Este trabalho foi financiado por doações do American Heart Association Maior filial Sudeste (09GRNT2060421), a Associação Médica Americana, e da Universidade da Flórida Clínica e Translacional Science Institute. Adam Meca é um NIH / NINDS, companheiro NRSA predoctoral (F30 NS-060335). Robert Regenhardt recebeu apoio comunhão predoctoral da Universidade da Flórida Programa de Formação Multidisciplinar em Hipertensão (T32 HL-083810).

Este protocolo foi aprovado pelo Animal Care Institucional e Comitê de Uso (IACUC) da Universidade da Flórida e está em conformidade com o &quot;Guia para o Cuidado e Uso de Animais de Laboratório&quot; (oitava edição, a National Academy of Sciences, 2011). Materiais <ol><li> Animais: Oito semanas de idade, macho, Sprague Dawley (Farms Charles River, Wilmington, MA, EUA) pesando 250-300 g no momento da cirurgia. </li><li> Anestesia <ol class="lalpha"><li> Sistema de anestesia por inalação (VetEquip Inc., Pleasanton, CA, EUA) </li><li> Anestésico isoflurano (Baxter Farmacêutica, em Deerfield, Illinois, EUA) </li></ol></li><li> Estereotáxico sistema (David Kopf Instruments, Tujunga, CA, EUA) <ol class="lalpha"><li> Sistema estereotáxico de pequenos animais </li><li> Não-ruptura de ouvido barras para ratos </li><li> Gás titular cabeça anestesia para ratos </li></ol></li><li> Regulação da temperatura<ol class="lalpha"><li> BAT-12 termômetro microssonda (Precision Mundial Instruments, Inc., Sarasota, Flórida, EUA) </li><li> T / BOMBA, TP600 manta térmica (Gaymar Industries, Inc., Orchard Park, NY, EUA) </li></ol></li><li> Instrumentos cirúrgicos <ol class="lalpha"><li> Cabo de bisturi e lâmina n º 11, íris fórceps, fórceps Graefe, afastadores braçadeira bulldog, chave de fenda, 10 seringas ul com agulha de calibre 26 chanfrado (Instrumentos de Precisão World, Inc., Sarasota, Flórida, EUA) </li><li> Micromotor e broca titular estereotáxica, Quintessential Estereotáxica Injector (Stoelting, Wood Dale, IL, EUA) </li><li> 1,0 milímetros broca de perfuração redonda, 1,0 mm cone invertido de perfuração da broca (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EUA) </li></ol></li><li> Material cirúrgico <ol class="lalpha"><li> Parafusos de montagem 0-80 X 3/32 com 2,4 comprimento do eixo mm, 21 de calibre da cânula guia [4 milímetros longa abaixo do pedestal] e manequim cânula (Plásticos, uma Roanoke, VA, EUA) </li><li> Jet acrílico dentadura e líquido (Lang Dental Manufacturing Co., Inc., Wheeling, IL, EUA) </li><li> 3,0 nylon sutura (Oasis, Mettawa, IL, EUA) </li><li> Cotonetes, pomada Puralube (Fisher Scientific, Pittsburg, PA, EUA) </li><li> Elétrica de cortar cabelo (Oster, Providence, RI, EUA) </li></ol></li><li> Produtos químicos <ol class="lalpha"><li> A endotelina-1 (American Peptide, Sunnyvale, CA, EUA) </li><li> Clorexidina 2% (Agrilabs, St. Joseph, MO, EUA) </li><li> Buprenorfina HCl (Hospira Inc., Lake Forest, Illinois, EUA) </li></ol></li><li> Equipamento de visualização <ol class="lalpha"><li> Microscópio cirúrgico (Seiler Instrumento e Manufatura; St. Louis, MO, EUA) </li><li> Iluminador de fibra óptica (TechniQuip Corp, Livermore, CA, EUA) </li></ol></li><li> Laser Doppler fluxometria sistema (ADInstruments, Inc., Colorado Springs, CO, EUA) <ol class="lalpha"><li> StSonda Lápis andard </li><li> Suporte da sonda </li><li> Fluxômetro sangue </li><li> Powerlab 4/30 com 7 LabChart </li></ol></li><li> Medição do volume de enfarte <ol class="lalpha"><li> De cérebro