Name: the rabbit model of accelerated atherosclerosis: a methodological perspective of the iliac artery balloon injury
Uploaded: 2017-10-03T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury at JoVE.com

Este trabalho foi apoiado pela Suíça nacional Science Foundation Grant 150271.

este protocolo experimental foi aprovado pelo veterinário Cantonal, Friburgo e o suíço Federal veterinária Office, Switzerland (FR-2015/58).
 Nota: foram utilizados coelhos NZW masculino com peso entre 2,8 a 3,2 kg. Os animais estavam alojados em condições convencionais (12 h luz e ciclo escuro, fornecida comida e água ad libitum). Antes de desnudamento do balão, os animais foram aclimatados para 1 semana, durante o qual eles foram alimentados com dieta de comida normal. Após 1 semana de aclimatação, coelhos foram trocados para aterogênico dieta consistindo de alto teor de gordura (8,6%) e ácidos graxos saturados com dieta rica em colesterol (1%) 205 mg/kg para a duração inteira de estudo. Lesão de balão na artéria ilíaca esquerda foi realizada 1 semana após o início da dieta e os animais foram sacrificados após 2 semanas ou 4 semanas de lesão balão.
 1. procedimentos pré-operatórios <ol><li>esterilizar todos os instrumentos cirúrgicos antes do uso com um esterilizador de grânulo de vidro ou outro instrumento apropriado.</li>
	<li>Preparar e verificar o conjunto de cateter de balão.
	<ol><li>Anexar um conector luer-lock 1 mL seringa preenchida com solução salina normal para a parte do conector luer-lock do cateter balão. Monitorize cuidadosamente a ausência de ar. Verifique os vazamentos e garantir a inflação do balão apropriado pressionando o êmbolo da seringa.</li>
	</ol></li> <li>Pesar o coelho e ligar o thermopad a 37 ° C.</li>
	<li>Usar uma solução de buprenorfina em uma concentração de 0,3 mg / ml. injetar uma dose de 0,01 mg/kg por via subcutânea.</li>
	<li>ANESTHETIZE o coelho com 5% de isoflurano e 5 L/min O 2 em uma câmara de indução de 10-15 min.</li>
	<li>Coloque o coelho anestesiado sobre a almofada de aquecimento, mantida na plataforma cirúrgica. Coloque o patch e clipes para monitorar a temperatura, respiração e eletrocardiograma.</li>
	<li>Anexar o focinho do coelho para uma máscara facial conectada a uma máquina de anestesia adequada. Manter a anestesia com isoflurano (4,0% com 2,5 L/min O 2). Confirmar anesthetization adequada (indicado por falta de tônus muscular e perda de reflexos de mordaça e transformam).</li>
	<li>Aplicar a pomada oftálmica de ambos os olhos para impedir que as córneas de secagem. Cubra o coelho com uma folha cirúrgica estéril com apenas o membro inferior exposto.</li>
</ol> 2. Protocolo cirúrgico <ol><li>remover o cabelo da zona ventral logo abaixo as articulações de joelho, usando a máquina de cortar cabelo animal.</li>
	<li>Limpe a área com desinfetante apropriado para limpar a pele e remover pelos soltos.</li>
	<li>Localizar a artéria safena e faça uma incisão de pele pequena de cerca de 1,5 cm de comprimento com um bisturi.</li>
	<li>Expor uma pequena porção da artéria safena com pequenas pinças curvas sem danificar a veia femoral e o nervo femoral.</li>
	<li>Coloque dois loops de ligadura solto (seda 5-0) sob a artéria safena e amarre um laço de ligadura em direção a extremidade distal da artéria. Coloque uma pinça microvascular acima a ligadura para parar o fluxo de sangue da artéria ilíaca.</li>
	<li>Aplicar topicamente uma gota de papaverina para dilatar a artéria e prevenir vasoespasmo.</li>
	<li>Levante a artéria safena com a ajuda da ligadura amarrada e fazem uma incisão pequena arteriotomia usando um 24 agulha de calibre.</li>
	<li>Elevar as abas da incisão com pinça fina e inserir lentamente uma veia picareta ou uma guia de agulha no lúmen da artéria.</li>
