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 (WorldPrecision 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> Metzenbaum tesoura, íris fórceps, afastadores braçadeira bulldog, seringa de 10 ul com agulha de calibre 26 chanfrado, Bovie cautério kit (Precision Mundial Instruments, Inc., Sarasota, Flórida, EUA) </li><li> Micromotor broca (Stoelting, Wood Dale, IL, EUA) </li><li> 0,8 milímetros rodada broca broca (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EUA) </li><li> STORZ Bonn Sutura Pinças (Bausch e Lomb, Inc., Rochester, NY, EUA) </li></ol></li><li> Material cirúrgico <ol class="lalpha"><li> 3,0 nylon sutura (Oasis, Mettawa, IL, EUA) </li><li> Cotonetes, pomada Puralube (Fisher Scientific, Pittsburg, PA, EUA) </li><li> Pontos cirúrgicos e tiras (Medtronic Xomed, Inc., Jacksonville, FL, EUA) </li><li> Elétrica de cortar cabelo (Oster, Providence, RI, EUA) </li></ol></li&gt; <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></ol></li><li> Equipamento de visualização <ol class="lalpha"><li> Microscópio cirúrgico (Seiler Instrumento e Manufatura; St. Louis, MO, EUA) </li><li> Sony Handycam HDR-SR12 (Sony, Minato, Tokyo, Japão) </li><li> Iluminador de fibra óptica (TechniQuip Corp, Livermore, CA, EUA) </li></ol></li><li> Medição do diâmetro do vaso <ol class="lalpha"><li> VLC media player (Paris, França) </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. </li><li> O corvo<html> n da cabeça é raspada com cortar cabelo elétrica. </li><li> O rato é colocado numa posição de bruços sobre uma almofada absorvente encontra-se sobre uma superfície de funcionamento com temperatura controlada (manta térmica) e 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> Pomada lubrificante oftálmico é aplicada em ambos os olhos e as pálpebras são fechadas para evitar ressecamento dos olhos 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 do rato anestesiado firmemente mantido no dispositivo estereotáxico da área cirúrgica é lavada com a alternância de clorexidina e solução salina três vezes. </li></ol> 2. Pré-craniana Preparação Janela ONTEÚDO &quot;&gt; Antes de criar uma janela craniana, o rato deve estar preparado para todas as experiências de implantação de hardware necessário outro e deve receber quaisquer procedimentos necessários cirúrgicos. Para este protocolo, já implantada uma cânula guia para endotelina-1 (ET- 1) induzida OACM como mostra uma publicação companheiro intitulado &quot;A endotelina-1 Induzida Médio modelo de oclusão da artéria cerebral para AVC isquêmico com Laser Orientação dopplerfluxometria em rato.&quot; 3. Criação da Janela craniana Após a colocação de uma cânula guia ou equipamento necessário para a experimentação, uma janela craniana é criado para visualizar diretamente porções proximais da artéria cerebral média, durante um procedimento de acidente vascular cerebral. <ol><li> Em primeiro lugar, são utilizadas tesouras a incisão da pele que cobre o músculo temporal e medialmente começando a trabalhar lateralmente. </li><li> O músculo temporal é cortada utilizando o eletrocautério, e depois recuou 3,0 utilizando fio de náilon ao vi<html> lize a escama do osso temporal. </li><li> Um quadrado de aproximadamente 3-4 milímetros é desenhada na parte escamosa do osso temporal caudal para a órbita e superior para a base do processo zigomático uma vez que reflecte para fora do osso temporal. </li><li> Uma broca é utilizada para reduzir gradualmente o pedaço de osso esboçada livre a partir do osso temporal. Deve ser tomado cuidado para evitar a aplicação de pressão excessiva ao perfuração uma vez que é possível danificar a dura ou no córtex cerebral. </li><li> Lavagens freqüentes com solução salina estéril são realizadas para melhorar a visualização do campo cirúrgico e evitar o superaquecimento do crânio. </li><li> A partir de um canto solto, o pedaço de osso temoporal é cuidadosamente removido usando finas rato de dentes fórceps enquanto tomando cuidado para não rasgar os vasos associados com a dura. </li><li> A dura-máter é deixada intacta e detritos é lavado com solução salina estéril. </li></ol> 4. Gravação de constrição da artéria cerebral &gt; tenda &quot;Para demonstrar como a captura de imagens em tempo real, um rato que está passando por ET-1 induzida OACM é usado para esse protocolo. <ol><li> Antes da aplicação de compostos vasoactivos, uma linha de base de vídeo deve ser registada durante pelo menos 1 min. Para ET-1 induzida por MCAO, a gravação é feita referência, uma vez que a agulha tenha sido baixada para dentro do parênquima do cérebro, mas antes da injecção de ET-1. </li><li> A bomba de seringa é iniciado por injecção e registados durante 1 hora ou até que o ponto final desejado. A agulha é deixada no local durante a gravação de video, a fim de evitar perturbações no plano focal. </li><li> O rato deve ser profundamente anestesiados e sacrificados de acordo com um protocolo aprovado seguindo este procedimento. </li></ol> 5. Análise de Imagem Diâmetro do vaso pode ser determinada para qualquer parte do MCA visualizado. Como exemplo, vamos usar um ramo da MCA para medir o diâmetro dos vasos em tempo-pontos antes e depois da injeção de ET-1. Still quadros do vídeo são capturados em intervalos de 1 min usando VLC media player (VideoLAN). <ol><li> VLC player está instalado e aberto. </li><li> Ao selecionar ferramentas e, em seguida, as preferências, o cenário é alterado para &quot;Todos&quot; em &quot;Mostrar configurações&quot;. </li><li> O menu &quot;vídeo&quot; é ampliado na barra do lado esquerdo e &quot;Módulos de saída&quot; é então expandido. &quot;Filtros de Cena&quot; é selecionado para abrir o menu de vídeo cena filtro. </li><li> &quot;Scene&quot; é digitado na caixa de prefixo do nome. </li><li> Um prefixo caminho do diretório está definido. Este é o lugar onde ainda quadros são salvos. </li><li> Um rácio de gravação é seleccionado com base na taxa desejada de captura de quadros. Se um vídeo foi capturado em 29 frames por segundo, em seguida, &quot;1749&quot; (29 frames / seg x 60 seg / min) deve ser inserido na caixa para salvar uma ainda enquadrar cada 1 min. Todas as mudanças são salvos. </li><li> O vídeo desejado é então aberto em player VLC para salvar automaticamente quadros ainda uma vez a cada 1 min. </li><li> Diâmetro do vaso é então medida utilizando estesimagens. Em primeiro lugar, o software ImageJ (NIH) é aberta. </li><li> Em seguida, os quadros ainda que você vai medir são abertos no ImageJ. </li><li> Ao selecionar a &quot;analisar&quot; menu &quot;, definir medidas&quot; é então aberto e todas as caixas estão desmarcadas. </li><li> Em seguida, a ferramenta de linha reta é selecionada. </li><li> O atalho &quot;Ctrl +&quot; é usado para fazer zoom conforme necessário e uma linha é colocado perpendicularmente ao caminho vaso para medir o diâmetro do vaso. </li><li> Finalmente, comprimento do vaso é obtida selecionando a &quot;analisar&quot; no menu e, em seguida, &quot;medida&quot; (ctrl + M) para obter comprimento do vaso. </li><li> Este processo é repetido pelo menos três vezes, e calculada a média para cada recipiente de medição medidos em cada fotograma. </li><li> Diâmetro do vaso em cada ponto de tempo é normalizado para o diâmetro do vaso de linha de base de modo a que possam ser feitas comparações múltiplas utilizando ratos. Para fazer isso, utilizar a fórmula diâmetro diâmetro / corrente de linha de base x 100% para calcular o diâmetro da linha de base% de cada vaso. </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>

