Os autores agradecem Christiane Pahrmann por sua assistência técnica. Este estudo foi financiado pela Deutsche Stiftung fuer Herzforschung (F / 28/14). DW foi apoiado pelo prêmio de viagem da Sociedade Internacional de Transplante Cardíaco. TD recebeu o Else Kröner Excellence Bolsa do Else-Kröner-Fresenius-Stiftung (2012_EKES.04). SS recebeu bolsas de pesquisa do Deutsche Forschungsgemeinschaft (DFG; DE2133 / 2-1, TD e SCHR992 / 3- 1, SCHR992 / 4-1, SS).

Os animais receberam cuidado humano de acordo com o Guia para os Princípios de Animais de Laboratório, preparados pelo Instituto de Recursos Animais de laboratório e publicado pelo National Institutes of Health. Todos os protocolos com animais foram aprovados pela autoridade local responsável ( &quot;Amt für Gesundheit und Verbraucherschutz, Hansestadt (Departamento da Saúde e Defesa do Consumidor) Hamburg&quot;). 1. Animal Care <ol><li> Obter ratos ratos Lewis (LEW / CRL) e Rosa / ratos transgênicos luciferase-LEW pesando 300-350 g do Instituto de Animais de Laboratório. </li><li> Manter os ratos sob condições convencionais em armários ventilados e alimentá-los ração e água ad libitum autoclavado. </li><li> Executar um transplante de enxerto utilizando os / luciferase-LEW ratos transgênicos ROSA como os doadores e os singênicos ratos LEW / CRL como os destinatários. </li></ol> 2. Preparação do animal doador <ol><li> Use um indutocâmara de ionização para anestesiar um rato com isoflurano (2,5-3%). </li><li> Colocar o rato na sua parte de trás e manter a anestesia com uma máscara que cobre o nariz e a boca. Verifique se há profundidade suficiente de anestesia por beliscar as patas traseiras e verificar a ausência de reflexos. Aplique um pouco de pomada veterinário para os olhos para evitar a secura e sob anestesia. </li><li> Espalhe as pernas traseiras e fixar sua posição usando fita. </li><li> Raspar o cabelo inguinal com um aparador de pêlos e desinfectar a área inteira usando iodopovidona seguido por 80% de etanol. Repita o passo desinfecção duas vezes. NOTA: A área cirúrgica, gazes e instrumentos cirúrgicos devem ser esterilizados. Manter um campo esterilizado durante todo o procedimento e desgaste de uso único, luvas cirúrgicas estéreis, máscaras e bonés. </li><li> Sob um microscópio, realizar uma incisão vertical ao longo das inguinalis linea. Use duas pinças para separar delicadamente os tecidos subcutâneos e expor a veia epigástrica superficial de sua origin na veia femoral. Cuidadosamente isolar a veia epigástrica superficial dos tecidos circundantes. </li><li> Parar o fluxo de sangue na veia epigástrica superficial usando dois micro grampos. </li><li> Colheita um segmento de cerca de 0,5 a 1 cm da veia levantando cuidadosamente da veia isolado com uma pinça e o corte através do vaso com microtesouras. Deixar as micro grampos sobre o coto recipiente para evitar a perda de sangue. Coloque o pedaço removido da veia em gaze estéril. Cuidadosamente colocar uma agulha 30 G de uma extremidade interior da veia colhida e lavar o reactor com heparina (50 unidades / ml). NOTA: Pega a veia com cuidado e evitar danos durante a elevação, corte e descarga. Certifique-se de lavar o enxerto com a quantidade adequada de heparina. </li><li> Manter o segmento do vaso de lidocaína a 1% em gelo até que o transplante para o receptor de rato para prevenir um espasmo navio. </li><li> Eutanásia do rato doador, aumentando a anestesia com isoflurano a 5%. Depois de 2-3 min, open abdômen ao longo da linha alba, cortar através do diafragma, e remover o coração parar circulação. </li></ol> 3. Preparação do Rato Destinatário <ol><li> Anestesiar ratos e fixar o recipiente da mesma maneira como o rato dador. </li><li> Raspar o lado medial das pernas com um aparador de pêlos e desinfectar três vezes usando iodopovidona e 80% de etanol. </li><li> Monitorar a profundidade da anestesia e garantir que ele é suficiente, verificando a ausência de reflexos quando beliscar as patas traseiras. </li><li> Realizar uma incisão femoral médio do joelho até a dobra inguinal. Sob um microscópio, utilizar 2 pinça para separar a artéria femoral a partir dos seus arredores. </li><li> Use micro grampos para parar o fluxo de sangue. Coloque a braçadeira proximal em primeiro lugar, seguido pela pinça distal. </li><li> Corte um pequeno segmento da artéria femoral apertada com microtesoura e descartá-lo. Encurtar