Os autores gostariam de agradecer o apoio técnico da Alegria Yao e Dr. Karen Alt, bem como o financiamento do NHMRC e NHF.

Todos os experimentos envolvendo animais foram aprovados pelo Alfred Pesquisa Médica e da Educação Comissão de Ética Animal Precinct (E / 1534/2015 / B). Todas as manipulações cirúrgicas foram realizadas sob anestesia geral e os animais não apresentaram dor em qualquer fase. Todas as experiências descritas são não-recuperação. 1. Preparação <ol><li> Cortar bandas finas de papel de filtro (1 mm x 2 mm). </li><li> Recentemente preparar duas soluções de cloreto férrico, de 4% (w / v) e 6% (w / v) diluída em água desionizada. Preparar a solução de rodamina 6G 0,3% em PBS, filtrou-se através de 0,22 um. </li><li> Corte um pequeno pedaço (5 mm x 1 cm) a partir da seringa de plástico branco de embalagem. </li></ol> 2. Mesentery arteríola formação de trombos Observado por Microscopia intravital <ol><li> Pesar de 10-12 semanas de idade C57BL / 6 de tipo selvagem do rato e anestesiar em conformidade com uma mistura de cetamina (100 mg / kg) e xilazina (10 mg / kg), embora a injecção intraperitoneal. Monitore a profundidade da anestesia com a resposta aos pés, cauda e / ou pitada pele, reatividade de pitada córnea e da pálpebra, e ausência de movimento bigodes. Se necessário, injectar uma segunda dose de cetamina (50 mg / kg) para manter a anestesia do animal. Aplicar pomada veterinário sobre os olhos para evitar a secura sob anestesia. Colocar o rato numa pequena placa de petri colocado sobre uma almofada de aquecimento ajustada a 37 ° C. Nota: Embora a injecção IP pode resultar em dor para os animais, a duração deste desconforto será mínimo (menos de 3 segundos). Dor e desconforto associado com injecções irá ser minimizado através da utilização de pessoal experiente e competentes e tamanho da agulha adequada (25 L). Após todos os procedimentos, todos os animais a eutanásia, usando uma overdose de cetamina e xilazina, seguido por deslocação cervical. </li><li> Executar uma incisão na linha média abdominal de cerca de 3 cm na pele e corte cuidadosamente o peritônio. </li><li> Colocar o rato no lado do animal de estimaçãori prato, exteriorizar suavemente seus intestinos e espalhar cuidadosamente o mesentério com 2 cotonetes para trazer um recipiente adequado para a superfície da placa de Petri. Secar bem com um limpador delicado. NOTA: Para limitar o movimento dos vasos mesentéricos, a papaverina pode ser utilizado para inibir o peristaltismo intestinal. </li><li> Embeber a cauda do rato em água morna para dilatar os vasos e injectar 30 jil de Rodamina 6G (0,3%) na veia da cauda do rato com uma seringa 29 G para marcar leucócitos e plaquetas. </li><li> Coloque a placa de Petri sob um microscópio invertido e foco no arteriole escolhido utilizando o canal de campo claro. </li><li> Embebe-se um papel de filtro de banda com 6% (w / v) de ferro (III) e cloreto de aplicar o papel de filtro sobre a arteríola com duas pinças; a primeira a realizar o papel de filtro, o segundo ao pressioná-lo suavemente sobre a área de interesse. Observar a formação de trombos nos primeiros 10 segundos após a deposição do papel de filtro. NOTA: É muito commpara ferir a microvasculatura rodeado e a precisão da deposição do papel de filtro e a pressão é, por conseguinte, importante suavemente para limitar este problema. </li><li> Observe a formação de trombos por microscopia de fluorescência (TRITC canal: excitação de pico 557 nm, 576 nm pico de emissão), por meio do papel de filtro. Observe os leucócitos circulantes e plaquetas, que têm tido até o Rodamina 6G