A técnica cirúrgica foi originalmente adquirida no laboratório do Dr. William D. Hill, do Medical College of Georgia. Os autores também gostariam de agradecer ao Dr. David A. Rempe e Landa Prifti para o uso da câmera dissecção. Esta pesquisa foi suportada pelo NIH NS041744, NS051279, F31 NS064700 e 30815697D AHA.

Este protocolo foi aprovado pela Universidade de Rochester comitê dedicado ao uso ético de animais na pesquisa (UCAR). Técnicas de assepsia devem ser seguidas durante o protocolo. O uso de luvas estéreis e máscara é obrigatório. Todos os equipamentos, materiais, produtos químicos e ferramentas que são usadas durante o protocolo são descritos na Tabela 1. 1. Preparação pré-cirúrgica <ol><li> Injetar camundongos por via subcutânea com buprenorfina (0.05mg/kg) 2 h antes da cirurgia, imediatamente após a cirurgia e depois a cada 3-5 h nas 24 h primeiro período pós-operatório. </li><li> Esterilizar todos os instrumentos cirúrgicos, esponjas de gaze, e aplicadores com ponta de algodão por autoclave. Manter instrumentos cirúrgicos em etanol 70% durante a cirurgia e ar seco em gaze esterilizada direito antes de usar. Pulverizar a área de trabalho com álcool 70%. </li><li> Anestesiar o rato com uma mistura de gás de 3% isofluorano-20% de oxigênio, usando um vaporizador anestésico. Ajustar a quantidade de oxigênio com o medidor de vazão. Testar o nível de anestesia com pés ou beliscar a cauda (que deveria ser indiferente). Manter o nível de anestesia com 2% (v / v) isofluorano. </li><li> Aplicar lágrimas artificiais para os olhos do rato e tome cuidado para não danificar o olho durante o procedimento cirúrgico. Posicione o mouse em seu lado direito em uma posição lateral. </li><li> Raspar a área do lado esquerdo entre o olho esquerdo e na base da orelha esquerda usando o clippers animal. Limpar a área pela alternância entre uma solução de betadine e etanol a 70%, utilizando aplicadores de algodão ponta e esfregar levemente a área. Repita conforme necessário. </li><li> Posicione o mouse sobre uma almofada de aquecimento ligado a uma sonda retal para manter a temperatura corporal a 37 ° C. Introduzir a sonda retal, utilizando óleo mineral. </li><li> Posicione o mouse em seu lado direito sob o microscópio e prenda com fita adesiva. Cortar uma janela em uma gaze e cubra área cirúrgica. </li></ol> 2. Procedimento cirúrgico e MCA Ligadura <ol><li> Faça uma incisão vertical entre o olho esquerdo e na base da orelha esquerda usando uma tesoura fina reta. Use hemostats curvas para manter a área de cirurgia aberta. </li><li> Faça uma incisão horizontal sobre o músculo temporal usando uma tesoura primavera e ligeiramente separada do músculo temporal do crânio por lentamente puxando com fórceps. </li><li> Fazer uma craniotomia pequena subtemporal na junção do arco zigomático e osso esquamosal usando uma agulha de 18G, mantendo o maxilar com uma pinça curva. </li><li> Expor o MCA, removendo os pequenos pedaços de crânio com fórceps óssea. Do arco zigomático e conteúdo orbital não deve ser danificado durante este processo. Se a secagem excessiva de tecido ocorre durante este processo, aplique PBS estéril com ponta de algodão aplicadores. </li><li> Ligadura da porção distal do MCA usando um cauterizador de pequenos vasos. </li><li> Coloque o músculo temporal de volta para sua posição original e feche o local da incisão cirúrgica com nylon 5-0. </li><li> Descontinuar a anestesia e remover a sonda retal. Injetar o mouse com uma 2 ª dose de buprenorfina (0.05mg/kg) por via subcutânea. Devolver o mouse para a gaiola que foi mantida a 37 ° C com um painel de aquecimento. </li><li> Acompanhar de perto o mouse para os próximos 24 h para qualquer desconforto (diminuição do consumo de apetite / água, postura encurvada, respiração aumentada, pilo-erigido cabelo). Injetar o mouse com buprenorfina por via subcutânea a cada 3-5 h pós-operatório até 24h. Fornecer o mouse com gel de recuperação durante este período. </li></ol> 