	<li>Inserir um cateter de embolectomia arterial 2 francês Fogarty a artéria safena. Remover os grampos microvasculares e escolher veia.</li>
	<li>Avançar o cateter até a marca de sexto (20-25 cm) corresponde a uma posição de aproximadamente 2 a 5 cm acima da bifurcação ilíaca.</li>
	<li>Insuflar o balão com soro fisiológico normal de 0,1 mL utilizando uma seringa de 1 mL ou a uma pressão nominal de 6 atm usando um compressor de ar manual regulamentado conforme descrito em 16 , 22.</li>
	<li>Mantenha o cateter de balão com fórceps e arraste volta por 6cm através da ilíaca arterytoward o ponto de inserção, enquanto girar o cateter.</li>
	<li>Desinsuflar o balão puxando o êmbolo da seringa.</li>
	<li>Repita os passos 2.10 a 2.13 três vezes para garantir o completo desnudamento endotelial.</li>
	<li>Remover o cateter e imediatamente amarrar o laço de ligadura logo acima o site arteriotomia para parar o sangramento.</li>
	<li>Aplicar todo anti-séptico adequado em torno da periferia da ferida e limpar afastado os coágulos de sangue. Fechar a incisão na pele com uma sutura de 5-0 e desinfectar o local da cirurgia com solução de iodo-povidona.</li>
	<li>Repetir passos 2.1 a 2.16 sobre o contralateral ilíaca utilizando um cateter novo.</li>
	<li>a pomada oftálmica de olhos um esfregaço.</li>
</ol> 3. Cuidados pós-operatórios <ol><li>administrar sulfadoxine 40 mg/kg e trimetoprim 8 mg/kg ou a qualquer outro antibiótico adequado imediatamente após o procedimento cirúrgico.</li>
	<li>Durante o período de recuperação de anestesia, manter o coelho sobre uma almofada de calor colocada em uma gaiola limpa autoclavada.</li>
	<li>Remover o patch monitoramento e clipes.</li>
	<li>Após a recuperação, retornar os coelhos para jaulas em casa. Injectar por via subcutânea buprenorfina 0,05 - 0,1 mg/kg pós - operatório cada 6-12 h por 48 h. continuar aterogênico dieta por mais duas semanas ou quatro semanas.</li>
</ol> 4. Colheita de tecido e análise da composição da placa <ol><li>depois de duas semanas (para cedo placa fina) ou três semanas de lesão de balão, anestesiar o coelho utilizando isoflurano em uma maneira similar como descrito acima.</li>
	<li>Abrir a cavidade torácica e eutanásia os coelhos por exsanguinação intracardíaca.</li>
	<li>Isolar as artérias ilíacas, conforme descrito em 23.
	<ol><li>Brevemente, abrir o abdômen e expor o retroperitônio. Trace a aorta em direção a bifurcação ilíaca e amarrá-la acima da bifurcação. Retire com cuidado os tecidos circunvizinhos para expor e isolar ambas as artérias ilíacas.</li>
	</ol></li> <li>Dissecar fora de ambas as artérias ilíacas e mergulhe-os em solução salina gelada tamponada de fosfato. Remova os coágulos com a ajuda de pinças. Divida cada artéria ilíaca em 4-6 segmentos para caracterizar a espessura da placa ao longo da artéria.</li>
	<li>Imediatamente incorporar os segmentos arteriais em um molde contendo a temperatura ideal de corte composta, snap-congelamento com nitrogênio líquido e mantê-lo em-70 ° C. preparar 5 µm espessura seções usando um criostato, conforme descrito em 24.</li>
	<li>Executar histologia, imunofluorescência ou imuno-histoquímica mancha para morfometria, placa lipido e conteúdo celular conforme descrito em 10 , 25. 
	Nota: Brevemente, enxágue as seções arteriais com solução salina tamponada fosfato (PBS) e permeabilize usando 0,2% Triton. Enxagúe as seções com PBS e bloquear sites não específicas com albumina de soro bovino 2% por 30 min. Incubar as secções para 1 h a 37 ° C com anti actina α-SM (1: 200) ou anticorpo RAM11 (1: 200). Enxagúe as seções com PBS e incube-os com anticorpo secundário apropriado para 30 min a 37 ° C. Lave novamente com PBS e adicionar Hoechst (5 µ g/mL) por 10 min detectar núcleos.</li>
</ol>