ea sua agregação para o trombo é, portanto, fácil de identificar. </li><li> Aqui o papel de filtração após 1 min de exposição a ferro (III) e cloreto de continuar a monitorizar a formação do trombo. Lavar o recipiente com PBS. </li><li> Observar e registar a formação dinâmica do trombo em destaque com a rotulagem de plaquetas e leucócitos com a Rodamina 6G. Capturar as imagens e medir o tamanho do trombo. As imagens aqui apresentados foram obtidos com um microscópio intravital invertido, embora uma objectiva de 4X, no canal de fluorescência TRITC. </li><li> Fepois todos os procedimentos, a eutanásia dos animais usando uma overdose de cetamina e xilazina, seguido por deslocação cervical. </li></ol> 3. Formação Carótida Artéria Thrombus avaliada pela medição do fluxo de sangue Velocity <ol><li> Pesar de 10-12 semanas de idade C57BL / 6 mouse e anestesiar em conformidade com uma mistura de cetamina (100 mg / kg) e xilazina (10 mg / kg), embora a injecção intraperitoneal. Aplicar pomada veterinário sobre os olhos para evitar a secura sob anestesia. </li><li> Corrigir o mouse sob um microscópio cirúrgico usando fita adesiva nas pernas, em uma almofada de aquecimento ajustado para 37 ° C. </li><li> Faça um pequeno travesseiro para fora de um limpador e gravá-lo sob a cabeça do rato para elevar a cabeça ligeiramente. Use um loop fio com pinças para puxar o focinho para baixo (use dentes superiores). Isto irá expor a região da artéria carótida para fácil acesso. </li><li> Realize uma pequena 5 mm de profundidade da incisão da pele logo abaixo da mandíbula, até o esterno. </li><li> Dissecar a fascia e isolar um fragmento de qualquer artéria carótida comum esquerda ou para a direita acima da bifurcação. </li><li> Introduzir cuidadosamente pinças em-entre a artéria eo nervo para separá-los. Não perturbe o nervo que perto da artéria e evitar tocar muito da artéria carótida, uma vez que pode causar danos ao navio. Isolar uma secção de pelo menos 5 mm da artéria. </li><li> Seca-se a área da artéria adequadamente com escovas para evitar que qualquer líquido que interfere com o FeCl 3. </li><li> Coloque o pequeno pedaço de plástico branco sob a parte isolada da carótida comum, de modo a FeCl3 não mergulhe nos tecidos circundantes. Para esta finalidade, utilizar um segundo uma pinça para trazer a peça para o primeiro, em seguida, lentamente deslizar o papel plástico sob a artéria. </li><li> Embebe-se um pedaço de papel de filtro com 4% (w / v) ou 6% (w / v) de cloreto férrico e colocá-lo em redor da artéria. </li><li> Após 3 min de exposição, retirar o papel de filtro, lavar com PBS e secar a área wlimpadores de pára-om. </li><li> Coloque a sonda de fluxo Doppler em torno do navio no local da lesão e começar a gravar as mudanças no fluxo. Na carótida comum saudável de ratos adultos, o fluxo é normalmente cerca de 1 mL / min. Cuidado! contacto da sonda com cloreto férrico vai danificar a sonda de modo que qualquer contacto deve ser evitado. Os dados aqui apresentados foram obtidos com um medidor de fluxo Transonic System Inc., perivascular TS420 módulo equipado com uma sonda de fluxo Doppler Nano 0,5 PBS. NOTA: A concentração de cloreto férrico pode modificar as cinéticas de formação de trombo, resultando em diferentes tempos de oclusão. Assim, uma exposição a 6% (w / v) de cloreto férrico dá uma oclusão mais rápido do que a exposição a 4% (w / v) de cloreto férrico. </li><li> Seguindo todos os procedimentos, eutanásia todos os animais usando uma overdose de cetamina e xilazina seguido por deslocamento cervical e limpar cuidadosamente a sonda Doppler. </li></ol>