o coto arterial restante com microtesoura, criando uma lacuna que é1-2 mm maior do que o enxerto de veia. Lave o coto arterial com heparina usando uma agulha 30 G. NOTA: Se a adventícia sai ligeiramente para além do coto embarcação, utilize uma pinça para puxá-lo um pouco sobre a extremidade do navio e retirar um pedaço. </li><li> Coloque a veia colhida a partir do passo 2.8 entre os cotos arteriais e ajustar o comprimento de modo que ele se encaixa adequadamente na abertura. Note-se a direcção da veia. </li><li> Realização da anastomose proximal primeiro usando uma sutura 10-0 prolene. Realizar pontos individuais na ordem mostrada (Figura 1D). Comece com uma sutura em cada lado lateral antes de adicionar mais três suturas no lado ventral. Em seguida, colocam três pontos no lado dorsal do vaso para completar a anastomose. </li><li> Ligar os vasos distais, com o enxerto utilizando a mesma técnica para a anastomose proximal descrito no passo 3.8. Mais uma vez, comece com uma sutura em cada lado lateral, e em seguida, coloque três suturas na sid ventralE e o lado dorsal. </li><li> Carregar duas seringas de 1 ml com componente de cola de fibrina 1 e 2. Levante cuidadosamente o enxerto com uma pinça e soltar cerca de 100 mL de componente cola de fibrina 1 sob o enxerto, seguida pela componente 2. NOTA: Certifique-se de que os componentes 1 e 2 são aplicados na proporção de 1: 1. </li><li> Coloque o enxerto na sua posição para trás e soltar um adicional de 100 ul de componentes 1 e 2 em cima do enxerto. Certifique-se de que a cola abrange tanto o enxerto e anastomose, a fim de evitar a insuficiência da anastomose e sobre-distensão do enxerto de veia. </li><li> abrir a pinça distai cuidadosamente, seguido pela proximal. </li><li> Confirmar uma cirurgia bem-sucedida verificando o pulso visível na veia transplantada e artéria distal do enxerto. </li><li> Remover a colagem excessiva, o que impede o fechamento da pele. Utilize uma pinça para levantar a cola curada e remover o excesso com microtesouras. Feche as camadas da pele com 5-0 prolene. </li><li> Injetar 4-5 mg / kg carprofeno via subcutânea antes de permitir que o rato para acordar. Não deixe o animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Manter o animal em uma única gaiola até que esteja totalmente recuperado. </li><li> Adicionar Metamizolo à água de beber (50 mg Metamizolo por 100 ml) na forma de medicação para a dor para os 3 dias seguintes e monitorar o animal diariamente. </li></ol> 4. Duplex ultra-sonografia NOTA: Use sonografia duxplex para visualizar o fluxo sanguíneo não invasivo em ratos 14. <ol><li> Anestesiar uma ratazana numa câmara de indução (isoflurano a 2%). Coloque o rato sobre as suas costas e manter a anestesia com uma máscara cobrindo o nariz. </li><li> Use cortadores de cabelo e creme de depilação para remover os pêlos ao redor da área da coxa. </li><li> Aplique o gel de ultra-som na coxa. Certifique-se de que não existem bolhas de ar. Adquirir imagens ultra-sonografia duplex usando um MS 400 transdutor (frequência central: 30 MHz) com uma taxa de quadros de 230-400 frames / seg. </li></ol> 5. O exame histopatológico NOTA: Colheita e manchar o navio com coloração de Masson para análises morfométricas 15. <ol><li> Corrigir o vaso colhida em 4% de paraformaldeído durante a noite e desidratar em concentrações crescentes de etanol. Incorporar a amostra em parafina e corte-o em 5 mm fatias grossas usando um micrótomo. NOTA: O paraformaldeído é tóxico e deve ser manuseada com cuidado especial. </li><li> Desparafinar os slides antes da coloração-los com tricromo solução de coloração. Desidratar as lâminas coradas, limpá-las com xileno, e montá-los no meio de montagem. Depois de secar os slides, ver as amostras com um microscópio. </li></ol> 6. A bioluminescência Imagem (BLI) NOTA: O enxerto pós-operatória foi monitorado ao longo do tempo in vivo através da medição do sinal bioluminescente 16. <ol><li> Dissolve-se 1 g de sal de potássio de D-luciferina em 22 ml de PBS e injectada por via intraperitoneal em ratos (375 mg / kg de peso corporal). Esperar 15 min para a luciferina a circular no animal. </li><li> Colocar o rato dentro de um sistema bioluminescente quantificação em tempo real e aceder ao sinal de bioluminescência. </li></ol>

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The authors have nothing to disclose.