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A., Mosse, P. R., Safstrom, J. A., Young, S. B., Campbell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon+catheter+injury+to+rabbit+carotid+artery.+I.+Changes+in+smooth+muscle+phenotype.">Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype.</a> Arteriosclerosis. 9, 289-298 (1989).</li><li>Miyake, 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=Prevention+of+neointimal+formation+after+angioplasty+using+nuclear+factor-kappaB+decoy+oligodeoxynucleotide-coated+balloon+catheter+in+rabbit+model.">Prevention of neointimal formation after angioplasty using nuclear factor-kappaB decoy oligodeoxynucleotide-coated balloon catheter in rabbit model.</a> Circ Cardiovasc Interv. 7, 787-796 (2014).</li><li>Fulcher, J., Patel, S., Nicholls, S. J., Bao, S., Celermajer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+serial+in+vivo+imaging+of+aortic+plaque+in+the+rabbit:+a+preliminary+experience.">Optical coherence tomography for serial in vivo imaging of aortic plaque in the rabbit: a preliminary experience.</a> Open Heart. 2, e000314 (2015).</li><li>Abela, O. 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=Plaque+Rupture+and+Thrombosis:+the+Value+of+the+Atherosclerotic+Rabbit+Model+in+Defining+the+Mechanism.">Plaque Rupture and Thrombosis: the Value of the Atherosclerotic Rabbit Model in Defining the Mechanism.</a> Curr Atheroscler Rep. 18, 29 (2016).</li><li>Yamashita, A., Asada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rabbit+model+of+thrombosis+on+atherosclerotic+lesions.">A rabbit model of thrombosis on atherosclerotic lesions.</a> J Biomed Biotechnol. 2011, 424929 (2011).</li></ol>

Os autores declaram não concorrentes interesses financeiros.

O modelo de aterosclerose de artéria ilíaca coelho é amplamente utilizado na investigação de aterosclerose. Com este protocolo os coelhos rapidamente desenvolveram placas mais avançadas e graves em comparação com lesões espontâneas desenvolvidas com apenas dieta rica em colesterol. Importante, os animais se recuperar rapidamente da cirurgia.
O principal estímulo para atherogenesis é os danos mecânicos causados pelo cateter de balão que fere o endotélio e distende o vaso parede<s...

Ruptura das placas ateroscleróticas vulneráveis é uma das principais causas de morte no nações industrializadas1. Embora pesquisas nas últimas décadas se desenrolou vários mecanismos moleculares e celulares envolvidos na progressão da placa, continuou ainda são necessários esforços não só para desvendar o complexo mecanismo de progressão da doença, mas também para testar novas terapêuticas aproxima-se. Vários modelos animais têm sido propostos para estudar a aterosclerose. Manipulação genética, ferimentos de endotélio de alimentação ou mecânica de colesterol são as estratégias padrão compartilhadas por modelos mais animais da aterosclerose, incluindo ratos, coelhos ou miniporcos. Entre estes, coelhos NZW são sensíveis à dieta rica em colesterol, enquanto ratos e ratos normais não absorvem significativamente colesterol dietético2,3,4. Coelhos, espontaneamente, desenvolver lesões da aorta ricas em macrófagos com algum componente fibroso quando alimentados com colesterol dieta rica5,6. No entanto, o tempo tempo preparatório de 4-8 meses para induzir aterosclerótica plaquesby alimentação colesterol dieta sozinho6,7 é uma grande desvantagem para a maioria das