<ol>
	<li>Leadley, R. J., Chi, L., Rebello, S. S., Gagnon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+in+vivo+models+of+thrombosis+to+the+discovery+and+development+of+novel+antithrombotic+agents.">Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents.</a> J Pharmacol Toxicol Methods. 43 (2), 101-116 (2000).</li><li>Johnson, G. J., Griggs, T. R., Badimon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+animal+models+in+the+preclinical+study+of+interventions+to+prevent+human+coronary+artery+restenosis:+analysis+and+recommendations.+On+behalf+of+the+Subcommittee+on+Animal,+Cellular+and+Molecular+Models+of+Thrombosis+and+Haemostasis+of+the+Scientific+and+Standardization+Committee+of+the+International+Society+on+Thrombosis+and+Haemostasis.">The utility of animal models in the preclinical study of interventions to prevent human coronary artery restenosis: analysis and recommendations. On behalf of the Subcommittee on Animal, Cellular and Molecular Models of Thrombosis and Haemostasis of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis.</a> Thromb Haemost. 81 (5), 835-843 (1999).</li><li>Day, S. M., Reeve, J. L., Myers, D. D., Fay, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+thrombosis+models.">Murine thrombosis models.</a> Thromb Haemost. 92 (3), 486-494 (2004).</li><li>Sachs, U. J. H., Nieswandt, B. <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+thrombus+formation+in+murine+models.">In vivo thrombus formation in murine models.</a> Circ Res. 100 (7), 979-991 (2007).</li><li>Furie, B., Furie, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+thrombus+formation.">Mechanisms of thrombus formation.</a> N Engl J Med. 359 (9), 938-949 (2008).</li><li>Pierangeli, S. S., Liu, X. W., Barker, J. H., Anderson, G., Harris, E. N. <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+thrombosis+in+a+mouse+model+by+IgG,+IgM+and+IgA+immunoglobulins+from+patients+with+the+antiphospholipid+syndrome.">Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobulins from patients with the antiphospholipid syndrome.</a> Thromb Haemost. 74 (5), 1361-1367 (1995).</li><li>Kikuchi, S., Umemura, K., Kondo, K., Saniabadi, A. R., Nakashima, M. <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+endothelial+injury+in+the+mouse+as+a+screening+model+for+inhibitors+of+vascular+intimal+thickening.">Photochemically induced endothelial injury in the mouse as a screening model for inhibitors of vascular intimal thickening.</a> Arterioscler Thromb Vasc Biol. 18 (7), 1069-1078 (1998).</li><li>Rosen, E. D., Raymond, S., 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=Laser-induced+noninvasive+vascular+injury+models+in+mice+generate+platelet-+and+coagulation-dependent+thrombi.">Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi.</a> Am J Pathol. 158, 1613-1622 (2001).</li><li>Kurz, K. D., Main, B. W., Sandusky, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+arterial+thrombosis+induced+by+ferric+chloride.">Rat model of arterial thrombosis induced by ferric chloride.</a> Thromb Res. 60 (4), 269-280 (1990).</li><li>Eckly, A., Hechler, B., 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=Mechanisms+underlying+FeCl3-induced+arterial+thrombosis.">Mechanisms underlying FeCl3-induced arterial thrombosis.</a> J Thromb Haemost. 9 (4), 779-789 (2011).</li><li>Darbousset, R., 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=Involvement+of+neutrophils+in+thrombus+formation+in+living+mice.">Involvement of neutrophils in thrombus formation in living mice.</a> Pathol Biol (Paris). 62 (1), 1-9 (2014).</li><li>Denis, C., Methia, N., 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=A+mouse+model+of+severe+von+Willebrand+disease:+defects+in+hemostasis+and+thrombosis).">A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis).</a> Proc Natl Acad Sci U S A. 95 (16), 9524-9529 (1998).</li><li>Wang, X., Hagemeyer, C. E., 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=Novel+single-chain+antibody-targeted+microbubbles+for+molecular+ultrasound+imaging+of+thrombosis:+validation+of+a+unique+noninvasive+method+for+rapid+and+sensitive+detection+of+thrombi+and+monitoring+of+success+or+failure+of+thrombolysis+in+mice.">Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis: validation of a unique noninvasive method for rapid and sensitive detection of thrombi and monitoring of success or failure of thrombolysis in mice.</a> Circulation. 125 (25), 3117-3126 (2012).</li><li>Wang, X., Smith, P. L., Hsu, M. -. Y., Ogletree, M. L., Schumacher, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+ferric+chloride-induced+vena+cava+thrombosis:+evidence+for+effect+of+potato+carboxypeptidase+inhibitor.">Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor.</a> J Thromb Haemost. 4 (2), 403-410 (2006).</li><li>Karatas, H., Erdener, S. E., 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=Thrombotic+distal+middle+cerebral+artery+occlusion+produced+by+topical+FeCl(3)+application:+a+novel+model+suitable+for+intravital+microscopy+and+thrombolysis+studies.">Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies.</a> J Cereb Blood Flow Metab. 31 (6), 1452-1460 (2011).</li><li>Jirouskov&#225;, M., Chereshnev, I., V&#228;&#228;n&#228;nen, H., Degen, J. L., Coller, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+blockade+or+mutation+of+the+fibrinogen+gamma-chain+C-terminus+is+more+effective+in+inhibiting+murine+arterial+thrombus+formation+than+complete+absence+of+fibrinogen.">Antibody blockade or mutation of the fibrinogen gamma-chain C-terminus is more effective in inhibiting murine arterial thrombus formation than complete absence of fibrinogen.</a> Blood. 103 (6), 1995-2002 (2004).</li><li>Dubois, C., Panicot-Dubois, L., Merrill-Skoloff, G., Furie, B., Furie, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+VI-dependent+and+-independent+pathways+of+thrombus+formation+in+vivo.">Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo.</a> Blood. 107 (10), 3902-3906 (2006).</li><li>Navarrete, A. -. M., Casari, 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=A+murine+model+to+characterize+the+antithrombotic+effect+of+molecules+targeting+human+von+Willebrand+factor.">A murine model to characterize the antithrombotic effect of molecules targeting human von Willebrand factor.</a> Blood. 120 (13), 2723-2732 (2012).</li><li>Rayes, J., Hollestelle, 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=Mutation+and+ADAMTS13-dependent+modulation+of+disease+severity+in+a+mouse+model+for+von+Willebrand+disease+type+2B.">Mutation and ADAMTS13-dependent modulation of disease severity in a mouse model for von Willebrand disease type 2B.</a> Blood. 115 (23), 4870-4877 (2010).</li><li>Lamrani, L., Lacout, 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=Hemostatic+disorders+in+a+JAK2V617F-driven+mouse+model+of+myeloproliferative+neoplasm.">Hemostatic disorders in a JAK2V617F-driven mouse model of myeloproliferative neoplasm.</a> Blood. 124 (7), 1136-1145 (2014).</li><li>Zhang, Y., Ye, 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=Increased+platelet+activation+and+thrombosis+in+transgenic+mice+expressing+constitutively+active+P2Y12.">Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12.</a> J Thromb Haemost. 10 (10), 2149-2157 (2012).</li><li>Sch&#228;fer, K., Konstantinides, S., 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=Different+mechanisms+of+increased+luminal+stenosis+after+arterial+injury+in+mice+deficient+for+urokinase-+or+tissue-type+plasminogen+activator.">Different mechanisms of increased luminal stenosis after arterial injury in mice deficient for urokinase- or tissue-type plasminogen activator.</a> Circulation. 106 (14), 1847-1852 (2002).</li><li>Wang, X., Palasubramaniam, 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=Towards+effective+and+safe+thrombolysis+and+thromboprophylaxis:+preclinical+testing+of+a+novel+antibody-targeted+recombinant+plasminogen+activator+directed+against+activated+platelets.">Towards effective and safe thrombolysis and thromboprophylaxis: preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets.</a> Circ Res. 114 (7), 1083-1093 (2014).</li><li>Decrem, Y., 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=Ir-CPI,+a+coagulation+contact+phase+inhibitor+from+the+tick+Ixodes+ricinus,+inhibits+thrombus+formation+without+impairing+hemostasis.">Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis.</a> J Exp Med. 206 (11), 2381-2395 (2009).</li><li>Ma, D., 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=Desmolaris,+a+novel+factor+XIa+anticoagulant+from+the+salivary+gland+of+the+vampire+bat+(Desmodus+rotundus)+inhibits+inflammation+and+thrombosis+in+vivo.">Desmolaris, a novel factor XIa anticoagulant from the salivary gland of the vampire bat (Desmodus rotundus) inhibits inflammation and thrombosis in vivo.</a> Blood. 122 (25), 4094-4106 (2013).</li><li>Lei, X., 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=Anfibatide,+a+novel+GPIb+complex+antagonist,+inhibits+platelet+adhesion+and+thrombus+formation+in+vitro+and+in+vivo+in+murine+models+of+thrombosis.">Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis.</a> Thromb Haemost. 111 (2), 279-289 (2014).</li><li>Waisberg, M., 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=Plasmodium+falciparum+infection+induces+expression+of+a+mosquito+salivary+protein+(Agaphelin)+that+targets+neutrophil+function+and+inhibits+thrombosis+without+impairing+hemostasis.">Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis.</a> PLoS Pathog. 10 (9), e1004338 (2014).</li><li>Owens, A. P., Lu, Y., Whinna, H. C., Gachet, C., Fay, W. P., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+standardization+of+the+murine+ferric+chloride-induced+carotid+arterial+thrombosis+model.">Towards a standardization of the murine ferric chloride-induced carotid arterial thrombosis model.</a> J Thromb Haemost. 9 (9), 1862-1863 (2011).</li><li>Wang, X., Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+murine+model+of+ferric+chloride-induced+arterial+thrombosis+for+thrombosis+research.">An optimized murine model of ferric chloride-induced arterial thrombosis for thrombosis research.</a> Thromb Res. 115 (1-2), 95-100 (2005).</li><li>Tseng, M. T., Dozier, A., Haribabu, B., Graham, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transendothelial+migration+of+ferric+ion+in+FeCl3+injured+murine+common+carotid+artery.">Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery.</a> Thromb Res. 118 (2), 275-280 (2006).</li><li>Bonnard, T., 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=Leukocyte+mimetic+polysaccharide+microparticles+tracked+in+vivo+on+activated+endothelium+and+in+abdominal+aortic+aneurysm.">Leukocyte mimetic polysaccharide microparticles tracked in vivo on activated endothelium and in abdominal aortic aneurysm.</a> Acta Biomater. 10 (8), 3535-3545 (2014).</li><li>Boulaftali, Y., Lamrani, L., 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=The+mouse+dorsal+skinfold+chamber+as+a+model+for+the+study+of+thrombolysis+by+intravital+microscopy.">The mouse dorsal skinfold chamber as a model for the study of thrombolysis by intravital microscopy.</a> Thromb Haemost. 107 (5), 962-971 (2012).</li><li>Konstantinides, S., Sch&#228;fer, K., Thinnes, T., Loskutoff, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+and+its+cofactor+vitronectin+stabilize+arterial+thrombi+after+vascular+injury+in+mice.">Plasminogen activator inhibitor-1 and its cofactor vitronectin stabilize arterial thrombi after vascular injury in mice.</a> Circulation. 103 (4), 576-583 (2001).</li><li>Li, W., McIntyre, T. M., Silverstein, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+murine+carotid+arterial+injury:+A+model+of+redox+pathology.">Ferric chloride-induced murine carotid arterial injury: A model of redox pathology.</a> Redox Biol. 1 (1), 50-55 (2013).</li></ol>