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Em resumo, esta técnica de preparação de janela craniana é muito versátil uma vez que podem ser alterados para satisfazer as necessidades das várias experiências com pequenas modificações, 4, 5. Por exemplo, conseguimos monitorado o fluxo sanguíneo cerebral em ramos específicos MCA usando a dopplerfluxometria a laser para se concentrar diretamente em uma artéria cerebral visualizado através de uma janela de crânio (Meca AP 2009 e 2011). Além disso, uma preparação semelhante com a incisão dur...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Sistema de anestesia por inalação </td><td> VetEquip Inc., Pleasanton, CA, EUA </td><td> 901806 </td><td></td></tr><tr><td> Anestésico isoflurano </td><td> Pharmaceutics Baxter, Deerfield, IL, EUA </td><td> 1001936060 </td><td></td></tr><tr><td> Sistema estereotáxico de pequenos animais </td><td> David Kopf Instruments, Tujunga, CA, EUA </td><td> 900 </td><td></td></tr><tr><td> Não-ruptura barras de ouvido de ratos, </td><td> David Kopf Instruments, Tujunga, CA, EUA </td><td> 957 </td><td></td></tr><tr><td> Rat gás suporte da cabeça anestesia </td><td> David Kopf Instruments, Tujunga, CA, EUA </td><td> 1929 </td><td></td></tr><tr><td> BAT-12 termômetro microssonda </td><td> Precisão mundo Instruments, Inc., Sarasota, FL, EUA </td><td> BAT-12 </td><td&gt; </td></tr><tr><td> T / BOMBA, manta térmica </td><td> Gaymar Industries, Inc., Orchard Park, NY, EUA </td><td> T / BOMBA, TP600 </td><td></td></tr><tr><td> Metzenbaum Tesoura </td><td> Precisão mundo Instruments, Inc., Sarasota, FL, EUA </td><td> 501254 </td><td></td></tr><tr><td> Iris fórceps </td><td> Precisão mundo Instruments, Inc., Sarasota, FL, EUA </td><td> 15915 </td><td></td></tr><tr><td> Afastadores bulldog braçadeira </td><td> Precisão mundo Instruments, Inc., Sarasota, FL, EUA </td><td> 14119-G </td><td></td></tr><tr><td> Seringa de 10 ul de 26 gaugue </td><td> Precisão mundo Instruments, Inc., Sarasota, FL, EUA </td><td> SGE010RNS </td><td></td></tr><tr><td> Bovie, kit cautério alta temperatura </td><td> Precisão mundo Instruments, Inc., Sarasota, FL, EUA </td><td> 500392 </td><td></td></tr><tr><td> Dente rato fórceps 0,12 </td><td> Stotz </td><td> E1811 </td><td></td></tr><tr><td> Broca Micromotor </td><td> Stoelting,Wood Dale, IL, EUA </td><td> 51449 </td><td></td></tr><tr><td> 0,8 broca de perfuração milímetros rodada </td><td> Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, EUA </td><td> RS-6280C-1 </td><td></td></tr><tr><td> STORZ Bonn sutura fórceps </td><td> Bausch e Lomb, Inc., Rochester, NY, EUA </td><td></td><td></td></tr><tr><td> Nylon de sutura, de tamanho 3,0 </td><td> Oasis, Mettawa, IL, EUA </td><td> MV-663 </td><td></td></tr><tr><td> Cotonetes </td><td> Fisher Scientific, Pittsburg, PA, EUA </td><td> 22-029-488 </td><td></td></tr><tr><td> Puralube pomada </td><td> Fisher Scientific, Pittsburg, PA, EUA </td><td> NC0138063 </td><td></td></tr><tr><td> Elétrica de cortar cabelo </td><td> Oster, Providence, RI, EUA </td><td> 78005-301 </td><td></td></tr><tr><td> ET-1 diluído a 80 μ M de concentração em PBS </td><td> American Peptide, Sunnyvale, CA, EUA </td><td> 88-1-10A </td><td></td></tr><tr><td> Clorexidina, 2% </td&gt; <td> Agrilabs, St. Joseph, MO, EUA </td><td> 1040, Rev. 6-06, NAC n º: 10580322 </td><td></td></tr><tr><td> Microscópio cirúrgico </td><td> Seiler Instrumento e Manufatura, St. Louis, MO, EUA </td><td> Evolução XR6 </td><td></td></tr><tr><td> Sony Handycam </td><td> Sony, Minato, Tokyo, Japão </td><td> HDR-SR12 </td><td></td></tr><tr><td> Iluminador de fibra óptica </td><td> TechniQuip Corp, Livermore, CA, EUA </td><td> FO1-150 </td><td></td></tr><tr><td> VLC Media Player </td><td> (Paris, França) </td><td></td><td></td></tr><tr><td> Image J software </td><td> EUA National Institutes of Health, Bethesda, MA, EUA </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

