Os autores Christiane Pahrmann agradecer a assistência técnica.
D.W. foi apoiado por Max Kade Foundation. T.D. recebido concede a outra Kröner Fondation (2012_EKES.04) e a Deutsche Forschungsgemeinschaft (DE2133/2-1. _. Bolsas de investigação S. S. recebeu o Deutsche Forschungsgemeinschaft (DFG; SCHR992/3-1, SCHR992/4-1).

Os animais receberam atendimento humano em conformidade com o guia para os princípios de animais de laboratório, preparado pelo Instituto de recursos animais de laboratório e publicado pela National Institutes of Health. Todos os protocolos de animais foram aprovados pela autoridade local competente (' Amt für Gesundheit und Verbraucherschutz, (serviço de saúde e defesa do consumidor) de Hansestadt Hamburg ').
1. cateter preparação
Nota: Consulte a Tabela de materiais , para obter informações sobre o cateter.
<ol><li>Retire o cateter o titular do cateter.</li>
	<li>Puxe o fio-guia do lúmen através do orifício distal para fora.</li>
	<li>Colocar em uma gota de adesivo de cianoacrilato na extremidade distal do cateter. Cuidado: Use luvas de látex ou nitrilo.</li>
	<li>Coloque o fio-guia na porta RX do cateter e avançá-lo através do lúmen para o porto distal. Depois, puxe o fio-guia ligeiramente para deixar um espaço de ~ 5 mm até o fim.</li>
	<li>Espere 5 minutos para permitir que o adesivo secar.</li>
	<li>Mover o fio-guia, deve ser fixado. Se é ainda móvel, repita os passos 1.3-1.6.</li>
	<li>Encher uma seringa de 3 mL com soro fisiológico a 0,9% 1,5 mL e conectá-lo à porta de insuflação do balão.</li>
	<li>Empurre o êmbolo da seringa para teste de insuflação do balão. Deixe 0,6 mL de soro fisiológico na seringa.</li>
</ol>2. preparação do rato
<ol><li>Obter camundongos machos na idade de 14 semanas, pesando aproximadamente 30 g. Nota: Usamos animais obtidos a partir do Instituto de animais de laboratório. Aqui usamos C57BL/6J.</li>
	<li>Use uma câmara de indução para anestesiar o mouse com isofurane de 2.0-2.5% (taxa de fluxo de oxigênio de 500 mL/min).</li>
	<li>Coloque o mouse sobre as costas de uma almofada de aquecimento e manter anestesia com uma máscara cobrindo a boca e o nariz do mouse. Verifique se há suficiente profundidade da anestesia beliscar as patas e cauda para verificar a ausência de reflexos.</li>
	<li>Remova o cabelo abdominal usando uma máquina de cortar cabelo.</li>
	<li>Espalhe as patas traseiras e fixar sua posição usando a fita.</li>
	<li>Desinfete a área abdominal usando iodo-povidona, seguido por 80% de etanol. Repita mais duas vezes.</li>
	<li>Use um pano cirúrgico para garantir local cirúrgico não ser contaminado. Abra as camadas de pele e músculo ao longo da linea alba com uma tesoura (ou bisturi) para expor os órgãos abdominais.</li>
	<li>Colocar os intestinos em uma luva de hidratada salina 0,9% e enrole para mantê-los húmida.</li>
	<li>Use dois pinça fina para remover o tecido adiposo acima a aorta abdominal.</li>
	<li>Usando uma seringa de insulina (30G), injetar 250 µ l de solução de heparina (50 U/mL) para a veia Cava Inferior (VCI) e espere 3 min para sua distribuição sistêmica. Heparina poderá suprimir a hemostasia e evitar coagulação indesejada durante a cirurgia.</li>
	<li>Usando duas pinças, disse a aorta infra-renal até sua bifurcação e seus vasos de ramo a saída.</li>
	<li>Ligar os vasos colaterais, que são esperados para ser colocado na área do preso, com um cauterizador de alta temperatura.</li>
	<li>Interromper o fluxo de sangue, fixando a aorta infra-renal diretamente abaixo das artérias renais.</li>
	<li>Coloque uma segunda pinça em uma posição distal logo acima da bifurcação da aorta.</li>
	<li>Realize uma pequena incisão horizontal usando a tesoura no ponto médio entre os grampos ao longo do navio. Nota: O tamanho da incisão deve ser equivalente a 1/3 da circunferência do vaso.</li>
	<li>Introduza uma seringa com uma agulha (30G) da incisão e liberar a aorta com 250 µ l de solução heparina (50 U/mL).</li>
	<li>Utilizar suturas de 10-0, coloque um único nó de cada lado da incisão.</li>
	<li>Dilatação da aorta inserindo um dilatador de vaso a incisão e espalhar o recipiente ligeiramente. Repita a dilatação de 2 a 3 vezes.</li>
	<li>Hidrate o cateter-balão com soro fisiológico a 0,9%.</li>
	<li>Insira o cateter-balão achatado na aorta e avançá-lo em direção a pinça proximal na aorta.</li>
	<li>Quando chegar a pinça proximal, cuidadosamente Abra a pinça proximal e insuflar o balão para evitar o vazamento de sangue, através da injeção de ~0.6 mL de solução salina. Nota: A relação do balão inflado de navio é 1.5: 1.</li>
	<li>Avance o cateter retrógrado para aproximadamente 2 cm.</li>
	<li>Puxe o cateter expandido, enquanto desinflar o balão ligeiramente liberando a seringa.</li>
	<li>Quando o cateter chega a incisão da aorta, recolocar a pinça proximal. Esvazie o balão completamente e removê-lo.</li>
