Agradecemos Annalisa Buffo para sugestões e comentários perspicazes, e Maurizio Grassano, Marina Boido e Ermira Pajaj para o tiroteio. Este trabalho foi financiado pelo FP7-MC-214003-2 (Marie Curie Initial Training Rede AXREGEN) e da Compagnia di San Paolo, o projeto gliarep.

1. Pré-cirurgia <ol><li> Pesar Rose Bengal em um tubo de 1,5 ml e dissolvem-se em solução salina estéril até atingir uma concentração final de 15 mg / ml. Filtrar através de um filtro de esterilização de 0,2 um e armazenar no escuro à temperatura ambiente até dois meses. </li><li> Esterilizar todos os instrumentos cirúrgicos em autoclave. A área cirúrgica deve ser higienizado menos de uma hora antes do início da cirurgia. </li><li> Anote o peso do corpo do mouse para ajustar a dose de Rosa Bengala para ser injetado. Nós injetado 10 ml / g de peso de animais em mulheres camundongos CD1 12 semanas de idade, no presente protocolo. A quantidade de Rosa de Bengala necessária para produzir o tamanho de lesões corticais desejada pode ser facilmente determinada de um conjunto separado de experiências preliminares, testando diferentes doses (tipicamente de 2 ul / g, 5 ul / g ou 10 ml / g de peso corporal). Note-se que a quantidade de Rosa de Bengala a ser injectado é altamente dependente das condições da experiência, em particular do tipo de fonte de luzutilizado e da duração da exposição à luz. No presente estudo, 10 ul / g (150 ug / g), a dose foi considerado necessário para induzir fototrombose após exposição à luz durante 15 minutos, ao passo que outros grupos relataram que, ou 50 ug / g de 6,8 e 100 mg / g 10 de injecção intraperitoneal era suficiente para induzir uma lesão photothrombotic. </li></ol> 2. Procedimento anestésico <ol><li> Anestesiar ratos com isoflurano numa câmara de indução transparente (3,5-4% para indução, 1,5-2% para manutenção) em 50% (v / v) oxygen/50% (v / v) de mistura de gás de monóxido de diazoto. Anestesia gasosa permite uma rápida wake up dos animais eo nível de gás anestésico pode ser ajustado facilmente. Alternativamente, os ratinhos podem também ser anestesiados com uma mistura de cetamina-xilazina. </li><li> Quando é atingida anestesia profunda, remover o animal anestesiado e a partir da câmara de indução, coloque-o na moldura estereotáxica e manter a anestesia utilizando uma máscara facial. Ajuste o isoflurane dose para atingir um nível de anestesia adequada. Monitorar a taxa de respiração durante todo o procedimento e verifique se ele é constante (40 - 60 respirações por minuto). </li><li> Use pitadas toe, a fim de assegurar que o animal é anestesiado profundamente. </li><li> Aplicar pomada ocular, a fim de evitar que os olhos sequem. </li><li> Suavemente inserir a sonda rectal para monitorizar a temperatura durante os procedimentos cirúrgicos. Definir a almofada de aquecimento controlada por realimentação associado para manter a temperatura do corpo do rato, a 37 ± 0,5 ° C. </li></ol> 3. Cirurgia para iluminação da área de destino <ol><li> Raspar o couro cabeludo do rato com um barbeador elétrico. </li><li> Fixe