Name: vascular balloon injury and intraluminal administration in rat carotid artery
Uploaded: 2014-12-23T13:00:00.000+00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery at JoVE.com

We are grateful to Dr. Clowes for first developing and describing this method. We are also thankful to Dr. Tulis for his detailed protocol which has been fundamentally helpful to our previous, current and future work. This work was supported by grants R01HL097111 and R01HL123364 from the NIH to M.T., and by American Heart Association grant 14GRNT18880008 to M.T.
We would like to thank Rachel Newton for her expert technical support and for her valuable help during the filming process.

NOTA: O uso de animais para os seguintes experimentos tem sido revisado e aprovado pelo cuidado e uso Comitê Institucional Animal (IACUC). 1. Procedimentos pré-operatórios <ol style="margin-left: 40px;"><li> Esterilizar instrumentos cirúrgicos antes de usar. <ol><li> Autoclave todos os instrumentos cirúrgicos 24 horas ou menos antes da cirurgia. Se várias cirurgias são realizadas no mesmo dia, esterilizar os instrumentos por um esterilizador talão seca entre cirurgias. </li></ol></li><li> Filtro-esterilizar a solução salina antes da utilização. </li><li> Pesa-se o rato e o cálculo da dose de anestésicos (cetamina 80 mg / kg de xilazina e 7 mg / kg). </li><li> Administrar as drogas anestésicas intra-peritoneal (ip). <ol><li> Verificar a adequação da sedação por toe pitada de 5-10 min. Administrar uma pequena dose adicional de drogas (cetamina 7 mg / kg e xilazina 0,6 mg / kg), se a sedação não está completa. </li><li> Certifique-se de que a agulha é profundo o suficiente para entrega de drogas intra-peritoneally porque falha na entrega de toda a solução de um fármaco para a cavidade peritoneal irá causar sedação inadequada. NOTA: As úlceras e perda de cabelo na pele no local da injecção será visíveis vários dias após a cirurgia, devido a inadvertida por via subcutânea (sc) de injecção de solução de fármaco. </li></ol></li><li> Injectar 3 ml de solução salina estéril solução por via subcutânea (sc). Usando uma mecha de algodão estéril, aplicar uma pequena quantidade de pomada oftálmica para ambos os olhos para evitar as córneas de secar. </li><li> Prepare o equipamento de aquecimento. Almofadas de calor Pré-quentes por microondas ou banho-maria. </li><li> Coloque o animal indolentemente na plataforma cirúrgica. <ol><li> Retire o cabelo na região do pescoço ventral. Aplique o removedor de pêlos com um cotonete, espere 30 segundos e limpe completamente com gaze. </li><li> Esfregue o pescoço com esfoliação iodopovidona e 70% de etanol. </li></ol></li><li> Utilizar equipamento de proteção pessoal, incluindo vestido, capa de cabelo, máscara cirúrgica e óculos. Coloque em sterile luvas cirúrgicas no final antes de manusear os instrumentos cirúrgicos estéreis e suprimentos. </li><li> Armar o rato com um lençol cirúrgico estéril com apenas a região do pescoço exposto. </li></ol> 2. Procedimentos Cirúrgicos <ol style="margin-left: 40px;"><li> Durante o procedimento cirúrgico, verifique a profundidade da sedação de subprodutos animais pitada toe em cada 15 min. Se um animal responde a toe pitada, adicionar pequena dose adicional (10% da dose inicial) da cetamina e xilazina. </li><li> Dissecar artéria carótida comum esquerda (CCA) <ol><li> Usar um bisturi para fazer uma incisão longitudinal recta no meio do pescoço. O comprimento aproximado de incisão é de 1,5-2 cm, com a finalidade de isolar uma porção de 1,5-2 cm da artéria. O comprimento pode variar depende da finalidade do estudo. </li><li> Bluntly dissecar o tecido conjuntivo da pele. Mantenha as dicas fórceps-se e certifique-se para não perfurar a pele ou o tecido subjacente. </li><li> Dissecar camadas musculares longitudinalmente Along do lado esquerdo da traquéia. </li><li> Ao abrir-se a camada muscular, visualize a CCA esquerda com o nervo vago intimamente ligados. Sem rodeios dissecar ao lado da artéria carótida esquerda com extrema cautela para separar o nervo vago com o mínimo de alongamento. </li><li> Dissecar CCA distal até a bifurcação. Dissecar cuidadosamente a bifurcação e dois ramos - artéria carótida interna (ACI) e artéria carótida externa (ECA). </li><li> Manter dissecando em torno do CCA, até aproximadamente uma porção de 1,5-2 cm da artéria é isolado a partir dos tecidos circundantes. </li></ol></li><li> Lesão por balão <ol><li> Permanentemente fazer uma ligadura sobre o ECA em aproximadamente 5 mm de distância da bifurcação. Permanentemente ligadura do ramo occipital da ECA, que é próximo à bifurcação da ECA e ICA. Também ligar permanentemente outros ramos, se houver, localizando entre a bifurcação e ECA ligadura - por exemplo, o ramo da tireóide superior. Sutura que é utilizado para todas as