	<li>Enxágue a aorta com 250 µ l de solução heparina (50 U/mL), utilizando uma seringa de 30G.</li>
	<li>Feche a incisão da aorta usando suturas de 10-0. Lugar interrompido pontos de cada lado lateral, seguido por um ou dois pontos no lado ventral.</li>
	<li>Abra a pinça distal. Em caso de sangramento, fechar a pinça novamente e colocar pontos adicionais.</li>
	<li>Abra a pinça proximal com cuidado.</li>
	<li>Coloque dois cotonetes na sutura para apoiá-lo e parar qualquer sangramento.</li>
	<li>Coloque hemostatos absorvíveis na sutura para sustentá-lo. Nota: Um pulso aórtico deve ser visível distal da incisão.</li>
	<li>Coloque os intestinos para o abdômen.</li>
	<li>Enxaguar a cavidade abdominal com solução salina 0,9%, que foi previamente aquecido a 37 ° C.</li>
	<li>Feche a camada muscular abdominal usando suturas execução de 6-0.</li>
	<li>Feche a pele com suturas de execução de 5-0.</li>
	<li>Injete o carprofeno de 4-5 mg/kg por via subcutânea antes de permitir que o mouse para acordar. Monitorar o animal até ganhou consciência, manter a prostração esternal. Manter o animal em uma gaiola até recuperação completa.</li>
	<li>Adicionar o metamizol à água potável (50 mg/100 mL) como medicação para a dor por 3 dias e monitorar o animal diariamente. Geralmente os ratos como o doce sabor de dipirona e começam a beber imediatamente após a cirurgia. Se preferirem, agentes injetáveis de liberação sustentada podem ser usados em vez de dipirona. Nota: O período de observação para este modelo é de 28 dias.</li>
</ol>3. histopatologia
<ol><li>Colheita da aorta com balão-ferido depois de 28 dias, preparando os ratos como descrito nos passos 2.2 a 2.9.</li>
	<li>Use a tesoura para remover a balão-ferido da aorta (entre a bifurcação e 0,3 mm acima dos vasos renais) e eutanásia o mouse cortando seu coração.</li>
	<li>Irrigue o lúmen do vaso com 0,9% NaCl.</li>
	<li>Consertar o navio colhido em paraformaldeído 4% (PFA) durante a noite e desidratá-lo em concentrações crescentes de etanol, começando com etanol a 70% por 2 horas, 80% de etanol por 1 hora, etanol 95% por 2 horas e 100% de etanol por 5 horas. Então, Incube as amostras em xilol durante 2 horas 3 vezes, antes de me infiltrar as amostras com parafina. Nota: em vez de liberar o navio colhido com 0,9% NaCl, ele pode ser liberado com 4% PFA. Cuidado: PFA e xileno são tóxicos e devem ser manuseados com cuidado especial.</li>
	<li>Incorporar a amostra em parafina e cortados em fatias de espessura de 5 µm usando um micrótomo.</li>
	<li>Deparaffinize os slides com xileno 3 vezes durante 5 minutos.</li>
	<li>Reidratar slides de tecido usando uma série decrescente de etanol. Comece com 100% de etanol 2 vezes por 5 min, seguido de 3 minutos de etanol 95%, 80% e 70%.</li>
	<li>Mancha os slides com coloração tricromo de Masson como descrito18.</li>
	<li>Desidrate corrediças manchadas em etanol 100% 2 vezes por 10 min cada. Limpar com xilol 2 vezes por 10 min cada e montar no meio de montagem.</li>
	<li>Visualizar os slides com um microscópio de campo claro. Use uma lente com 5x ampliação e uma abertura numérica de 0,12 por imagens uma visão ou uma lente com 20 ampliações de x com uma abertura numérica de 0.35 para observação detalhada.</li>
</ol>4. microscopia de imunofluorescência
<ol><li>Reidratar slides de tecido usando uma série decrescente de etanol. Comece com 100% de etanol 2 vezes por 5 min, seguido por 3 min de etanol 95%, 80% e 70%.</li>
	<li>Execute recuperação de antígeno aquecendo as lâminas em solução de recuperação do antígeno em um navio a vapor por 20 min.</li>
	<li>Deixe arrefecer o slides até à temperatura.</li>
	<li>Depois de lavar os slides por três vezes com solução salina tamponada fosfato (PBS), aplica solução de bloqueio de antígeno em seções por 30 min.</li>
	<li>Lave slides três vezes por 5 min com PBS.</li>
	<li>Incube as seções com anticorpo primário diluído em diluente de anticorpo primário. Nota: O momento de concentração e a incubação deve ser escolhido separadamente para cada anticorpo.</li>
	<li>Lave slides três vezes por 5 min com PBS para remover anticorpos não acoplado.</li>
	<li>Incube as seções com um anticorpo secundário conjugado previamente diluído em diluente de anticorpo secundário. Nota: O momento de concentração e a incubação deve ser escolhido separadamente para cada anticorpo.</li>
	<li>Remova anticorpos desacoplados lavando os slides por 5 min, três vezes.</li>
	<li>Counterstain os núcleos de célula usando 4', 6-diamidino-2-phenylindole (DAPI) por 15 min; a concentração final de DAPI deve ser de 350 nM.</li>
	<li>Monte as lâminas na imunofluorescência compatível com solução de montagem. Nota: Usando a solução de montagem errada pode obscurecer o sinal de fluorescência.</li>
	<li>Exibição de slides com um microscópio de fluorescência. Use uma lente de ampliação de 40x com uma abertura numérica de 1.3.</li>
</ol>