3. TTC Coloração e Determinação do Volume Sistólico <ol><li> Vinte e quatro horas após a cirurgia profundamente anestesiar o mouse, transcardially perfundir do mouse com um TTC 4% (w / v) em tampão fosfato salino (PBS) por 15 min e depois com uma solução de paraformaldeído a 4% por 10 min usando uma mini-bomba peristáltica de vazão média. Remover o cérebro e colocá-lo na solução de paraformaldeído 4% durante a noite. </li><li> Posição do cérebro dentro do bloco de corte. Fatia do cérebro em fatias 1 mm de espessura, utilizando lâminas de barbear. </li><li> Coloque as fatias ao lado de uma régua de escala milimétrica. Fotografar as fatias usando uma câmera digital conectada ao microscópio de dissecação. </li><li> Calcular a área acidente vascular cerebral, subtraindo a área não-infartada do site ipsilateral da área total do site contralateral utilizando Software Image J (http://rsbweb.nih.gov/ij/). Calcular o volume de ejeção pelo empilhamento da área golpe de 22 fatias. </li></ol> 4. Instruções para usar o software Image J <ol><li> Abra o arquivo de imagem a ser analisada no software Image J, clicando no menu arquivo. </li><li> Clique na guia &quot;linha reta&quot; no programa. Desenhar uma linha reta entre as duas margens na régua. Clique em &#39;analisar&#39; menu e selecione &quot;escala set &#39;. Definir a distância conhecida como 1, unidade de comprimento como mm. </li><li> Clique na guia &#39;círculo à mão livre &quot;. Desenhar um círculo para delinear o hemisfério contralateral. Clique em &#39;analisar&#39; menu e selecione &quot;medida&quot;. A janela de cálculo irá aparecer mostrando a área do círculo desenhado. </li><li> Desenhar outro círculo em torno da área do acidente vascular cerebral ipsilateral excluindo hemisfério (branco). Medir a área, como indicado no passo 4.3. O novo valor será adicionado à janela de cálculo. A diferença entre os valores 1 e 2 representa a área do infarto, que é indicado como A na fórmula abaixo. </li><li> Calcular a área acidente vascular cerebral em cada fatia, repetindo os passos 4,2-4,4. Tome somatória da área curso calculado em cada fatia (ΣA n). Isso representa volume de curso o fato de saber que a espessura de cada fatia é de 1mm. ΣA n x 1mm (espessura de cada fatia) = volume do curso </li></ol> 5. Resultados representativos: Os infartos obtidos por ligadura MCA permanente no rato são principalmente cortical. No entanto, é possível obter lesões subcorticais se o MCA é ligada ao ramo proximal lenticulostriate. Os volumes de acidente vascular cerebral após MCA ligadura pode variar de 10 milímetros a 35mm ³ ³ 19, 23. O volume sistólico calculado com TTC coloração em Moyanova et al. 19 estão entre 10mm a ³ ³ 22 milímetros, enquanto os volumes de acidente vascular cerebral determinada a partir de imagens de ressonância magnética em Filiano et al 23 têm entre 20mm a ³ ³ 35mm. A possível razão para estas diferenças talvez a localização exata da ligadura e os diferentes métodos utilizados para medir os volumes de acidente vascular cerebral. Na Figura 1, um manchado TTC cérebro 24 horas pós ligadura MCA permanente em um rato selvagem tipo C57/BL6 é mostrado. O site do curso é branco na aparência (Fig. 1A, B). Figura 1A e B ilustram o site infarto de diferentes ângulos. A Figura 2 mostra fatias 1 mm de espessura do cérebro TTC manchada acidente vascular cerebral no sentido antero-posterior (Fig. 2A-G). Volume de infarto foi calculada 24 horas pós-cirurgia. O volume do infarto é ~ ³ 23mm. <img src="/files/ftp_upload/3039/3039fig1.jpg" alt="Figura 1"/> Figura 1. Representação do site acidente vascular cerebral. AB, TTC manchado imagens do cérebro inteiro, 24 h após a ligadura MCA. Área branca representa o infarto. <img src="/files/ftp_upload/3039/3039fig2.jpg" alt="Figura 2"/> Figura 2. Resultados representativos. (A G&gt;), as seções do cérebro TTC manchada 1mm, 24 h após Mcal. Fatias estão alinhados no sentido antero-posterior. Área branca representa o infarto.

