los vasos extracraneales que suministran sangre al cerebro, así como gradual reperfusien las tarifas que más estrechamente imita la reperfusión en los seres humanos 5-7. Por otro lado, el modelo ET-1 tiene desventajas que incluyen la necesidad de una craneotomía, así como una mayor variabilidad en el volumen de eyección 8. Esta variabilidad se puede reducir con el uso de la flujometría láser Doppler (LDF) para verificar la isquemia cerebral durante la infusión de ET-1. Factores que afectan la variabilidad apoplejía incluyen precisión de la infusión y el lote de la ET-1 usado 6. Otra consideración importante es que a pesar de la reperfusión es una ocurrencia común en el ictus humano, la duración de la oclusión para ET-1 inducida por MCAO no puede imitar estrechamente la de apoplejía humana donde muchos pacientes tienen reperfusión parcial durante un período de horas a días después de la oclusión 9, 10. Este protocolo se describen en detalle la ET-1 inducida por MCAO modelo de accidente cerebrovascular isquémico en ratas. También se hará referencia a consideraciones especiales e inconvenientes potenciales durante el procedimiento.

1. Post-Op evaluación neurológica Después de que el animal recobre el conocimiento, una amplia variedad de pruebas se puede utilizar para evaluar los déficits neurológicos, incluyendo el equilibrio, la fuerza de agarre, colocación pata, la asimetría postural y la escalera de escalada. La tarea de semilla de girasol es una evaluación bruto de la función motora y sensorial que tiene una correlación significativa con el volumen de infarto 7, 12. Durante esta tarea, las ratas s...

Watch this Scientific Journal Video about Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat at JoVE.com

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

Varios modelos animales de isquemia cerebral han sido desarrollados para simular la condición humana de accidente cerebrovascular. Este protocolo describe la endotelina-1 (ET-1) inducida por la oclusión de la arteria cerebral media (MCAO) modelo de accidente cerebrovascular isquémico en ratas. Además, las consideraciones importantes, ventajas y deficiencias de este modelo se discuten.

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 ...

endothelin-1-induced-middle-cerebral-artery-occlusion-model-for

Research

JoVE Journal

Medicine

19.8K vistas.  University of Florida. Varios modelos animales de isquemia cerebral han sido desarrollados para simular la condición humana de accidente cerebrovascular. Este protocolo describe la endotelina-1 (ET-1) inducida por la oclusión de la arteria cerebral media (MCAO) modelo de accidente cerebrovascular isquémico en ratas. Además, las consideraciones importantes, ventajas y deficiencias de este modelo se discuten.

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

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminal la arteria cerebral media de oclusión (MCAO) Modelo para el ictus isquémico con Orientación flujometría láser Doppler en ratones

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

Bilateral Común oclusión de la arteria carótida como un estímulo de preacondicionamiento adecuado para inducir la tolerancia isquémica precoz de isquemia cerebral focal

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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

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 performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

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 ...

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 ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Carrera Modelado en ratones: Coagulación permanente de la arteria cerebral media Distal

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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 ...

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 ...