Criação de uma janela craniana é um método que permite a visualização directa das estruturas sobre a superfície cortical do cérebro 1-3. Esta técnica pode ser realizada em diversos locais do cérebro de rato que se sobrepõem, mas é mais facilmente realizado através da criação de uma craniectomia sobre os ossos de fácil acesso frontal ou parietal. Mais frequentemente, temos usado esta técnica, em combinação com o modelo de oclusão endotelina-1 na artéria cerebral média de AVC isquémico para quantificar as alterações no diâmetro dos vasos da artéria cerebral média, que ocorrem com a injecção de endotelina-1 para o parênquima cerebral adjacente ao MCA proximal 4, 5. A fim de visualizar a porção proximal da MCA, durante endotelina -1 induzida MCAO, usamos uma técnica para criar uma janela craniana no osso temporal na face lateral do crânio de rato (Figura 1). Artérias cerebrais pode ser visualizada quer com a dura intacta ou com a dura-máter e incisa RetraCTED. Mais comumente, deixamos a dura intacta durante a visualização desde a endotelina-1 induzida por MCAO envolve a entrega do péptido vasoconstritor para o parênquima cerebral. Isto evita a necessidade de incisão na dura directamente sobre os vasos visualizado para a entrega da droga. Este protocolo irá descrever como criar uma janela craniana para visualizar artérias cerebrais de uma forma faseada, bem como a forma de evitar muitas das armadilhas potenciais referentes a este método.