de rato matriz (Zivic-Miller Lab., Inc., Allison Park, PA, EUA) </li><li> 2,3,5-cloreto de trifeniltetrazólio (Sigma-Aldrich Co., St. Louis, MO, EUA), diluída a 0,05% em PBS </li><li> Scanner de mesa (Epson Perfection V30, Epson America, Inc., Long Beach, CA, EUA) </li><li> Image J software (ImageJ 1.42q software, EUA National Institutes of Health, Bethesda, MA, EUA) </li></ol></li></ol> 1. Pré-cirúrgicos Passos <ol><li> Antes da cirurgia, os ratos são alojados sob um ciclo de luz / escuridão 12:12, com livre acesso à água e à ração de roedores. </li><li> A anestesia é induzida com 4% de isoflurano em 100% de mistura de gás O2, em uma câmara de indução até que o rato já não retiradas para aperto da pata traseira. </li><li> Atécnica séptico deve ser mantida durante este processo, incluindo a utilização de luvas estéreis, instrumentos cirúrgicos estéreis, e uma cortina cirúrgica estéril 11. </li><li> A coroa da cabeça é raspada com cortar cabelo elétrica. </li><li> O rato é colocado na posição de decúbito ventral com uma almofada absorvente que encontra-se sobre uma superfície de funcionamento com temperatura controlada (manta térmica). </li><li> A cabeça é colocada no aparelho estereotáxico de partida com a colocação da máscara de gás anestésico. </li><li> Em seguida, as barras de orelha são inseridos e apertados. </li><li> Durante o procedimento de anestesia é mantida com isoflurano a 2% em 100% de mistura de gás de O 2. </li><li> Unguento oftálmico lubrificante é aplicado a ambos os olhos, e as pálpebras estão fechadas para evitar a dessecação do olho durante o procedimento cirúrgico. </li><li> Uma sonda de temperatura rectal é inserido para manter uma temperatura constante de animais núcleo de 37 ± 0,5 ° C. </li><li> Com a cabeça firmemente mantido no estereotáxicodispositivo, a área cirúrgica é lavada com alternância de clorexidina a 2% e solução salina, três vezes. </li></ol> 2. Passos cirúrgicos <ol><li> Utilizando um bisturi, de uma incisão na linha média é feita sobre a pele que recobre o calvarium dos aspectos mais caudais dos olhos (násio) para entre as orelhas (linha nucal superior). </li><li> A pele é então retraída lateralmente com 3 pinças bulldog. </li><li> O tecido conectivo é removida do crânio com cotonetes de algodão seco, de modo que as estruturas múltiplas pode ser vista claramente. Estes incluem bregma, a sutura coronária, e a crista do crânio lateral direito. Cotonetes de algodão são usadas para remover o sangue do campo cirúrgico. </li><li> Usando o microscópio cirúrgico, bregma está localizado, e os manipuladores estereotáxicas são ajustados até que a broca broca 1,0 milímetros rodada é zerada em bregma. </li><li> A broca de perfuração é, então, movido para 1,6 mm anterior e 5,2 mm lateral à bregma. </li><li> Um buraco broca que penetra no crânio é perfurado para cannula colocação (Figura 1). Excesso de detritos e sangue são continuamente eliminados com cotonetes. </li></ol> Neste ponto, uma cânula de guia pode ser inserida (passo 7) ou uma injecção de ET-1 directo através do orifício de broca pode ser realizada (avançar directamente para o passo 16). <ol start="7"><li> Em seguida, os furos de broca de 3 parafusos de fixação são perfurados através de espessura parcial do crânio utilizando um cone invertido 1,0 milímetros de perfuração da broca (Figura 1). Um buraco é perfurado em cada osso frontal de cerca de 1-2 mm anterior ao da sutura coronária com 1-2 mm lateral à