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Este artigo demonstra um modelo murino para estudar o desenvolvimento de hiperplasia myointimal e permite a exploração dos processos patológicos subjacentes e os testes de novas drogas ou opções terapêuticas.
O passo mais crítico neste protocolo é o desnudamento da aorta. Cuidado especial deve ser dada durante esta etapa como denudação excessiva conduzirá à formação de aneurisma e fracasso do modelo. Por outro lado, se a denudação é executada insuficientemente, myointima muito...

Estenose arterial nas artérias coronárias e periféricas tem um grande efeito sobre a morbidade e mortalidade de pacientes1. Um mecanismo patológico subjacente é hiperplasia myointima (MH), que é caracterizada pelo aumento da proliferação, migração e síntese de proteínas da matriz extracelular do músculo liso vascular células (SMC)2. SMC estão localizados na camada de mídia do navio e migrar a estimulação para a superfície do lúmen. Sinais estimulatórios incluem fatores de crescimento, citocinas, contato célula-célula, lipídeos, componentes de matriz extracelular e cisalhamento mecânico e estendem as forças3,4,5,6. Lesões da parede do vaso, patológica ou iatrogênica, causam células endoteliais e danos nas células musculares lisas e estimulam reações inflamatórias e assim levam a MH7.
Diferentes modelos animais estão atualmente disponíveis para estudar a lesão arterial e hiperplasia myointima. Grandes animais como porcos ou cães têm a vantagem de compartilhar uma artéria semelhante e anatomia coronariana com seres humanos e são especialmente adequados para estudos investigando angioplastia dispositivos, procedimento e técnicas8. No entanto, modelos de porco têm a desvantagem da maior trombogenicidade9,10, enquanto os cães só têm uma resposta suave ao navio lesão11. Além disso, todos os grandes modelos animais exigem especial habitação, equipamentos e funcionários, que estão conectados com custos elevados e não estão sempre disponíveis na instituição. Pequenos modelos animais incluem ratos e camundongos. Em comparação com ratos, os ratos têm as vantagens de custo mais baixo e a existência de uma variedade de nocaute do modelos. O modelo descrito neste vídeo pode ser combinado com ApoE-/-os ratos alimentados com uma dieta ocidental pròxima imitar o ambiente clínico de angioplastia em vasos aterosclerótica12. Modelos anteriores induzido por lesão vascular via fio lesão13, fluido dessecação14, Primavera15ou de lesão do manguito16. Desde que a natureza da lesão afetará grandemente o desenvolvimento e a constituição de MH, utilizar um cateter balão para induzir lesões no vaso é a melhor maneira para imitar o ambiente clínico.
Neste artigo nós descrevemos um método novo para induzir MH com um cateter de balão em camundongos. O uso de um cateter balão (1,2 x 6 mm) com um RX-Port (figura 1A) permite a raspagem da camada da íntima e, ao mesmo tempo, a indução de uma hiperdistensão do navio. Ambos esses fatores são gatilhos importantes para o desenvolvimento do MH. O tempo de observação para este modelo é de 28 dias17.