<ol>
	<li>Britton, M., Rafols, J., Alousi, S., Dunbar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+middle+cerebral+artery+occlusion+on+central+nervous+system+apoptotic+events+in+normal+and+diabetic+rats.">The effects of middle cerebral artery occlusion on central nervous system apoptotic events in normal and diabetic rats.</a> Int J Exp Diabesity Res. 4, 13-20 (2003).</li><li>Ciceri, P., Rabuffetti, M., Monopoli, A., Nicosia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+leukotrienes+in+a+model+of+focal+cerebral+ischaemia+in+the+rat.">Production of leukotrienes in a model of focal cerebral ischaemia in the rat.</a> Br J Pharmacol. 133, 1323-1329 (2001).</li><li>Jin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+transplantation+of+human+neural+precursor+cells+improves+outcome+from+focal+cerebral+ischemia+in+aged+rats.">Delayed transplantation of human neural precursor cells improves outcome from focal cerebral ischemia in aged rats.</a> Aging Cell. 9, 1076-1083 (2010).</li><li>McCaig, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+GADD34+expression+after+focal+cerebral+ischaemia.">Evolution of GADD34 expression after focal cerebral ischaemia.</a> Brain Res. 1034, 51-61 (2005).</li><li>Shirotani, T., Shima, K., Chigasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+studies+of+extracellular+metabolites+in+the+striatum+after+distal+middle+cerebral+artery+occlusion+in+stroke-prone+spontaneously+hypertensive+rats.">In vivo studies of extracellular metabolites in the striatum after distal middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats.</a> Stroke. 26, 878-884 (1995).</li><li>Wu, Y. P., Tan, C. K., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Fos-like+immunoreactivity+in+the+brain+and+spinal+cord+of+rats+following+middle+cerebral+artery+occlusion.">Expression of Fos-like immunoreactivity in the brain and spinal cord of rats following middle cerebral artery occlusion.</a> Exp Brain Res. 115, 129-136 (1997).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J Cereb Blood Flow Metab. 1, 53-60 (1981).</li><li>Bederson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion:+evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.</a> Stroke. 17, 472-476 (1986).</li><li>Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+gender+influences+on+sensitivity+to+focal+cerebral+ischemia+in+the+stroke-prone+spontaneously+hypertensive+rat.">Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat.</a> Hypertension. 33, 681-685 (1999).</li><li>Mary, V., Wahl, F., Uzan, A., Stutzmann, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enoxaparin+in+experimental+stroke:+neuroprotection+and+therapeutic+window+of+opportunity.">Enoxaparin in experimental stroke: neuroprotection and therapeutic window of opportunity.</a> Stroke. 32, 993-999 (2001).</li><li>Menzies, S. A., Hoff, J. T., Betz, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+in+rats:+a+neurological+and+pathological+evaluation+of+a+reproducible+model.">Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model.</a> Neurosurgery. 31, 100-107 (1992).</li><li>Iaci, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+growth+factor+2+promotes+functional+recovery+with+treatment+initiated+up+to+7+days+after+permanent+focal+ischemic+stroke.">Glial growth factor 2 promotes functional recovery with treatment initiated up to 7 days after permanent focal ischemic stroke.