sutura sagital. Um buraco é perfurado no osso parietal de cerca de 2-3 mm posterior à sutura coronal e 2-3 mm lateral da sutura sagital ipsilateral ao orifício guia da broca cânula. Três 0-80 x 3/32 parafusos são colocados nestes buracos broca e fornecerá suporte para o cimento que na cânula. Os parafusos devem apenas ser avançado 2 ou 3 voltas de modo a não danificar a dura-máter. </li&gt; <li> A cânula guia é colocada no suporte da cânula estereotáxico e bregma está localizado. </li><li> Os manipuladores estereotáxicas são ajustados até que a cânula guia foi zerada em bregma. A cânula é movida para o furo de broca localizado 1,6 milímetros anterior e 5,2 mm lateral à bregma. </li><li> Finalmente, a cânula guia foi baixada para dentro do furo de broca com a posição de ponta final de 4,5 mm ventral a bregma. </li><li> Laser Doppler colocação da sonda de fluxo (opcional) <ol class="lalpha"><li> A fim de controlar o fluxo de sangue cerebral utilizando LDF, um suporte da sonda pode ser colocada em posição antes da fixação da cânula guia com cimento dental. </li><li> A base de suporte da sonda é aparado rente ao pedestal com exceção de guia em forma de cunha pequena. </li><li> O suporte da sonda é então colocada posterior à cânula guia e apenas medial ao rebordo lateral do crânio com a guia orientado medialmente (Figura 1). </li><li> O titular da sonda e guide cânula estão afixadas em conjunto com cimento dental. </li></ol></li><li> Cimento dentário é então utilizado para fixar a cânula no lugar. O cimento está em contacto com os três parafusos e rodeia toda a base da cânula. </li><li> O cimento deve ser completamente seca antes da remoção do suporte da cânula. Isto demora cerca de 5 min. </li></ol> Depois destes passos, a incisão cirúrgica pode ser fechada e que o boneco cânula pode ser aparafusado na cânula guia. Alternativamente, a ET-1 induzida por MCAO pode ser realizada no rato, após um período de recuperação a partir da cirurgia de implante da cânula. Por este método, o passo 19 deve ser realizada em seguida e os passos 14-18 pode ser realizada em um momento posterior. Para realizar o implante da cânula guia e ET-1 a injecção durante a mesma cirurgia, passo 14 deve ser realizada em seguida. <ol start="14"><li> A seringa de infusão é carregado com ET-1 (diluído a 80 uM em PBS) e, em seguida, montado no injector estereotáxica. </li><li> Omanipuladores estereotáxicas são ajustados até que a ponta da agulha é colocado em zero na borda da cânula guia. </li><li> A ponta da agulha é abaixada através da cânula guia para uma posição de 17,2 milímetros ventral para o aro da cânula guia. Se uma cânula guia não é utilizada, a ponta da agulha é colocado a zero no bregma e abaixada através do orifício de broca de um milímetro posição ventral 8,7 a bregma. </li><li> 3 ul de 80 mM ET1 é infundida a uma taxa de 1 ml por min. </li><li> A seringa é deixada no lugar durante 3 minutos após a conclusão da perfusão e então lentamente removido. </li><li> A incisão é fechada com sutura de nylon 3,0 e o manequim cânula é aparafusado na cânula. </li><li> Uma dose de analgésico apropriado (isto é, a buprenorfina a 0,05-0,1 mg / kg) deve ser utilizado após a cirurgia, para minimizar a dor e desconforto durante o período de recuperação. </li><li> O rato é removido do conjunto cirúrgico e colocadas numa área de recuperação quente, seco para evitar a hipotermia, com acesso livre, fácil de alimentos moles e água. </li&gt; </ol>