configurações de experimentais. Na busca para induzir lesões em tempo relativamente curto, uma combinação de colesterol alto dieta e balão prejuízo foi desenvolvida pela Baumgarter e Studer8. O objetivo geral desta técnica é induzir o ateroma composta de células de espuma (semelhantes a raia gorda em seres humanos) em coelhos hipercolesterolêmicos dentro de 2 semanas. A presente técnica descreve o procedimento de ferimento de parede arterial com base no método do Baumgarter utilizando um cateter balão avançado nas artérias ilíacas de coelhos hipercolesterolêmicos NZW.
Em conjunto com uma dieta rica de colesterol, danos que resultem de endotelização balão induzida vão levar a aterosclerose. Lesão de balão acelera a formação de lesões ateroscleróticas e produz placa de tamanho uniforme e distribuição. Espessamento da íntima aumenta ao longo de um período de tempo e célula da íntima infiltração começa dentro de alguns dias após lesão. Listras gordas com macrófagos substanciais começam a aparecer após 7-10 dias de lesão de balão e são representadas como lesão tipo II, de acordo com a classificação pela American Heart Association. Lesão de balão em coelho é frequentemente realizado na aorta para estudar a composição da placa bacteriana. O endotélio neointimal expressa altos níveis da molécula de adesão intercelular. As placas estão associadas com dissecção medial e alterações adventícia. Lesões ateroscleróticas são compostas de lipídios, proliferação células musculares lisas (SMCs), fibras colágenas e células inflamatórias que se acumulam sob o endotélio regenerado e são principalmente tipo II na natureza. A distribuição topológica de placas coelho foi semelhante à relatada na aorta humana 9,10 , em princípio, a aorta é maior no tamanho em comparação com as artérias ilíacas e produziria placa de maior comprimento. No entanto, a grande vantagem de usar a artéria ilíaca como o site da aterosclerose em coelhos é sua acessibilidade, sua similaridade no conteúdo muscular de coronária humana11, lesão uniforme desenvolvimento12, fator tecidual elevada atividade13 e consistente navio dimensão comparável ao humana artéria coronária, permitindo a avaliação dos dispositivos fabricados comercialmente morfométricos e angiográficos pontos de extremidade. Métodos invasivos e não-invasivos têm sido investigados para analisar as placas nas artérias ilíacas de coelho no animal vivo. Relatórios anteriores descrevem o uso de ressonância magnética (MRI) com a ajuda de um senhor de 2,35-tesla sistema 14 além disso, o ultra-som intravascular (USIV) ou cateteres de coerência óptica (OCT) a tomografia podem ser devidamente aplicado à imagem ateroma nas artérias ilíacas coelho. A artéria ilíaca é acessível por ultra-sonografia quando usando uma ecografia de alta resolução e a aorta também pode ser explorada com esta técnica.
Na última década, este modelo de coelho de lesão de balão tem ajudado a entender melhor os mecanismos de progressão de placa15e regressão de placa16. Além disso, o modelo tem sido usado para estudar a influência de novos agentes terapêuticos como estatinas, agentes antiplaquetários padrão, antioxidante agentes17,18 e stents farmacológicos tais como everolimus ou eluidor de Zotarolimus stent19,20 em espessamento neointimal. Este modelo também serviu para investigar a imagem intravascular de fluorescência de infravermelho próximo da imagem latente do cateter21.