de forma segura a cabeça e coloque as barras de ouvido em meato externo. Tenha cuidado para não danificar os tímpanos. </li><li> Desinfectar a pele sobre a superfície com furtos de etanol a 70% e betadine usando cotonetes alternada. </li><li> É um bisturi para fazer uma incisão na linha média do olho level até o pescoço. Aplicar afastadores para manter a pele do crânio exposta. </li><li> Suavemente retrair o periósteo às bordas do crânio com um bisturi e deixar secar a superfície do crânio com cotonetes de algodão estéril. Identificar o bregma e lambda. Coloque uma micropipeta de vidro no bregma como ponto de referência, em seguida, movê-lo para as coordenadas da sua região de interesse. A região de interesse escolhido para este artigo é aproximadamente em forma redonda, centrada aproximadamente 2 mm lateral ao bregma, e que cobre uma área de cerca de 30 mm 2, que inclui uma grande parte do córtex sensorimotor de acordo com o atlas do cérebro do rato e por Franklin Paxinos 11. </li><li> Marque a posição de interesses com pontos de referência e colocar uma fibra óptica em estreito contacto com a superfície do crânio para evitar o espalhamento de luz, mas preste atenção para não exercer pressão sobre ele. A área de iluminação pode ser limitada pela aplicação sobre o crânio de uma máscara com uma pequena abertura ou uma tampa na extremidade da fibra óptica guide. </li></ol> 4. Rosa Bengala Injection e Ativação <ol><li> Carregar a solução Rosa Bengala, numa seringa de 1 ml e calcular a quantidade a ser injectado de acordo com uma dose de 10 ml / g de peso corporal. </li><li> Proceder a uma injeção intraperitoneal lento. </li><li> Deixe o difuso corante e entrar na corrente sanguínea. Após 5 min, ligar o iluminador luz fria. Evitar a iluminação do animal por qualquer outra fonte de luz. Usamos iluminador de fibra óptica de 150 W intensidade. </li><li> Após 15 min de iluminação, parar a exposição à luz e suturar a ferida. Cinco minutos de iluminação já produzem infarto e 10 min é provável que suficiente para obter um efeito máximo 12. A fim de minimizar a variabilidade, que escolhemos para iluminar por 15 min. </li></ol> 5. Sutura <ol><li> Remova os afastadores de pele e aplicar solução salina estéril para evitar a desidratação. </li><li> Fechar a ferida usando needl corte reversoe de seda ou fio de sutura nylon. </li><li> Interromper a entrega de anestesia, retire cuidadosamente o mouse do aparelho estereotáxico e colocá-lo em uma almofada de aquecimento pré-aquecido até que esteja totalmente desperto, em seguida, devolvê-lo à sua jaula. A temperatura do corpo devem ser cuidadosamente monitorizados durante os procedimentos para limitar a variabilidade da extensão do infarto. De acordo com a concentração e a via de administração, Rosa Bengala pode ainda ser detectado na corrente sanguínea, durante várias horas após a injecção 13. Prefere o cobertor de aquecimento para lâmpada de aquecimento para evitar possíveis danos secundários (Rosa Bengala absorção de comprimento de onda está no espectro verde). </li></ol>