ligaduras é 4-0 pretoseda. Grampo na extremidade proximal do CCA e a extremidade distal da ICA. NOTA: Agora, o fluxo sanguíneo foi interrompido permanentemente através da ligadura (on ECA) ou temporariamente por meio de recorte (no CCA e ICA). Conteúdo luminal na área de bifurcação ter sido isolado a partir da circulação sistémica. </li><li> Faça uma incisão arteriotomy no ECA por pequenos produtores, micro tesoura. Certifique-se de que a incisão fica perto do nó de sutura distal. Sangue limpo com soro fisiológico e cotonetes. </li><li> Insira o cateter balão insuflado 2F para o lúmen do TCE. Avançar o cateter balão proximal ao lúmen CCA. Mantenha o avanço do cateter proximal até sua ponta chega onde o clipe fica. </li><li> Conectar o dispositivo de insuflação do balão para um luer lock fêmea num torneira de 3 vias e ligar o bloqueio Luer macho da torneira de 3 vias para o cateter de balão. </li><li> Lentamente inflar o balão com aproximadamente 1,5 atm de pressão, de modo a distender a artéria carótida para 1,5 vezes o diâmetro. </li&gt; <li> Puxe cuidadosamente o balão rotativo de volta para a bifurcação. </li><li> Desinflar o balão e avançá-lo de volta para a extremidade proximal. Encha-o novamente e repita o procedimento puxando-back mais duas vezes. </li><li> Retirar o cateter balão do lúmen da artéria. </li></ol></li><li> Administração Intraluminal de reagentes (por exemplo, siRNA, drogas) NOTA: Aqui, o reagente utilizado foi uma solução contendo partículas lentivirais que codificam quer estromais da molécula alvo shRNA Interacção 1 (STIM1) ou não-alvo de controlo de shRNA. STIM1 é um retículo endoplasmático (ER) única proteína transmembranar que é um sensor de ER Ca 2+ controlando a activação da membrana plasmática canais de Ca2 + e é regulado para cima durante a desdiferenciação do músculo liso vascular em um fenótipo proliferativo migratório 12,30-33. <ol><li> Anexar um cateter intravascular sobre-a-agulha de uma seringa (24 L, 1,6 cm). Aspirar solução de 30 ul do ensaio reagentos. Inserir o cateter na mesma incisão no ECA. </li><li> Avance a ponta do cateter no CCA e amarrar um pedaço de fio de sutura com um único nó no ECA para fixar o cateter e fechar a incisão temporariamente. </li><li> Injectar a solução reagente de teste para o lúmen do CCA. Conservar a solução do lúmen do vaso para 30 min. Mantenha o tecido exposto úmido com solução salina e cobri-lo com um pedaço de gaze molhada. </li><li> Após a incubação, aspirar a solução restante. Solte que nó simples e retirar o cateter. </li><li> Amarre o ECA com um pedaço de fio de sutura proximal à incisão arteriotomy. Adicione o nó tão próximo quanto possível da bifurcação. </li></ol></li><li> Feche acima da ferida. <ol><li> Uma vez que a lesão por balão e introdução do cateter pode causar um vazamento ou punção do vaso sanguíneo, remover o clipe na ICA e verifique se há algum vazamento. Se o sangramento for observado, aplicar um pedaço de gaze e aplique uma leve pressão para parar o sangramento. </li><li> Removero outro clipe no CCA. </li><li> Certifique-se de que não há sinais de hemorragia, e depois remover todas as braçadeiras e outros instrumentos cirúrgicos. Corte o excesso de suturas. </li><li> Fechar ferida com suturas de pele (4-0 seda preta). Cotonete em todos os lados da ferida fechada com o Povidon-iodo ou outros agentes anti-séptico / agente bactericida / virucida. Injectar 3 ml de solução salina estéril sc </li></ol></li></ol> 3. Os procedimentos pós-operatórios <ol style="margin-left: 40px;"><li> Mantenha rato na almofada de calor após a cirurgia. Administrar uma dose de 0,05 mg / kg de buprenorfina por via de injecção intramuscular (im) para o rato. Durante o processo de recuperação, mantenha os olhos ea boca dos animais úmido. Monitorar o animal até que ele está acordado e ambulante. </li><li> Colocar o animal em uma gaiola limpa, sem qualquer roupa de cama até que esteja totalmente recuperado. Após a recuperação, o animal será devolvido para a sala de animais e alojados individualmente. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods In Molecular Medicine. 139, 1-30 (2007).</li><li>Zhang, W., Trebak, M., Szallasi, A., B&#237;r&#243;, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon+Injury+in+Rats+as+a+Model+for+Studying+TRP+Channel+Contribution+to+Vascular+Smooth+Muscle+Remodeling.">Balloon Injury in Rats as a Model for Studying TRP Channel Contribution to Vascular Smooth Muscle Remodeling.</a> T TRP Channels in Drug DiscoveryMethods in Pharmacology and Toxicology. , 101-111 (2012).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+morphometric+analyses+for+rat+carotid+balloon+injury+model).">Histological and morphometric analyses for rat carotid balloon injury model).</a> Methods In Molecular Medicine. 139, 31-66 (2007).</li></ol>