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A ET-1 induzida OACM é um modelo estabelecido de acidente vascular cerebral isquêmico experimental que é regularmente utilizado em várias linhagens de ratos. Muitas variáveis, tais como a estirpe de ratos, a idade do animal, temperatura corporal, método de anestesia, e experiência do operador pode levar a aumento da variabilidade nos volumes de infarto ao usar este modelo de 5, 14. No entanto, vários investigadores têm mostrado que as vantagens deste modelo incluem a abordagem relativamente não inv...

<table border="1"><tbody><tr><td></td><td></td><td></td><td><ol><li> Animais: Oito semanas de idade, macho, Sprague Dawley (Farms Charles River, Wilmington, MA, EUA) pesando 250-300 g no momento da cirurgia. </li><li> Anestesia <ol class="lalpha"><li> Sistema de anestesia por inalação (VetEquip Inc., Pleasanton, CA, EUA) </li><li> Anestésico isoflurano (Baxter Farmacêutica, em Deerfield, Illinois, EUA) </li></ol></li><li> Estereotáxico sistema (David Kopf Instruments, Tujunga, CA, EUA) <ol class="lalpha"><li> Sistema estereotáxico de pequenos animais </li><li> Não-ruptura de ouvido barras para ratos </li><li> Gás titular cabeça anestesia para ratos </li></ol></li><li> Regulação da temperatura <ol class="lalpha"><li> BAT-12 termômetro microssonda (Precision Mundial Instruments, Inc., Sarasota, Flórida, EUA) </li><li> T / BOMBA, TP600 manta térmica (Gaymar Industries, Inc., Orchard Park, NY, EUA) </li></ol></li><li> Instrumentos cirúrgicos <ol class="lalpha"><li> Cabo de bisturi e lâmina n º 11, íris fórceps, fórceps Graefe, afastadores braçadeira bulldog, chave de fenda, 10 seringas ul com agulha de calibre 26 chanfrado (Instrumentos de Precisão World, Inc., Sarasota, Flórida, EUA) </li><li> Micromotor e broca titular estereotáxica, Quintessential Estereotáxica Injector (Stoelting, Wood Dale, IL, EUA) </li><li> 1,0 milímetros broca de perfuração redonda, 1,0 mm cone invertido de perfuração da broca (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EUA) </li></ol></li><li> Material cirúrgico <ol class="lalpha"><li> Parafusos de montagem 0-80 X 3/32 com 2,4 comprimento do eixo mm, 21 de calibre da cânula guia [4 milímetros longa abaixo do pedestal] e manequim cânula (um Plásticos, Roanoke, VA, EUA) </li><li> Jet acrílico dentadura e líquido (Lang Dental Manufacturing Co., Inc., Wheeling, IL, EUA) </li><li> 3,0 nylon sutura (Oasis, Mettawa, IL, EUA) </li><li> Cotonetes, pomada Puralube (Fisher cientificamentec, Pittsburg, PA, EUA) </li><li> Elétrica de cortar cabelo (Oster, Providence, RI, EUA) </li></ol></li><li> Produtos químicos <ol class="lalpha"><li> A endotelina-1 (American Peptide, Sunnyvale, CA, EUA) </li><li> Clorexidina 2% (Agrilabs, St. Joseph, MO, EUA) </li><li> Buprenorfina HCl (Hospira Inc., Lake Forest, Illinois, EUA) </li></ol></li><li> Equipamento de visualização <ol class="lalpha"><li> Microscópio cirúrgico (Seiler Instrumento e Manufatura; St. Louis, MO, EUA) </li><li> Iluminador de fibra óptica (TechniQuip Corp, Livermore, CA, EUA) </li></ol></li><li> Laser Doppler fluxometria sistema (ADInstruments, Inc., Colorado Springs, CO, EUA) <ol class="lalpha"><li> Sonda lápis padrão </li><li> Suporte da sonda </li><li> Fluxômetro sangue </li><li> Powerlab 4/30 com 7 LabChart </li></ol></li><li> Medição do volume de enfarte <ol class="lalpha"><li> De cérebro de ratomatriz (Zivic-Miller Lab., Inc., Allison Park, PA, EUA) </li><li> 2,3,5-cloreto de trifeniltetrazólio (Sigma-Aldrich Co., St. Louis, MO, EUA), diluída a 0,05% em PBS </li><li> Scanner de mesa (Epson Perfection V30, Epson America, Inc., Long Beach, CA, EUA) </li><li> Image J software (ImageJ 1.42q software, EUA National Institutes of Health, Bethesda, MA, EUA) </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