The authors have nothing to disclose.

O modelo de trombose cloreto férrico induzida é uma excelente ferramenta de pesquisa. Como se mostra neste estudo, é extremamente fácil de implementar e, quando utilizado em combinação com a microscopia intravital ou medidor de fluxo de Doppler, que fornece uma boa monitorização em tempo real de formação de trombos. Ajustando o tempo de exposição e a concentração do FeCl 3, como também oferece a possibilidade de produzir ou trombos oclusivos ou não-oclusivos. No en...

O estudo dos mecanismos envolvidos no desenvolvimento de trombose e a avaliação da eficácia de fármacos anti-trombóticos bem estabelecida requer modelos animais experimentais. Grandes modelos animais foram os primeiros a ser utilizado uma vez que proporcionam grandes vasos mais semelhantes aos seres humanos do que roedores 1. No entanto, de alto custo, as instalações maiores necessários e da dificuldade em manipulá-las geneticamente são os principais inconvenientes para a sua utilização e animais de grande porte são agora limitados a estudos pré-clínicos final uma vez ensaios preliminares em roedores têm dado resultados conclusivos 2. Com a disponibilidade de largura de estirpes transgénicas e knockout e seu pequeno tamanho que minimiza a quantidade de drogas antitrombóticas necessárias para os ensaios in vivo, os ratinhos são utilizados principalmente para a investigação trombose. Portanto, vários modelos de doenças trombóticas tem sido desenvolvido em murganhos 3. Muitos modelos de trombose estabelecidos perturbar os intimuma camada da parede do vaso, seguido pela exposição da matriz extracelular sub-endotelial para o fluxo sanguíneo induzindo a formação de coágulos sanguíneos 4. Os trombos podem resultar da exposição de colagénio, que provoca activação de plaquetas e / ou a partir da exposição do factor tecidual que activa a cascata de coagulação 5. Várias técnicas são então utilizados para realizar a lesão inicial navio. Pierangeli et ai. Desenvolveram um modelo de ruptura mecânica com uma ferramenta de microcirurgia na veia femoral 6. Kikushi et ai. Descreveu um modelo que consiste na administração de um composto reactivo foto (Rosa de Bengala) que se acumula na bicamada lipídica de células endoteliais, seguido por excitação específica da parede do vaso de interesse com luz verde (540 nm) 7. A lesão, também pode ser induzida por uma alta intensidade de iluminação curto pulso de laser 8. Outra técnica, em primeiro lugar estabelecido na artéria carótida de ratosconsiste na aplicação tópica de cloreto férrico (FeCl3) 9. Neste caso, os resultados denudação vaso de radicais livres gerados por FeCl 3, que faz com que a peroxidação lipídica e destruição de células endoteliais 10. A lesão induz a expressão de várias moléculas de adesão que desencadeiam a adesão e agregação de plaquetas, bem como o recrutamento de leucócitos. Demonstrou-se que os leucócitos, principalmente os neutrófilos, desempenham um papel crucial na activação da cascata de coagulação do sangue levando a trombose 11. Este método está bem adaptado para reproduzir a cascata de coagulação; os investigadores devem ter em mente que, neste modelo de ratinho, a trombose é tipicamente induzido em vasos saudáveis ​​enquanto a trombose em seres humanos está ocorrendo principalmente na ex doente. vasos ateroscleróticos. Como este modelo é muito simples de implementar e é também eficaz em camundongos, é agora o modo de trombose usado na maior partel para pequenos animais em estudos in vivo. Além disso, esta técnica oferece a possibilidade de induzir a formação de trombos numa variedade de vasos. Navios-alvo pode ser artérias ou veias de grande diâmetro (carótida, femoral, veia cava) ou de pequeno diâmetro (mesentério, cremaster) 12-14. Mais recentemente, foi também utilizado na artéria cerebral média proximal para desenvolver um modelo de acidente vascular cerebral 15. A formação da trombose pode ser directamente observada por meio de microscopia intravital após marcação fluorescente de plaquetas e leucócitos ou monitorizada medindo a diminuição do fluxo sanguíneo com uma sonda de temperatura ou uma sonda Doppler 12,16,17. Vários parâmetros, tais como tempo de oclusão, o tempo de formação de