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, 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=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Mathers, C. D., Boerma, T., Ma Fat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+causes+of+death.">Global and regional causes of death.</a> Br. Med. Bull. 92, 7-32 (2009).</li><li>Rosenblum, W. 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Neurol. 17, 497-504 (1985).</li><li>Bergeron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+photochemical+cortical+lesions+in+rat+brain.">Inducing photochemical cortical lesions in rat brain.</a> Curr. Protoc. Neurosci. Chapter 9, Unit 9 16 (2003).</li><li>Lee, J. K., 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=Photochemically+induced+cerebral+ischemia+in+a+mouse+model.">Photochemically induced cerebral ischemia in a mouse model.</a> Surg. Neurol. 67, 620-625 (2007).</li><li>Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D., Bethea, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+infarction.+I.+Early+microvascular+alterations.">Photochemically induced cerebral infarction. I. Early microvascular alterations.</a> Acta Neuropathol. 72, 315-325 (1987).</li><li>Schroeter, M., Jander, S., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J. Neurosci. Methods. 117, 43-49 (2002).</li><li>Kitagawa, K., 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=Cerebral+ischemia+after+bilateral+carotid+artery+occlusion+and+intraluminal+suture+occlusion+in+mice:+evaluation+of+the+patency+of+the+posterior+communicating+artery.">Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery.</a> J. Cereb. Blood Flow Metab. 18, 570-579 (1998).</li><li>Sigler, A., Goroshkov, A., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hardware+and+methodology+for+targeting+single+brain+arterioles+for+photothrombotic+stroke+on+an+upright+microscope.">Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope.</a> J. Neurosci. Methods. 170, 35-44 (2008).</li><li>Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Piao, M. S., Lee, J. K., Jang, J. W., Kim, S. H., Kim, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+photochemically+induced+spinal+cord+injury.">A mouse model of photochemically induced spinal cord injury.</a> J. Korean Neurosurg. Soc. 46, 479-483 (2009).</li><li>Silva, V. M., Corson, N., Elder, A., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+ear+vein+model+for+investigating+in+vivo+thrombogenicity+of+ultrafine+particles+(UFP).">The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP).</a> Toxicol. Sci. 85, 983-989 (2005).</li><li>Watson, B. D., Prado, R., Dietrich, W. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+spinal+cord+injury+in+the+rat.">Photochemically induced spinal cord injury in the rat.</a> Brain Res. 367, 296-300 (1986).</li><li>Van Reempts, J., Van Deuren, B., Van de Ven, M., Cornelissen, F., Borgers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flunarizine+reduces+cerebral+infarct+size+after+photochemically+induced+thrombosis+in+spontaneously+hypertensive+rats.">Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats.</a> Stroke. 18, 1113-1119 (1987).</li><li>Carmichael, S. T. <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+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2, 396-409 (2005).</li><li>Kleinschnitz, C., 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=Blocking+of+platelets+or+intrinsic+coagulation+pathway-driven+thrombosis+does+not+prevent+cerebral+infarctions+induced+by+photothrombosis.">Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis.</a> Stroke. 39, 1262-1268 (2008).</li><li>Porritt, M. J., 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=Photothrombosis-induced+infarction+of+the+mouse+cerebral+cortex+is+not+affected+by+the+Nrf2-activator+sulforaphane.">Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane.</a> PLoS One. 7, e41090 (2012).</li><li>Baskin, Y. K., Dietrich, W. D., Green, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J. Neurosci Methods. 129, 87-93 (2003).</li><li>Markgraf, C. G., 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=Comparative+histopathologic+consequences+of+photothrombotic+occlusion+of+the+distal+middle+cerebral+artery+in+Sprague-Dawley+and+Wistar+rats.">Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.</a> Stroke. 24, 286-292 (1993).</li><li>Wester, P., Watson, B. D., Prado, R., Dietrich, 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=A+photothrombotic+'ring'+model+of+rat+stroke-in-evolution+displaying+putative+penumbral+inversion.">A photothrombotic 'ring' model of rat stroke-in-evolution displaying putative penumbral inversion.</a> Stroke. 26, 444-450 (1995).</li><li>Hu, X., Wester, P., Brannstrom, T., Watson, B. D., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+and+reproducible+focal+cortical+ischemia+with+or+without+late+spontaneous+reperfusion+generated+by+a+ring-shaped,+laser-driven+photothrombotic+lesion+in+rats.">Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.</a> Brain Res. Brain Res. Protoc. 7, 76-85 (2001).</li><li>Maxwell, K. A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+focal+ischemic+lesions+in+neonatal+mice+by+photothrombosis.">Induction of reproducible focal ischemic lesions in neonatal mice by photothrombosis.</a> Dev. 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As modificações e substituições Por causa de seu pico de absorção em 562 nm, um laser de luz verde de uma lâmpada de arco de xenônio filtrada foi originalmente escolhido para irradiar o fotossensível Rosa Bengala. Apesar da excitação do laser ainda mediada recently5 foi utilizado, pode ser substituída pela lâmpada de luz fria também assegurar excitação corante 10,15. Fibras ópticas de luz fria são mais fáceis de manipular e menos dispendiosa do que as fontes de las...