<table>
	<tbody>
		<tr>
			<td>10-0 Ethilon suture</td>
			<td>Ethicon</td>
			<td>2814G</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL Syringe</td>
			<td>BD Medical</td>
			<td>309658</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 prolene suture</td>
			<td>Ethicon</td>
			<td>EH7229H</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 prolene suture</td>
			<td>Ethicon</td>
			<td>8706H</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Fuchsin</td>
			<td>Sigma-Aldrich</td>
			<td>F8129-25G</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Antigen retrieval solution</td>
			<td>Dako</td>
			<td>S1699</td>
			<td></td>
		</tr>
		<tr>
			<td>Azophloxin</td>
			<td>Waldeck</td>
			<td>1B-103</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Bepanthen Eye and Nose ointment</td>
			<td>Bayer</td>
			<td>1578675</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Charles River</td>
			<td>Stock number 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp applicator</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen 3</td>
			<td>abcam</td>
			<td>ab7778</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fischer</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG AF555</td>
			<td>Invitrogen</td>
			<td>A21432</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF488</td>
			<td>Invitrogen</td>
			<td>A11055</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF555</td>
			<td>Invitrogen</td>
			<td>A31572</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Th. Geyer</td>
			<td>2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Th. Geyer</td>
			<td>2295</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Th. Geyer</td>
			<td>2246</td>
			<td></td>
		</tr>
		<tr>
			<td>FAP</td>
			<td>abcam</td>
			<td>ab28246</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Forceps fine</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps standard</td>
			<td>Fine Science Tools</td>
			<td>11023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>537020</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Rotexmedica</td>
			<td>PZN 3862340</td>
			<td>25.000 I.E./mL</td>
		</tr>
		<tr>
			<td>High temperature cautery kit</td>
			<td>Bovie</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-iT FX Signal Enhancer</td>
			<td>Invitrogen</td>
			<td>I36933</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Light Green SF</td>
			<td>Waldeck</td>
			<td>1B-211</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Microsurgical clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Micro-Serrefine - 4mm</td>
		</tr>
		<tr>
			<td>MINI TREK Coronary Dilatation Catheter 1.20 mm x 6 mm / Rapid-Exchange</td>
			<td>Abbott</td>
			<td>1012268-06U</td>
			<td></td>
		</tr>
		<tr>
			<td>Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>1.00532.0100</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>NaCl 0,9%</td>
			<td>B.Braun</td>
			<td>PZN 06063042 Art. Nr.: 3570160</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12075-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>Serva Electrophoresis</td>
			<td>33427</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Primary antibody diluent</td>
			<td>Dako</td>
			<td>S3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Mounting solution</td>
			<td>Thermo Fischer</td>
			<td>P36930</td>
			<td>Mounting solution for immunofluorescence stained slides</td>
		</tr>
		<tr>
			<td>Replaceable Fine Tip</td>
			<td>Bovie</td>
			<td>H101</td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcin-Fuchsin Weigert</td>
			<td>Waldeck</td>
			<td>2E-30</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>400684.00.00</td>
			<td>Carprofen</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Vannas-style</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast acting Adhesive MINIS 3x1g</td>
			<td>UHU</td>
			<td>45370</td>
			<td>Cyanoacrylate</td>
		</tr>
		<tr>
			<td>Slide Rack</td>
			<td>Ted Pella</td>
			<td>21057</td>
			<td></td>
		</tr>
		<tr>
			<td>SM22</td>
			<td>abcam</td>
			<td>ab10135</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>abcam</td>
			<td>ab21027</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Staining dish</td>
			<td>Ted Pella</td>
			<td>21075</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabotamp fibrillar</td>
			<td>Ethicon</td>
			<td>431962</td>
			<td>Absorbable hemostat</td>
		</tr>
		<tr>
			<td>Transpore Surgical Tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin syringe</td>
			<td>BD Medical</td>
			<td>324825</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Dilator</td>
			<td>Fine Science Tools</td>
			<td>18603-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitro-Clud</td>
			<td>Langenbrinck</td>
			<td>04-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigerts iron hematoxylin Kit</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Th. Geyer</td>
			<td>3410</td>
			<td></td>
		</tr>
	</tbody>
</table>

balloon-based injury to induce myointimal hyperplasia in the mouse abdominal aorta