</a> Neuropharmacology. 59, 640-649 (2010).</li><li>Richard, M. J. P., Khan, B. V. C. B. J., Saleh, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+by+which+lipoic+acid+confers+protection+during+the+early+stages+of+cerebral+ischemia:+A+possible+role+for+calcium.">Cellular mechanisms by which lipoic acid confers protection during the early stages of cerebral ischemia: A possible role for calcium.</a> Neuroscience Research. , (2011).</li><li>Heiss, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+derangement+of+periinfarct+viable+tissue+in+ischemic+stroke.">Progressive derangement of periinfarct viable tissue in ischemic stroke.</a> J Cereb Blood Flow Metab. 12, 193-203 (1992).</li><li>Nedergaard, M., Gjedde, A., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+ischemia+of+the+rat+brain:+autoradiographic+determination+of+cerebral+glucose+utilization,+glucose+content,+and+blood+flow.">Focal ischemia of the rat brain: autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow.</a> J Cereb Blood Flow Metab. 6, 414-424 (1986).</li><li>Nowicki, J. P., Assumel-Lurdin, C., Duverger, D., MacKenzie, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+evolution+of+regional+energy+metabolism+following+focal+cerebral+ischemia+in+the+rat.">Temporal evolution of regional energy metabolism following focal cerebral ischemia in the rat.</a> J Cereb Blood Flow Metab. 8, 462-473 (1988).</li><li>Butcher, S. P., Bullock, R., Graham, D. I., McCulloch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+amino+acid+release+and+neuropathologic+outcome+in+rat+brain+following+middle+cerebral+artery+occlusion.">Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion.</a> Stroke. 21, 1727-1733 (1990).</li><li>Arlicot, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+remote+microglial+activation+in+rodent+models+of+focal+ischaemia+using+the+TSPO+radioligand+CLINDE.">Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE.</a> Eur J Nucl Med Mol Imaging. 37, 2371-2380 (2010).</li><li>Moyanova, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+for+type+4+metabotropic+glutamate+receptors+against+ischemic+brain+damage.">Protective role for type 4 metabotropic glutamate receptors against ischemic brain damage.</a> J Cereb Blood Flow Metab. , (2010).</li><li>Ortolano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+imaging+of+new+targets+for+pharmacological+intervention+in+stroke:+real-time+tracking+of+T-cells+in+the+ischaemic+brain.">Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain.</a> Br J Pharmacol. 159, 808-811 (2010).</li><li>O'Neill, M. J., A, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+cerebral+ischemia.">Rodent models of focal cerebral ischemia.</a> Current Protocols in Neuroscience. , 9.6.1-9.6.32 (2000).</li><li>Lin, T. N., He, Y. Y., Wu, G., Khan, M., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+brain+edema+on+infarct+volume+in+a+focal+cerebral+ischemia+model+in+rats.">Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats.</a> Stroke. 24, 117-121 (1993).</li><li>Filiano, A. J., Tucholski, J., Dolan, P. J., Colak, G., Johnson, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transglutaminase+2+protects+against+ischemic+stroke.">Transglutaminase 2 protects against ischemic stroke.</a> Neurobiol Dis. 39, 334-343 (2010).</li></ol>