No início do século 21, acidente vascular cerebral isquêmico é uma doença devastadora que representa a segunda causa de incapacidade a longo prazo e uma a segunda causa de mortalidade no mundo, no qual AVC foi responsável por aproximadamente 5,7 milhões de mortes em 2004 2. Apesar dos muitos esforços que foram colocadas em, ainda não existe um tratamento eficaz disponível para melhorar a recuperação funcional após o acidente vascular cerebral. Os modelos animais de acidente vascular cerebral são amplamente utilizados no campo da pesquisa do curso, pois permitem a modelagem da patofisiologia da lesão isquémica e testar a eficácia de diferentes estratégias neuroprotectoras in vivo. A maioria destes modelos visam induzir infartos extensos, interrompendo (temporariamente ou permanentemente) o fluxo de sangue dentro da artéria cerebral média, enquanto que outros modelos foram desenvolvidos para o estudo de lesões de pequeno porte em áreas específicas, normalmente o motor eo córtex somatossensorial. No entanto, vários fatores podem contribuir para gerar acertain grau de variabilidade no curso estudos experimentais, incluindo a estirpe de ratinhos utilizada, a idade e o sexo dos animais incluídos no estudo e, acima de tudo, a técnica utilizada para induzir o dano isquémico. No que diz respeito a este último ponto, a duração e invasividade da cirurgia (isto é, a necessidade de uma craniotomia), assim como a habilidade cirúrgica necessária para o operador para induzir lesão isquémica de forma confiável são determinantes críticos para o sucesso e imparcial em estudo in vivo AVC . O conceito de fototrombose foi inicialmente proposto por Rosenblum e El-Sabban em 1977 3 e tornou-se famoso pela sua aplicação em cérebro de rato por Watson et al, em 1985, 4 em que a técnica foi amplamente melhorado e definir a base do atual modelo 3. - 6. A abordagem photothrombotic visa induzir um infarto cortical através da foto-ativação de um corante sensível à luz anteriormente entregue no sistema sanguíneo, which resulta em trombose de vasos local nas áreas expostas à luz. Quando o corante é iluminado circulando no comprimento de onda apropriado através de uma fonte de luz fria, liberta energia para as moléculas de oxigénio, o que, por sua vez, geram uma grande quantidade de produtos de oxigénio singlet altamente reactivos. Estes intermediários de oxigénio induzem a peroxidação da membrana de células endoteliais, que conduz à adesão e agregação plaquetária, e, eventualmente, para a formação de trombos, que determina a interrupção do fluxo local cerebral 7. Fototrombose é um modelo de isquemia não-canônico que não obstruir ou quebrar apenas uma artéria como normalmente acontece em acidente vascular cerebral humano, mas provoca lesões nos vasos mais superficiais, o que resulta em interrupção seletiva do fluxo de sangue nas áreas expostas à luz. Por esta razão, esta abordagem pode ser adequada para estudos celulares e moleculares da plasticidade cortical. A principal vantagem desta técnica reside na sua simplicidade de execução.Além disso, fototrombose pode ser facilmente realizado em cerca de 40 minutos por animal, incluindo a vinte minutos de espera (3 min para a anestesia, 1 min para raspar o couro cabeludo; 3 a 5 min a colocar o animal no aparelho estereotáxico, 2 min para esfregar a couro cabeludo com solução anti-séptica, fazer uma incisão e limpar o crânio; 2 a 4 min para colocar a fibra de luz fria, 1 min para injetar a solução de rosa de bengala, 5 min-espera para a difusão intraperitoneal, 15 min de iluminação, e 5 min para limpar a ferida e sutura do animal). Além disso, nenhum perícia cirúrgica é necessário para realizar esta técnica quanto à lesão é induzida através de simples iluminação do crânio intactos. Ao contrário de oclusão arterial clássica, este método determina oclusões seletivos de microvasos pial e intraparenquimatosa dentro da zona irradiada e reduz a variabilidade entre as lesões que nenhum navio garantia é deixada para fornecer oxigênio na área alvo. Apesar de sua natureza particular, oações danos photothrombotic mecanismos essenciais que ocorrem em acidente vascular cerebral. Similarmente à oclusão da artéria, em acidente vascular cerebral humano, a agregação de plaquetas e formação de coágulos de determinar a interrupção do fluxo de sangue na área irradiada 7. Do mesmo modo, este modelo também partes essenciais de respostas inflamatórias em oclusão da artéria cerebral média 8. No entanto, por causa dos limites bem delimitada, a zona de penumbra, o que corresponde a uma área de metabolismo parcialmente preservada, é muito reduzida ou inexistente após uma lesão photothrombotic. Esta fronteira clara pode facilitar o estudo de respostas celulares dentro isquêmico ou intacto área cortical. Modelo do rato Indocianina é particularmente adequada para estudos de acidente vascular cerebral em uma variedade de animais transgénicos. Na verdade os modelos clássicos não pode caber para todas as cepas e estudos de longo período na linhagem C57BL / 6 rato relataram uma alta taxa de mortalidade que pode causar viés 9.