The authors declare that they have no competing financial interests.

O rato da lesão do balão carótida foi bem descrito por Tulis em 2007 34. Tem sido exaustivamente discutido todos os detalhes deste procedimento pelo Dr. Tulis. Os leitores que estão interessados ​​em realizar este procedimento são altamente recomendados para ler protocolo Tulis &#39;. No entanto, há uma coisa que não está concordando com o Dr. Tulis: Em vez de inflar o balão com soro fisiológico ou qualquer tipo de líquido, sugerimos para inflá-lo com o ar. De acordo com nossa experiência pessoal, inflando com l?...

Os cateteres de balão são dispositivos médicos utilizados no procedimento da angioplastia, com a finalidade de alargamento local obstruído (s) de ateroma ou trombo num vaso sanguíneo. Os lúmen do vaso estreitadas é forçado a abrir pelo balão inflado e fornecimento de sangue seria restaurada sequencialmente para aliviar os sintomas de isquemia a jusante, tais como angina, infarto do miocárdio e dor na perna. No entanto, o grande sucesso da angioplastia foi diminuída por complicações pós-operatórias, tais como resultados de força causando barotrauma vascular (lesão por balão), nomeadamente a remodelação da parede do vaso e, em muitos casos re-estreitamento da luz do vaso (reestenose) 1. Uma série de modelos animais têm sido desenvolvidas imitando o procedimento de angioplastia para ajudar os investigadores a compreender os mecanismos subjacentes a embarcação remodelação parede relacionados com o balão-lesão 2. Entre todas as espécies de animais utilizados para a modelagem, o rato é o mais utilizado. Compared a coelhos, cães e porcos, as vantagens de os ratos são seu baixo custo, sua relativa facilidade de uso e o conhecimento atual da fisiologia rato. Embora os ratos têm uma vantagem adicional em uma ampla gama de estirpes geneticamente manipuladas, o navio ratinhos é muito pequeno para inserir um cateter balão. Ao longo das últimas três décadas, os ratos experimentais têm permitido aos pesquisadores obter uma melhor compreensão dos mecanismos moleculares e celulares que sustentam a formação da neoíntima e remodelação vascular 3-6. Além da lesão do balão, o remodelamento vascular também estão envolvidos na maioria das doenças vasculares importantes, como a aterosclerose 7,8, hipertensão 9 e 10 de aneurisma. Assim, o conhecimento adquirido através do modelo de lesão balão é, em geral, benéfica para estudos globais da doença parede vascular. O objectivo geral do modelo de lesão por balão de rato não é apenas de compreender ainda as doenças vasculares, mas também para testar a potência de novos agentes parao controle da doença 11,12. Tratamento medicamentoso clínico atual para reestenose é aplicado por stents farmacológicos efectuadas através do lúmen do vaso logo após a angioplastia. Em modelos animais, uma forma eficiente ainda mais econômica para novos testes agente é um método de perfusão intraluminal locais bem desenvolvida. Os agentes candidatos que foram testados através deste método incluem drogas de moléculas pequenas 13,14, citocinas ou factores de crescimento 15,16, agentes de manipulação de genes (clones de cDNA, siRNA, etc.), 17-20, e novas formulações farmacêuticas 21,22. Até agora, o modelo de lesão balão rato continua sendo um dos modelos mais úteis para o estudo de doenças / distúrbios vasculares. É o passo fundamental da bancada de cabeceira, geralmente como o primeiro passo, no sentido de in vitro para in vivo, mas não deve ser o último. O resultado de experiências com ratos precisa ser deliberado e ainda caracterizado antes tradução humanautilização clínica, devido à diferença de leitos vasculares e a anatomia do vaso, bem como as diferenças intrínsecas entre as espécies humana e de rato 23-26. No entanto, ainda é uma ferramenta essencial na investigação médica translacional. Embora essa pesquisa utilizou a ser limitada pela falta de ratos geneticamente modificados, que deixou de ser um problema desde abordagens genômicas inéditas, como zinc-finger nucleases 27, 28 e TALENS CRISPR-Cas 29 fizeram ratos knockout facilmente acessível.