Não há conflitos de interesse declarados.

O método de ligadura permanente MCA dá volumes infarto altamente reprodutível e aumentou as taxas de sobrevivência pós-operatória em comparação com outros métodos disponíveis. A facilidade ea curta duração (~ 30 min) do processo de torná-lo ainda mais prático. O método é amplamente utilizado em ambos os ratos e ratazanas. Esta técnica requer uma cirurgia invasiva sob um estereomicroscópio. Portanto, a experiência em operar sob um microscópio e aperfeiçoando uma craniotom...

<table border="1"><tbody><tr><th> NOME </th><th> Da empresa e número CATÁLOGO </th></tr><tr><td> A. Soluções e Produtos Químicos </td><td></td></tr><tr><td> Iodo-povidona Solution, Scrub Cirúrgica </td><td> Aplicare, 82-209 </td></tr><tr><td> Etanol 70% </td><td> Koptec, V1001 </td></tr><tr><td> 2,3,5 cloreto trifeniltetrazólio (TTC) </td><td> Sigma, T8877 </td></tr><tr><td> Paraformaldeído 4% </td><td> Ciências Microscopia Eletrônica, 159SP </td></tr><tr><td> Tampão fosfato pH, Saline: 7.4 </td><td> Fez e esterilizados no laboratório. </td></tr><tr><td> Óleo mineral </td><td> PML Microbiologicals, R6570 </td></tr><tr><td> Isofluorano </td><td> A Companhia Butler, 029405 </td></tr><tr><td> Buprenorfina (0.3mg/ml) </td><td> Hospira, NDC 0409-2012-32 </td></tr><tr><td> Lágrimas artificiais </td><td> A Companhia Butler, 007312 </td></tr><tr><td> Gel de recuperação </td><td> Clara H 2 O, 72-01-5022 </td></tr><tr><td> B. Materiais e Equipamentos Cirúrgicos </td><td></td></tr><tr><td> C57/BL6 mouse, 10-12 semanas de idade </td><td> A colônia foi criada no biotério da universidade </td></tr><tr><td> 100 tanque de oxigênio% </td><td> Airgas </td></tr><tr><td> Vaporizador anestésico e medidor de fluxo </td><td> Surgivet, Modelo 100 </td></tr><tr><td> Pad de aquecimento com sonda retal </td><td> Multa Ferramentas Ciência, TR200 </td></tr><tr><td> Microscópio de dissecação </td><td> Zeiss, Stemi 2000 </td></tr><tr><td> Painel de aquecimento </td><td> Petco Services Ltd. 0307-013 </td></tr><tr><td> Definir cauterizador pequenos vasos </td><td> Multa Ferramentas Ciência, 18000-00 </td></tr><tr><td> Mini bomba peristáltica </td><td> Harvard Apparatus, MPII </td></tr><tr><td> Seringa de 1ml </td><td> BD, 329650 </td></tr><tr><td> Agulha de 18G </td><td> Becton Dickinson, 305196 </td></tr><tr><td> Sutura Nylon cirúrgica 5,0 </td><td> Ethilon, 698H </td></tr><tr><td> Seccionamento de bloco </td><td> Kent Scientific, um milímetro </td></tr><tr><td> Razor Blades Single Edge </td><td> Ciências Microscopia Eletrônica, 71962 </td></tr><tr><td> Tape rótulo </td><td> Scienceware, Bel-art Produtos F13463-2075 </td></tr><tr><td> Esponjas de gaze </td><td> Kendall gurança 2252, Johnson e Johnson 6415 </td></tr><tr><td> Dica Aplicadores de algodão </td><td> Puritan 806-WCL </td></tr><tr><td> Ferramentas C. Cirurgia </td><td></td></tr><tr><td> Tesoura cirúrgica Primavera </td><td> Multa Ferramentas Ciência 15003-08 </td></tr><tr><td> Forceps Microdissecting angular </td><td> Multa Ferramentas Ciência 5 / 45 </td></tr><tr><td> Forceps Microdissecting curvas </td><td> Belas Ciência Tools 5 Ti </td></tr><tr><td> Tesoura reta fina </td><td> Multa Ferramentas Ciência 14568-12 </td></tr><tr><td> Rongeur osso curvo </td><td> Multa Ferramentas Ciência 16021-14 </td></tr><tr><td> Hemostat Hartman Curved </td><td> Multa Ferramentas Ciência 13003-10 </td></tr></tbody></table> Tabela 1. A lista de materiais, ferramentas e produtos químicos utilizados para o procedimento de ligadura MCA. A. Soluções e produtos químicos, materiais cirúrgicos e B. Equipamentos, Ferramentas Cirurgia C..