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 <td>Rose Bengal </td>
 <td>Sigma, Italy </td>
 <td>330000 </td>
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 <td>Isoflurane Vet </td>
 <td>Merial </td>
 <td> 103120022 </td>
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 <td>Paraformaldehyde </td>
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 <td>Fluosorber Filter </td>
 <td>Havard apparatus </td>
 <td> 340415 </td>
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 <td>150W fiber optic illuminator</td>
 <td>Photonic</td>
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 <td>Temperature Controller for Plate TCAT-2DF </td>
 <td>Havard apparatus</td>
 <td>727561 </td>
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 <td>Stereotaxic Instrument </td>
 <td>Stoelting</td>
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 <td>Operating microscope </td>
 <td>Takagi </td>
 <td> OM8 </td>
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 <td>Oxygen and nitrogen gas</td>
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 <td>Optic fiber taps and mask are custom-made </td>
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photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies

O modelo de acidente vascular cerebral photothrombotic visa induzir um dano isquêmico dentro de uma determinada área cortical, por meio de foto-ativação de um corante sensível à luz anteriormente injetado. Após iluminação, o corante é activado e produz oxigénio singuleto que danifica os componentes das membranas das células endoteliais, com subsequente agregação de plaquetas e formação de trombos, a qual, eventualmente, determina a interrupção do fluxo sanguíneo local. Esta abordagem, inicialmente proposto por Rosenblum e El-Sabban em 1977, foi posteriormente melhorado por Watson em 1985, em cérebro de rato e definir a base do modelo atual. Além disso, o aumento da disponibilidade de linhas de camundongos transgênicos contribuiu ainda mais para aumentar o interesse no modelo fototrombose. Resumidamente, um corante fotossensível (Rosa de Bengala) é injectado intraperitonealmente e entra na corrente sanguínea. Quando iluminada por uma fonte de luz fria, o corante torna-se activada e induz lesão endotelial com a activação de plaquetas e trombose, resultando em locaisinterrupção do fluxo sanguíneo. A fonte de luz pode ser aplicada sobre o crânio intactos, sem necessidade de craniotomia, o que permite direccionamento de qualquer área cortical de interesse de uma forma reprodutível e não-invasiva. O rato é então suturada e permitiu acordar. A avaliação do dano isquémico pode ser rapidamente realizada por cloreto de trifenil-tetrazólio e de coloração com violeta cresil. Esta técnica produz enfarte de pequena dimensão e aos limites bem delimitadas, o que é altamente vantajosa para a caracterização de células precisas ou estudos funcionais. Além disso, é particularmente adequado para estudar as respostas celulares e moleculares subjacentes plasticidade cerebral em ratinhos transgénicos.