<table><tbody><tr><td>New Zealand White rabbits</td><td>Charles River laboratories,France</td><td>Cre:KBL(NZW)</td><td></td></tr><tr><td>Cholesterol rich diet</td><td>Ssniff spezialdi&amp;auml;ten</td><td>Ssniff EF K High Fat and Cholesterol</td><td></td></tr><tr><td>Glass bead sterilizer-Germinator 500</td><td>VWR, Leicestershire, UK</td><td>101326-488</td><td></td></tr><tr><td>Fogarty balloon embolectomy catheters, 2 French</td><td>Edwards Lifesciences, Switzerland</td><td>120602F</td><td>For single use only</td></tr><tr><td>Luer Lock Syringe</td><td>Becton, Dickinson and Company, USA</td><td>309628</td><td></td></tr><tr><td>Thermopad Type 226</td><td>Solis, Switzerland AG</td><td>397387</td><td></td></tr><tr><td>Buprenorphine- Temgesic</td><td>Reckitt Benckiser AG, Switzerland</td><td>7.68042E+12</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care, Inc, Bethlehem, PA 18017</td><td>2667-46-7</td><td></td></tr><tr><td>Anaesthesia machine-combi-vet Base Anesthesia System</td><td>Rothacher Medical GmbH, Switzerland</td><td>CV 30-301-A</td><td></td></tr><tr><td>Cardell touch veterinary vital signs monitor</td><td>Midmark, Ohio, USA</td><td>8013-001</td><td></td></tr><tr><td>Ophthalmic ointment-Humigel</td><td>Virbac, France</td><td></td><td></td></tr><tr><td>Animal hair clippers</td><td>Aesculap AG, Germany</td><td>GT420</td><td></td></tr><tr><td>Disinfectant-Betadine solution</td><td>MundipharmaMedicalCompany, Switzerland</td><td>14671-1203</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>FST Germany</td><td>11274-20</td><td></td></tr><tr><td>Medium and small microscissors</td><td>Medline International Switzerland S&amp;agrave;rl</td><td>UC4337</td><td></td></tr><tr><td>Microvascular clamps</td><td>FST, Germany</td><td>18051-28</td><td></td></tr><tr><td>Papaverine</td><td>ESCA chemicals, Switzerland</td><td>RE 356 803</td><td></td></tr><tr><td>Vein Pick</td><td>Harvard Apparatus, Cambridge, UK</td><td>72-4169</td><td>For single use only</td></tr><tr><td>Saline</td><td>Laboratorium Dr. G. Bichsel AG, , Switzerland</td><td>1330055</td><td></td></tr><tr><td>Polysorb 5-0 suture</td><td>Covidien AG, Switzerland</td><td>UL 202</td><td>Monofilament</td></tr><tr><td>Sulfadoxine and Trimethoprim-Trimethazol</td><td>Werner Stricker AG, Switzerland</td><td>Swissmedic Nr. 50&#039;361</td><td></td></tr><tr><td>Antiseptic- Octenisept</td><td>Sch&amp;uuml;lke &amp;amp; Mayr AG, Switzerland</td><td>GTIN: 4032651214068</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Roth</td><td>1058.1</td><td></td></tr><tr><td>Isobutanol-2-Methylbutane</td><td>Sigma-Aldrich, Switzerland</td><td>M32631-1L</td><td></td></tr><tr><td>Optimum Cutting Temperature compound-Tissue-Tek</td><td>VWR Chemicals, Belgium</td><td>25608-930</td><td></td></tr><tr><td>Cryostat</td><td>Leica, Glattbrugg, Switzerland</td><td>Leica CM1860 UV</td><td></td></tr><tr><td>Glass slide- Superfrost Plus</td><td>Thermo Scientific</td><td>4951PLUS4</td><td></td></tr><tr><td>Mayer&#039;s Haematoxylin</td><td>Sigma-Aldrich, Switzerland</td><td>MHS32-1L</td><td></td></tr><tr><td>Eosin 0.5% aq.</td><td>Sigma-Aldrich, Switzerland</td><td>HT110232-1L</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich, Switzerland</td><td>O0625-25G</td><td></td></tr><tr><td>&amp;alpha;-smooth muscle actin antibody</td><td>Abcam, UK.</td><td>ab7817</td><td></td></tr><tr><td>Macrophage Clone RAM11 antibody</td><td>DAKO, Switzerland</td><td>M063301</td><td></td></tr><tr><td>Hoechst</td><td>Abcam, UK.</td><td>ab145596</td><td></td></tr><tr><td>Goat polyclonal Secondary Antibody (Chromeo 546)</td><td>Abcam, UK.</td><td>ab60316</td><td></td></tr><tr><td>Alexa Fluor 488/547</td><td>Abcam, UK.</td><td></td><td></td></tr><tr><td>Glycergel Mounting Medium, Aqueous</td><td>DAKO, Switzerland</td><td>C056330</td><td></td></tr><tr><td>Hematoxylin for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>H3136-25G</td><td></td></tr><tr><td>Ferric chloride for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>157740-100G</td><td></td></tr><tr><td>Iodine for Movat staining</td><td>Sigma-Aldrich, Switzerland</td><td>207772-100G</td><td></td></tr><tr><td>Potassium iodide for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>60400-100G-F</td><td></td></tr><tr><td>Alcian blue for Movat staining</td><td>Sigma-Aldrich, Switzerland</td><td>A5268-10G</td><td></td></tr><tr><td>Strong Ammonia for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>320145-500ML</td><td></td></tr><tr><td>Brilliant crocein MOO for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>210757-50G</td><td></td></tr><tr><td>Acid Fuchsin for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>F8129-50G</td><td></td></tr><tr><td>Sodium Thiosulfate for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>72049-250G</td><td></td></tr><tr><td>Phosphotungstic acid for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>79690-100G</td><td></td></tr><tr><td>Crocin for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>17304-5G</td><td></td></tr><tr><td>EUKITT for Movat pentachrome staining</td><td>Sigma-Aldrich, Switzerland</td><td>03989-100ML</td><td></td></tr></tbody></table>