trombos ou tamanho do trombo pode então ser investigados. As diferenças fisiológicas entre os vasos investigado resultado em variações significativas na trombos obtido. Portanto, os pesquisadores geralmente selecionar o vaso-alvo de acordo com os parâmetros que eles querem measure e / ou a configuração que deseja investigar a doença. Normalmente, o modelo na artéria carótida é mais relevante para a pesquisa sobre aterotrombose relacionada ao infarto do miocárdio ou acidente vascular cerebral enquanto que estudos sobre a veia cava são mais relevantes para a pesquisa sobre trombose venosa profunda. A acessibilidade dos diferentes vasos também determina o método utilizado para medir o crescimento do trombo. Por exemplo, os vasos mesentéricos são de fácil acesso tornando este modelo adequado para observação microscópica intravital e o estudo da dinâmica de formação de trombos. A artéria carótida é menos acessível, mas maior, permitindo medições de fluxo de sangue e fornecer um excelente modelo para estudar a trombose oclusiva. O modelo de trombose induzida cloreto férrico tem prestado um enorme progresso na compreensão desta patologia. Ele tem sido usado em muitos estudos que incidem sobre o papel do factor de von Willebrand na formação da trombose 18,19. Combinado com modi genéticatécnicas ficação, permitiu a identificação de muitos genes específicos envolvidos em doenças trombóticas. Lamrani et ai. por exemplo, demonstraram que um knock-in do gene JAK2 V617F está associada com uma formação acelerada de coágulo instável 20. Zhang et ai. Investigaram a implicação fisiológica do receptor P2Y12 de plaquetas e demonstraram que os ratinhos transgénicos que sobre-expressam este receptor especificamente em plaquetas somente, apresentou uma formação de trombo mais rápida e estável na artéria mesentérica feridos com FeCl 3 21. O papel crucial de activador do plasminogénio tipo tecido (tPA) e uroquinase-tipo do activador de plasminogénio (uPA) no processo de degradação de fibrina, também tem sido investigada neste método 22. Além disso, este modelo também fornece um modo simples e preciso de testar as capacidades fibrinolíticas de muitas drogas novas in vivo. Por exemplo, Wang et al. Usaram este modelo de the validação pré-clínica de um romance ativador do plasminogênio recombinante dirigido contra plaquetas ativadas 23. Este método também permitiu a validação de proteínas terapêuticas isoladas a partir da saliva de morcegos vampiros, carrapatos e mosquitos, ou a partir do veneno de serpentes de identificação específico do alvo 24-27. Estes exemplos demonstram a versatilidade do modelo de cloreto férrico. Neste artigo, vamos nos concentrar em dois métodos e estudar cloreto férrico trombose induzida em dois tipo de navio diferente; vaso mesentérico e artéria carótida.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Whatman chromatography paper</td>
			<td>GE Healthcare</td>
			<td>3030917</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (III) chloride 40 % (w/v)</td>
			<td>VWR</td>
			<td>24212.298</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma</td>
			<td>R4127</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital black-and-white camera&#160;</td>
			<td>Olympus</td>
			<td>XM10</td>
			<td></td>
		</tr>
		<tr>
			<td>Doppler flowmeter</td>
			<td>Transonic</td>
			<td>TS420</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano-doppler flow probe</td>
			<td>Transonic</td>
			<td>0.5 PBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira&#160;</td>
			<td>0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rampun)</td>
			<td>Bayer</td>
			<td>75313&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1472</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (29 G)</td>
			<td>BD Ultra-Fine</td>
			<td>326103</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>BSN medical</td>
			<td>211827A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynek dysilk sutures</td>
			<td>Dynek Pty Ltd</td>
			<td>CS30100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline (PBS)</td>
			<td>Gibco life technologies</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Kirchner</td>
			<td>T60</td>
			<td></td>
		</tr>
	</tbody>
</table>