Este protocolo irá produzir uma lesão cortical que já é visível após dissecação do córtex para o olho nu (Figuras 1A-1C). A lesão photothrombotic desenvolve em superficiais e profundas camadas corticais em que o tecido é suficientemente transparente para permitir a foto-ativação da Rosa Bengala. Medição do grau de enfarte cerebral pode ser realizada rapidamente através de coloração histológica com cloreto de trifenil-tetrazólio (TTC) em tecido fresco ou de violeta cresil, após fixa?...

Watch this Scientific Journal Video about Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies at JoVE.com

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Fototrombose é uma técnica rápida, minimamente invasiva para induzir pequena e bem delimitada infarto em áreas de interesse de maneira altamente reprodutível. É particularmente adequado para o estudo de respostas celulares e moleculares subjacentes plasticidade cerebral em ratinhos transgénicos.

Isquemia Photothrombotic: Um Modelo de lesão cortical fotoquímica Minimamente Invasiva e reprodutível para mouse Estudos curso

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

photothrombotic-ischemia-minimally-invasive-reproducible

Research

JoVE Journal

Medicine

34.4K visualizações.  University of Turin. Fototrombose é uma técnica rápida, minimamente invasiva para induzir pequena e bem delimitada infarto em áreas de interesse de maneira altamente reprodutível. É particularmente adequado para o estudo de respostas celulares e moleculares subjacentes plasticidade cerebral em ratinhos transgénicos.

Vídeo: Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Um modelo murino de hemorragia subaracnóide

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Medicina

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lesão Explorer: Um guiada por vídeo, protocolo padronizado para Volumetrics MRI derivados precisos e confiáveis ​​na doença de Alzheimer e idosos normais

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve patient outcomes. We present our experience with a minimally invasive pterional approach for anterior circulation aneurysms performed in a major tertiary cerebrovascular center and compare the results with an aged matched dataset from the Nationwide Inpatient Sample (NIS). From August 2008 to December 2012, 22 elective aneurysm clippings on patients &#8804;55 years of age were performed by the same dual fellowship-trained cerebrovascular/endovascular neurosurgeon. One patient (4.5%) experienced transient post-operative complications. 18 of 22 patients returned for follow-up imaging and there were no recurrences through an average duration of 22 months. A search in the NIS database from 2008 to 2010, also for patients aged &#8804;55 years of age, yielded 1,341 hospitalizations for surgical clip ligation of unruptured cerebral aneurysms. Inpatient length of stay and hospital charges at our institution using the minimally invasive thumb-sized pterional technique were nearly half that of NIS (length of stay: 3.2 vs 5.7 days; hospital charges: $52,779 vs. $101,882). The minimally invasive thumb-sized pterional craniotomy allows good exposure of unruptured small and medium-sized supraclinoid anterior circulation aneurysms. Cerebrospinal fluid drainage from key subarachnoid cisterns and constant bimanual microsurgical techniques avoid the need for retractors which can cause contusions, localized venous infarctions, and post-operative cerebral edema at the retractor sites. Utilizing this set of techniques has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimally invasive thumb-sized pterional craniotomy for aneurysm clipping has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimamente Invasiva Thumb porte pterional craniotomia para pinça de aperto cirúrgica de Unruptured Anterior Aneurismas de circulação

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Um Modelo de acidente vascular cerebral trombótico Baseado em Transient isquemia-hipoxia cerebral

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Déficit motor reprodutível após oclusão aórtica em um modelo de rato da isquemia da medula espinhal

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Um modelo minimamente invasivo para analisar a fratura endocondral cura em ratos sob condições biomecânicas padronizadas

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

Localização do nódulo transtorácico de navegação eletromagnética para cirurgia torácica minimamente invasiva

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Um modelo pré-clínico do rato para o estudo da lesão da Isquemia-reperfusion na microcirurgia reconstrutiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Avaliação da compatibilidade e precisão da linha de amostragem da capnografia quando usado com um monitor de capnografia portátil

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

Um Método Não Invasivo para Gerar o Modelo de Lesão da Cartilagem Intra-Articular Induzida por Carga Cíclica do Joelho de Rato

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...