the application of permanent middle cerebral artery ligation in the mouse

Isquemia cerebral focal está entre o tipo mais comum de acidente vascular cerebral vista em pacientes. Devido à importância clínica, tem havido um esforço prolongado para desenvolver modelos animais adequados para o estudo dos eventos que acontecem durante o insulto isquêmico. Estas técnicas incluem transitória ou permanente, modelos de isquemia focal ou global usando muitos diferentes modelos animais, com os roedores mais comuns são. O método de ligadura permanente MCA que também é referido como pMCAo na literatura é usado extensivamente como um modelo de isquemia focal em roedores 1-6. Este método foi originalmente descrito para ratos por Tamura et al. em 1981 7. Neste protocolo uma craniotomia foi usado para acessar o MCA e as regiões proximal foram ocluídas por eletrocoagulação. Os infartos envolvem regiões principalmente cortical e às vezes estriado, dependendo da localização da oclusão. Esta técnica está agora bem estabelecida e usada por muitos laboratórios 8-13. Utilização precoce desta técnica levou à definição e descrição do &quot;core infarto&quot; e &quot;penumbra&quot; 14-16, e é freqüentemente usado para avaliar o potencial neuroprotetor compostos 10, 12, 13, 17. Embora os estudos iniciais foram realizados em ratos, ligadura MCA permanente tem sido usado com sucesso em camundongos com ligeiras modificações 18-20. Este modelo produz infartos reprodutível e aumento da sobrevivência pós-taxas. Aproximadamente 80% dos acidentes vasculares cerebrais isquêmicos em humanos acontecem na área MCA 21 e, portanto, este é um modelo muito relevante para os estudos acidente vascular cerebral. Atualmente, há uma escassez de tratamentos eficazes disponíveis para pacientes com AVC, e, portanto, há uma necessidade de bons modelos para testar o potencial de compostos farmacológicos e avaliar os resultados fisiológicos. Este método também pode ser usada para estudar mecanismos de resposta a hipóxia intracelular in vivo. Aqui, apresentamos a cirurgia de ligadura MCA em um rato C57/BL6. Descrevemos a preparação pré-cirúrgica, MCA cirurgia de ligadura e 2,3,5 trifeniltetrazólio cloreto de coloração (TTC) para quantificação dos volumes de infarto.

Watch this Scientific Journal Video about The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse at JoVE.com

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Ligadura da artéria cerebral média (MCA) é uma técnica para estudo da isquemia cerebral focal em modelos animais. Neste método, o da artéria cerebral média é exposto por craniotomia e ligados por cauterização. Este método dá volumes infarto altamente reprodutível e aumentou as taxas de sobrevivência pós-operatória em comparação com outros métodos disponíveis.

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 ...

the-application-permanent-middle-cerebral-artery-ligation

Research

JoVE Journal

Medicine

19.5K visualizações.  University of Rochester. Ligadura da artéria cerebral média (MCA) é uma técnica para estudo da isquemia cerebral focal em modelos animais. Neste método, o da artéria cerebral média é exposto por craniotomia e ligados por cauterização. Este método dá volumes infarto altamente reprodutível e aumentou as taxas de sobrevivência pós-operatória em comparação com outros métodos disponíveis.

Vídeo: The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

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 ...

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 ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Permanente oclusão vascular cerebral<em&gt; Via</em&gt; Dupla ligadura e transecção

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

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 ...

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, ...

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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Oclusão de artéria Cerebral média, permitindo a reperfusão através de reparação de artéria carótida comum em ratos

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modelo de infarto do miocárdio murino usando ligadura permanente da artéria coronária descendente anterior esquerda

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...