Este vídeo demonstra um modelo de interposição da veia de ratos para investigar a perda de enxerto de veia e para permitir a exploração dos processos patológicos subjacentes e teste de novas drogas ou opções terapêuticas. A anestesia é um aspecto crucial de procedimentos cirúrgicos. Um sistema de anestesia por inalação contínua é recomendado, pois este é um método seguro e fácil, especialmente durante operações prolongadas. Isto pode ser de grande importância durante a f...

doenças das artérias coronárias e suas complicações estão entre as principais causas de morte em todo o mundo. estratégias terapêuticas atuais concentrar em restabelecer o fluxo de sangue, seja através da dilatação do vaso estreitados ou através da criação de um bypass. Revascularização do miocárdio (RM) utilizando enxertos venosos foi descrita pela primeira vez em 1968 e tem sido aperfeiçoado ao longo dos anos. Para além da revascularização da artéria descendente anterior coronária, condutas de veia safena são mais comumente usados 1. No entanto, enxerto permeabilidade continua a ser o calcanhar de Aquiles da veia safena (SVG). Um ano após a cirurgia, enxerto de permeabilidade é de 85%, caindo para 61% após dez anos 2,3. Revelando os mecanismos fisiopatológicos e as causas da perda de permeabilidade SVG é, portanto, uma tarefa importante. Este vídeo demonstra um modelo de interposição da veia de ratos para investigar a perda de enxerto de veia. Os objetivos gerais deste método são para explorar a pathobiological subjacentee processos -physiological durante a progressão da doença e desenvolver um modelo adequado para drogas ou testes opção terapêutica. Transplantando a veia epigástrica superficial no sistema arterial, este modelo imita de perto a situação clínica de revascularização do miocárdio. O trauma cirúrgico, isquemia e estresse da parede são gatilhos importantes de alterações vasculares patológicos e são imitadas no modelo descrito. Diferentes modelos e espécies estão disponíveis para investigar a perda de permeabilidade do enxerto venoso. Grandes modelos animais, como porcos, ovelhas 4 5 6, cães e macacos 7, assemelham-se vaso humano e estruturas anatômicas e, assim, permitir estratégias terapêuticas complexas, como implante de stent de bypass ou novas técnicas cirúrgicas, a serem testadas 8. No entanto, especial habitação, equipamento e pessoal são necessários. Além disso, custos elevados e à necessidade de um anestesista adicional durante a cirurgia impedem a sua aplicação mais ampla. Smtodos os animais, incluindo ratos, são fáceis de manusear, não necessitam de alojamento especial, e têm custos controláveis. Em comparação com os modelos de rato 9,10, modelos de ratos têm a vantagem de uma melhor operacionalidade e, portanto, menor variabilidade no resultado. Os ratos são fisiológica e geneticamente mais semelhantes aos seres humanos do que os ratos 11,12. Além disso, a maioria dos camundongos selvagens só desenvolver myointima limitada 13, que fazem modelos de ratos propensos a erros do tipo II. A histologia das veias principais do rato, tal como a veia cava inferior, consiste apenas de algumas camadas de células e torna difícil a avaliação precoce 13. Uma outra desvantagem é a pequena quantidade de tecido disponível para análises subsequentes após a recuperação do enxerto. O modelo descrito neste vídeo é reprodutível, de baixo custo, e fácil de realizar, e pode ser estabelecida rapidamente e de forma fiável. É especialmente apropriado para a avaliação de agentes terapêuticos experimentais caros, tais como vectores viraispara terapia génica, de uma forma económica.