As imagens fixas tiradas do programa de vídeo captada que a mudança de diâmetro da artéria cerebral após a injecção de ET-1 pode ser facilmente observado utilizando esta técnica janela craniana (Figura 2). Poucos minutos após a injeção de ET-1, o navio começará a se contrair. Eventualmente, os vasos será difícil de visualizar o tecido cerebral e irá tornar-se clara. Após cerca de 20 min os efeitos de ET-1 irá diminuir e os vasos começará a dilatar gradualmente voltando ao diâmetro d...

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 artigo descreve um método para a visualização de artérias cerebrais de rato através de uma janela craniana via craniectomia temporal, a fim de visualizar partes proximais da artéria cerebral média (<strong&gt; Figura 1</strong&gt;). Este método versátil, pode ser combinada com várias técnicas de administração de fármacos para medir a reactividade da artéria cerebral<em&gt; In vivo</em&gt;.

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 ...

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

Research

JoVE Journal

Medicine

14.8K visualizações.  University of Florida. Este artigo descreve um método para a visualização de artérias cerebrais de rato através de uma janela craniana via craniectomia temporal, a fim de visualizar partes proximais da artéria cerebral média ( Figura 1). Este método versátil, pode ser combinada com várias técnicas de administração de fármacos para medir a reactividade da artéria cerebral In vivo.

Vídeo: 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.

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 ...

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.

A aplicação de ligadura da artéria Cerebral Média Permanente no 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 ...

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.

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 ...

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 ...

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 ...

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.

Quantificação de Proteção Neurovascular sequência repetitiva Hypoxic Pré-condicionamento e Transient Oriente oclusão da artéria cerebral em ratos

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.

Realizando Permanente Distal Oriente cerebral com carótida comum Oclusão da Artéria em ratos idosos para estudar Cortical Isquemia com Deficiência Sustentada

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.

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 ...