<table><tbody><tr><td>Fogarty balloon embolectomy catheters, 2 French&amp;nbsp;</td><td>Edwards Lifesciences, Germany</td><td>&amp;nbsp;120602F</td><td></td></tr><tr><td>Deltaphase Operating Board - Includes 2 Pads &amp;amp; 2 Insulators</td><td>Braintree Scientific, Inc.</td><td>39OP</td><td></td></tr><tr><td>LED light source</td><td>Fisher Scientific</td><td>12-563-501&amp;nbsp;</td><td></td></tr><tr><td>Hartmann Mosquito Forceps 4&amp;rdquo; curved</td><td>Apiary Medical, Inc. San Diego, CA</td><td>gS 22.1670</td><td></td></tr><tr><td>Crile Retractor 4&amp;rdquo; double ended</td><td>Apiary Medical, Inc.</td><td>gS 34.1934</td><td></td></tr><tr><td>Other surgical instruments</td><td>Roboz Surgical Instrument Company, Inc., Gaithersburg, MD</td><td></td><td></td></tr><tr><td>Peripheral Intravenous (I.V.) Cannula, 24 G</td><td>BD</td><td>381312</td><td></td></tr><tr><td>Ketamine HCl, 100 mg/ml, 10 ml</td><td>Ketaset- Patterson Vet</td><td>07-803-6637&amp;nbsp;</td><td></td></tr><tr><td>Xylazine (AnaSed), 20 mg/ml, 20 ml</td><td>Ketaset- Patterson Vet</td><td>07-808-1947</td><td></td></tr><tr><td>Buprenex, 0.3 mg/1 ml (5 Ampules/Box)</td><td>Ketaset- Patterson Vet</td><td>07-850-2280</td><td></td></tr><tr><td>Nair Baby Oil Hair Removal Lotion - 9 oz</td><td>Amazon/Walmart/CVS</td><td>N/A</td><td></td></tr><tr><td>Inflation Device</td><td>Demax Medical</td><td>DID30</td><td></td></tr><tr><td>D300 3-way Stopcock</td><td>B.Braun Medical Inc.</td><td>4599543</td><td></td></tr><tr><td>Artificial Tears Ointment&amp;nbsp;</td><td>Rugby Laboratories, Duluth, GA</td><td>N/A</td><td></td></tr></tbody></table>