AVC é a causa número um de deficiência e terceira principal causa de morte no mundo, com um custo estimado de US $ 70 bilhões em Estados Unidos, em 2009 1, 2. Vários modelos de isquemia cerebral têm sido desenvolvidos para mimetizar a condição humana de acidente vascular cerebral. Tem sido sugerido que até 80% de todo o resultado de danos isquémicos pancadas na artéria cerebral média (MCA) de área de 3. No início dos anos 1990, a endotelina-1 (ET-1) 4 foi utilizado para induzir isquemia, aplicando-a directamente adjacente à superfície da MCA após craniotomia. Mais tarde, este modelo foi modificado 5 utilizando uma injecção estereotáxica de ET-1 ao lado da MCA para produzir isquemia cerebral focal. As principais vantagens deste modelo incluem a capacidade de realizar o procedimento de forma rápida, a capacidade para controlar a constrição da artéria através da alteração da dose de ET-1 emitido, não há necessidade de manipular os vasos extracranianos que fornecem sangue ao cérebro, bem como reperfusi gradualem taxas que mais de perto imita a reperfusão em humanos 5-7. Por outro lado, o modelo ET-1 tem desvantagens que incluem a necessidade de uma craniotomia, bem como maior variabilidade no volume de curso 8. Esta variabilidade pode ser reduzida com a utilização de dopplerfluxometria a laser (LDF) para verificar se a isquemia cerebral durante a infusão de ET-1. Fatores que afetam a variabilidade AVC incluem precisão de infusão e lote do ET-1 utilizado 6. Outra consideração importante é que, apesar de reperfusão é uma ocorrência comum em acidente vascular cerebral humano, a duração da oclusão por ET-1 induzida por MCAO pode não imitam de perto a do acidente vascular cerebral humano onde muitos pacientes têm reperfusão parcial ao longo de um período de horas a dias após a oclusão 9, 10. Este protocolo irá descrever em detalhes a ET-1 induzida modelo OACM para AVC isquêmico em ratos. Será também chamar a atenção para as considerações especiais e inconvenientes potenciais durante todo o procedimento.

1. Pós-Op avaliação neurológica Depois de o animal recupera a consciência, uma grande variedade de ensaios pode ser utilizada para avaliar défices neurológicos incluindo o equilíbrio, a força de preensão, colocação da pata, assimetria postural e escada de escalada. A tarefa de semente de girassol é uma avaliação grosseira da função motora e sensorial que tem correlação significativa com infarto volume 7, 12. Durante essa tarefa, os ratos são cronometrados ao abrir...

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

Vários modelos animais de isquemia cerebral foi desenvolvido para simular a condição humana de acidente vascular cerebral. Este protocolo descreve a endotelina-1 (ET-1) induzida por oclusão da artéria cerebral média do modelo (MCAO) para acidente vascular cerebral isquêmico em ratos. Além disso, considerações importantes, vantagens e inconvenientes deste modelo são discutidos.

Endotelina-1 Induzida Médio modelo de oclusão da artéria cerebral para AVC isquêmico com Laser Orientação Fluxometria Doppler em ratos

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 visualizações.  University of Florida. Vários modelos animais de isquemia cerebral foi desenvolvido para simular a condição humana de acidente vascular cerebral. Este protocolo descreve a endotelina-1 (ET-1) induzida por oclusão da artéria cerebral média do modelo (MCAO) para acidente vascular cerebral isquêmico em ratos. Além disso, considerações importantes, vantagens e inconvenientes deste modelo são discutidos.

Vídeo: 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.

Curso de modelagem em Ratos - Oclusão da Artéria Cerebral Média com o 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 ...

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 do rato de Oclusão da Artéria Cerebral Média

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 artéria cerebral média Oclusão Model (MCAO) para Acidente Vascular Cerebral Isquêmico com Laser Doppler Fluxometria Orientação em Ratos

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.

Oclusão bilateral artéria carótida comum como um estímulo pré-condicionamento adequados para induzir a tolerância isquêmica início da 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 uma janela craniana para Visualizar a artéria cerebral média Durante endotelina 1 Induzida por oclusão da artéria cerebral média

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 otimizado para Monitorização de perfusão cerebral no modelo do rato do curso de oclusão da artéria cerebral média intraluminal

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.

Curso de modelagem em Ratos: Coagulação permanente da artéria distal Cerebral Média

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.

Embolic artéria cerebral média Oclusão (MCAO) para Acidente Vascular Cerebral Isquêmico com homólogos Coágulos de sangue em ratos

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.

Abordagem cirúrgica para oclusão da artéria cerebral média e reperfusão induzida por acidente vascular cerebral em ratos

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.

Transiente Artéria Cerebral Média Oclusão Modelo de AVC neonatal em ratos 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 ...