<table border="0" cellpadding="0" cellspacing="0" width="720">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:236px;">Rat LEW/Crl</td>
			<td style="width:183px;">Charles River</td>
			<td style="width:145px;">Stock number 004</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:236px;">Rat LEW-Tg(Gt(ROSA)26Sor- 1 
			luc)11Jmsk</td>
			<td style="width:183px;">Institute of laboratory animals, Kyoto University, Japan</td>
			<td style="width:145px;">NBPR rat number 0299</td>
			<td style="width:156px;">http://www.anim.med.kyoto-u.ac.jp/NBR/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td align="right">20%</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">hair clipper</td>
			<td>WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Forene</td>
			<td>AbbVie</td>
			<td style="width:145px;">PZN 10182054 Art.Nr.: B506</td>
			<td>Isoflurane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">microsurgical clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Micro-Serrefine - 4mm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">clamp applicator</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">hair removal creme</td>
			<td>Rufin cosmetic</td>
			<td align="right">27618</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Povidone-Iodine</td>
			<td>Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10-0 Ethilon suture</td>
			<td>Ethicon</td>
			<td>2814G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5-0 prolene suture</td>
			<td>Ethicon</td>
			<td>EH7229H</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rimadyl</td>
			<td>Pfizer</td>
			<td style="width:145px;">400684.00.00</td>
			<td>Carprofen</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heparin</td>
			<td>Rotexmedica</td>
			<td>PZN: 3862340</td>
			<td>25.000 I.E./mL</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylocain 1%</td>
			<td>AstraZeneca</td>
			<td>PZN: 1137907</td>
			<td>Lidocain</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EVICEL</td>
			<td>J&#38;J Med.Ethicon Biosur</td>
			<td style="width:145px;">PZN 7349697 Art. Nr.:EVK01DE</td>
			<td>fibrin glue</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">NaCl 0,9%</td>
			<td>B.Braun</td>
			<td style="width:145px;">PZN 06063042 Art. Nr.: 3570160</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:236px;">Vevo 770 high-resolution in vivo micro-imaging system</td>
			<td>VisualSonics</td>
			<td></td>
			<td>duplex sonography</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ecogel 100 ultrasound gel</td>
			<td>Eco-med</td>
			<td>30GB</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">D-Luciferin Firefly, potassium salt</td>
			<td>Biosynth</td>
			<td>L-8220</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xenogen Ivis 200</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>bioluminescence imaging</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Weigerts iron hematoxylin Kit</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Resorcine-Fuchsine Weigert</td>
			<td>Waldeck</td>
			<td>2.00E-30</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acid Fuchsin</td>
			<td>Sigma-Aldrich</td>
			<td>F8129-25G</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ponceau S solution</td>
			<td>Serva Electrophoresis</td>
			<td>33427</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Azophloxin</td>
			<td>Waldeck</td>
			<td>1B-103</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:236px;">Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>1.00532.0100</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Light Green SF</td>
			<td>Waldeck</td>
			<td>1B-211</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vitro-Clud</td>
			<td>Langenbrinck</td>
			<td>04-0001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td align="right">537020</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">37% HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylene</td>
			<td>Th. Geyer</td>
			<td align="right">3410</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol absolute</td>
			<td>Th. Geyer</td>
			<td align="right">2246</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol 96%</td>
			<td>Th. Geyer</td>
			<td align="right">2295</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol 70%</td>
			<td>Th. Geyer</td>
			<td align="right">2270</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Slide Rack</td>
			<td>Ted Pella</td>
			<td align="right">21057</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining dish</td>
			<td>Ted Pella</td>
			<td align="right">21075</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bepanthen Eye and Nose ointment</td>
			<td>Bayer</td>
			<td align="right">1578675</td>
			<td>Eye ointment</td>
		</tr>
	</tbody>
</table>

vein interposition model: a suitable model to study bypass graft patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

O modelo de interposição da veia rato é adequada para estudar o desenvolvimento de hiperplasia myointima e fracasso do enxerto venoso. Animais recuperar bem da cirurgia e mostrar excelente condição física pós-operação. A Figura 1 mostra os passos chave cirúrgicos. Após a incisão na pele ao longo das inguinalis linea, a veia superficial epigástrica e artéria femoral são identificados (Figura 1A). Colheita do enxerto deve ser realizada com cuidado, sem danificar o enxerto <...

Watch this Scientific Journal Video about Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency at JoVE.com

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Veia Interposição Modelo: Um modelo adequado para estudar Bypass Graft Perviedade

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

vein-interposition-model-suitable-model-to-study-bypass-graft

Research

JoVE Journal

Medicine

8.7K visualizações.  University Heart Center Hamburg. This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats. 

Vídeo: Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

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

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 ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

A Human<em&gt; Ex Vivo</em&gt; Placa aterosclerótica modelo para estudar Lesão Biologia

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Implantação de Veia Cava Inferior interposição de enxerto no Rato Modelo

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

Bypass cardiopulmonar em um modelo do rato: uma nova abordagem

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.

Modelo de suíno cirúrgico da isquemia cardíaca crônica, tratado pela cirurgia de Bypass coronário de bomba

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Um modelo de coelho da expressão do Transgene durável na veia Jugular para enxertos de interposição da artéria carótida comum

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Um modelo Crioferimento para estudar infarto do miocárdio no mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Um modelo de interposição venosa de coelho imitando cirurgia de revascularização usando enxertos de veia para avaliar hiperplasia intimal sob pressão arterial

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...