vascular balloon injury and intraluminal administration in rat carotid artery

O modelo de lesão por balão da artéria carótida em ratos tem sido bem estabelecida há mais de duas décadas. Mantém-se um método importante para estudar os mecanismos moleculares e celulares envolvidos na desdiferenciação do músculo liso vascular, a formação da neoíntima e remodelação vascular. Ratos machos Sprague-Dawley são os animais mais utilizados para este modelo. Ratas não são preferidos como hormônios femininos protegem contra doenças vasculares e, assim, apresentar uma variação para este procedimento. A carótida esquerda é tipicamente feridos com o carótida direita que serve como um controlo negativo. Lesão carótida esquerda é causada pelo balão insuflado que desnuda o endotélio e distender s parede do vaso. Após lesão, potenciais estratégias terapêuticas tais como a utilização de compostos farmacológicos e qualquer gene ou shRNA transferência pode ser avaliada. Tipicamente para gene ou transferência shRNA, a seção de feridos do lúmen do vaso é transduced localmente por 30 min com vIRAL partículas que codificam uma proteína ou shRNA ou para a entrega e expressão na parede do vaso lesionado. Espessamento neointimal que representa as células musculares lisas vasculares proliferativas normalmente picos a 2 semanas após a lesão. Os vasos são principalmente colhidas neste ponto de tempo para análise celular e molecular de vias de sinalização celular, bem como a expressão do gene e proteína. As embarcações também podem ser colhidas a pontos de tempo anteriores para determinar o início da expressão e / ou activação de uma proteína ou via específica, de acordo com os objectivos pretendidos experimentais. Os navios podem ser caracterizadas e avaliadas por meio da coloração histológica, imunohistoquímica, ensaios de proteína / mRNA, e ensaios de atividade. A artéria carótida direita intacto do mesmo animal é um controle interno ideal. Mudanças induzidas pelo prejuízo, em parâmetros moleculares e celulares pode ser avaliada comparando a artéria ferido à artéria controle direito interno. Da mesma forma, as modalidades terapêuticas pode ser avaliada comparando a firad e tratado artéria para o controle lesionado só artéria.

Duas semanas após a lesão, artérias carótidas são colhidas, seccionados e sujeitos a análise morfológica. As artérias são cross-seccionados e corados com H &amp; E (Figuras 1, 2B, C e 3). Da parede da artéria carótida de rato contém quatro camadas de lâmina elástica, que aparecem como linhas de rosa. A área entre a lâmina externa, lâmina elástica externa (EEL) e a lâmina mais interior, da lâmina elástica interna (IEL) é a camada de músculo liso meios (Figura 1). A...

Watch this Scientific Journal Video about Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery at JoVE.com

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

Este protocolo utiliza um cateter balão para causar uma lesão intraluminal na artéria carótida de rato e, doravante, provocar hiperplasia neointimal. Este é um modelo bem estabelecido para o estudo dos mecanismos de remodelação vascular em resposta a lesão. É também amplamente utilizada para determinar a validade de potenciais abordagens terapêuticas.

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 ...

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Research

JoVE Journal

Medicine

16.3K visualizações.  State University of New York College of Nanoscale Science and Engineering (SUNY CNSE).  Este protocolo utiliza um cateter balão para causar uma lesão intraluminal na artéria carótida de rato e, doravante, provocar hiperplasia neointimal. Este é um modelo bem estabelecido para o estudo dos mecanismos de remodelação vascular em resposta a lesão. É também amplamente utilizada para determinar a validade de potenciais abordagens terapêuticas. 

Vídeo: Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Treinar um técnica microcirúrgica sofisticado: Interposição de enxerto de veia jugular externa na artéria carótida comum em ratos

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Medicina

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2-oclusão do vaso / hipotensão: um modelo de rato de isquemia cerebral

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Vascular transferência de genes de Metallic Stent superfícies usando Adenoviral Vetores Tethered através hidrolisável reticulantes

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

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 ...

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

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

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

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

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

A 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 ...

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

Implantação de stents coronários de tamanho humano na aorta abdominal de rato usando um acesso trans-femoral

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of ...

Modelo de balão de venoplastia de roedores para estudos de tratamento de veias profundas

Rodent Inferior Vena Cava Venoplasty Balloon Model

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

Autor em destaque: Utilizando o modelo de balão de venoplastia em roedores para simular intervenções cirúrgicas para veias profundas

The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.