the rabbit model of accelerated atherosclerosis: a methodological perspective of the iliac artery balloon injury

Síndrome Coronariana Aguda resultante da oclusão coronária após ruptura e desenvolvimento da placa aterosclerótica é a principal causa de morte no mundo industrializado. Coelhos Nova Zelândia branco (NZW) são amplamente utilizados como modelo animal para o estudo da aterosclerose. Eles desenvolvem lesões espontâneas quando alimentados com dieta aterogênico; no entanto, isso requer tempo de 4 a 8 meses. Para mais, melhorar e acelerar o atherogenesis, uma combinação de dieta aterogênico e lesão endotelial mecânica é frequentemente empregada. O procedimento apresentado para indução de ateroma em coelhos usa um cateter balão para perturbar o endotélio na artéria ilíaca esquerda de coelhos NZW alimentados com dieta aterogênico. Tais danos mecânicos causados por cateter balão induz uma cadeia de reações inflamatórias, iniciando o acúmulo de lipídios neointimal em uma forma dependente do tempo. Placa aterosclerótica seguindo balão lesão mostrar neointimal aumento do volume com lipídios extensa infiltração, conteúdo de célula de alta de músculo liso e presença de macrófagos derivados de células de espuma. Esta técnica é simples, Reproduzível e produz placa de comprimento controlada dentro da artéria ilíaca. Todo o processo é concluído no prazo de 20-30 min. O procedimento é seguro, com baixa mortalidade e também oferece elevado sucesso na obtenção de lesões da íntima substanciais. O procedimento do cateter balão induzida resulta de lesão arterial em aterosclerose dentro de duas semanas. Este modelo pode ser usado para investigar a patologia da doença, diagnóstico por imagem e avaliar novas estratégias terapêuticas.

Lesão de balão da artéria ilíaca foi realizada com sucesso sem complicação (Figura 1). O tempo operacional total variou de 20 a 30 min para lesões executada apenas uma artéria ilíaca e 35 a 45 min para lesões em ambas as artérias. O coelho recuperado dentro de 1h após a lesão de balão. Todos os animais apareceram saudáveis sem perda significativa de peso. Sem infecção, edema ou trombose arterial foi encontrada. A área da ferida foi normal, a...

Watch this Scientific Journal Video about The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury at JoVE.com

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Modelos animais de aterosclerose são essenciais para entender o mecanismo e a investigar novos enfoques para evitar o desenvolvimento de placa ou ruptura, das principais causas de morte no mundo industrializado. Este protocolo utiliza uma combinação de lesão de balão e colesterol dieta rica para induzir ateroma na artéria ilíaca coelho.

O modelo de coelho da aterosclerose acelerada: uma perspectiva metodológica da artéria ilíaca balão lesão

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

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Research

JoVE Journal

Medicine

12.0K visualizações.  University of Fribourg. Modelos animais de aterosclerose são essenciais para entender o mecanismo e a investigar novos enfoques para evitar o desenvolvimento de placa ou ruptura, das principais causas de morte no mundo industrializado. Este protocolo utiliza uma combinação de lesão de balão e colesterol dieta rica para induzir ateroma na artéria ilíaca coelho.

Vídeo: The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

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

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared fluorescência de imagem intravascular (NIRF) Molecular da placa inflamatória, uma Abordagem Multimodal para imagem da Aterosclerose

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Induzir miointimais Hiperplasia Versus aterosclerose em camundongos: uma introdução de dois modelos válidos

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Reprodutível Arterial Desnudamento Lesão por aorta abdominal infrarrenal fixação num modelo murino

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Lesão baseada no balão para induzir a hiperplasia Myointimal da Aorta Abdominal de Mouse

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Uma lesão de balão segmental controlada por pressão da artéria de rato com aplicação terapêutica periadventicional

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of access to reliable sources of diseased human aortic valves. Among the various animal models, AVS rabbit models are one of the most commonly used in large animal studies. However, traditional AVS rabbit models require a long-term period of dietary supplementation and genetic manipulation to induce significant stenosis in the aortic valve, limiting their use in experimental studies. To address these limitations, a new AVS rabbit model is proposed, in which stenosis is induced by a direct balloon injury to the aortic valve. The present protocol describes a successful technique for inducing AVS in New Zealand white (NZW) rabbits, with step-by-step procedures for the preparation, the surgical procedure, and the post-operative care. This simple and reproducible model offers a promising approach for studying the initiation and progression of AVS and provides a valuable tool for investigating the underlying pathological mechanisms of the disease.

An appropriate animal model is needed to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) and to evaluate the efficacy of therapeutic interventions. The present protocol describes a new procedure for developing the AVS rabbit model via a direct balloon injury in vivo.

Modelo de estenose da valva aórtica de coelho induzida por lesão direta por balão

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of ...