ferric chloride-induced thrombosis mouse model on carotid artery and mesentery vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

A observação de microscopia intravital fluorescente do mesentério irá revelar a acumulação de Rodamina 6G marcado plaquetas e leucócitos ao longo da parede do vaso lesionado por FeCl 3. A formação progressiva de um trombo é monitorizado parcial num recipiente de 200 um mesentério (Figura 1). Um trombo aparece lentamente e é claramente identificável após o primeiro minuto de exposição à FeCl 3 (Figura 1, t = 60 segundos). 40 segundos após a remoç?...

Watch this Scientific Journal Video about Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel at JoVE.com

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Férrico Thrombosis Modelo rato na artéria carótida e Mesentery Vessel induzida por Chloride

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

ferric-chloride-induced-thrombosis-mouse-model-on-carotid-artery

Research

JoVE Journal

Medicine

17.4K visualizações.  Baker IDI Heart and Diabetes Institute. The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Vídeo: Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Um modelo de Disturbed Aterosclerose Flow-induzido em rato artéria carótida pela ligadura parcial e um método simples de isolamento de RNA a partir do endotélio Carótidas

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Medicina

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Modelo do mouse completa de Stasis Inferior Trombose da veia cava

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

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 ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Um modelo murino de implante de stent na artéria carótida para o Estudo da reestenose

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Adulto Rato venoso Hipertensão Modelo: artéria carótida comum para External Jugular Vein anastomose.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Lesão Balloon Vascular e Administração Intraluminal em Rat Carótida

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

A Rat carótida Balão Injury modelo para testar Therapeutics remodela Anti-vasculares

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Férricos induzida pelo cloreto modelos murinos Trombose

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Imagens de ressonância magnética fase contraste na artéria carótida comum de rato

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...