O uso de modelos animais é essencial para uma melhor compreensão do MH, uma das principais causas de estenose arterial. Neste artigo, vamos demonstrar um modelo de denudação murino de balão, que é comparável com os modelos de lesão navio estabelecido em animais de grandes porte. O modelo de desnudamento de aorta com cateteres balão imita o ambiente clínico e leva a mudanças fisiológicas e pathobiological comparáveis. Brevemente, após a realização de uma incisão horizontal na aorta abdominalis, um cateter balão será inserido dentro do vaso sanguíneo, inflado e introduziu retrogradely. Inflação do balão vai levar a lesão da íntima e hiperdistensão do navio. Depois de retirar o cateter, a incisão da aorta será fechada com pontos simples. O modelo mostrado neste artigo é reprodutível, fácil de realizar e pode ser estabelecida de forma rápida e confiável. É especialmente apropriado para avaliar caros agentes terapêuticos experimentais, que podem ser aplicados de forma econômica. Usando diferentes cepas de mata-mata-rato, pode ser avaliado o impacto de diferentes genes no desenvolvimento do MH.

Desnudamento do balão é um modelo adequado para estudar o desenvolvimento de MH em camundongos. Animais recuperar bem da cirurgia e mostram uma pós-operação condição física excelente. Nós estabelecemos este modelo em 50 ratos com menos de 3% de mortalidade devido a procedimento cirúrgico. Figuras 1B -C mostrar as principais etapas cirúrgicas. Após uma incisão de pele ao longo do linea alba, identificar a aorta abdomi...

Watch this Scientific Journal Video about Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta at JoVE.com

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

Este artigo demonstra um modelo murino para estudar o desenvolvimento de hiperplasia myointimal (MH) após lesão aórtica balão.

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 ...

balloon-based-injury-to-induce-myointimal-hyperplasia-mouse-abdominal

Research

JoVE Journal

Medicine

9.5K visualizações.  University Heart Center. Este artigo demonstra um modelo murino para estudar o desenvolvimento de hiperplasia myointimal (MH) após lesão aórtica balão.

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

The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL varies between 2.5 and 20% 2-5. Over the years, many strategies aimed at lowering the incidence of anastomotic leakage have been examined6, 7. 
The cause of AL is probably multifactorial. Etiological factors include insufficient arterial blood supply, tension on the anastomosis, hematoma and/or infection at the anastomotic site, and co-morbid factors of the patient as diabetes and atherosclerosis8. Furthermore, some anastomoses may be insufficient from the start due to technical failure.
Currently a new device is developed in our institute aimed at protecting the colorectal anastomosis and lowering the incidence of AL. This so called C-seal is a biofragmentable drain, which is stapled to the anastomosis with the circular stapler. It covers the luminal side of the colorectal anastomosis thereby preventing leakage.
The C-seal is a thin-walled tube-like drain, with an approximate diameter of 4 cm and an approximate length of 25 cm (figure 1). It is a tubular device composed of biodegradable polyurethane. Two flaps with adhesive tape are found at one end of the tube. These flaps are used to attach the C-seal to the anvil of the circular stapler, so that after the anastomosis is made the C-seal can be pulled through the anus. The C-seal remains in situ for at least 10 days. Thereafter it will lose strength and will degrade to be secreted from the body together with the gastrointestinal natural contents.
The C-seal does not prevent the formation of dehiscences. However, it prevents extravasation of faeces into the peritoneal cavity. This means that a gap at the anastomotic site does not lead to leakage.
Currently, a phase II study testing the C-seal in 35 patients undergoing (colo-)rectal resection with stapled anastomosis is recruiting. The C-seal can be used in both open procedures as well as laparoscopic procedures. The C-seal is only applied in stapled anastomoses within 15cm from the anal verge. In the video, application of the C-seal is shown in an open extended sigmoid resection in a patient suffering from diverticular disease with a stenotic colon.

The C-seal is a biofragmentable drain protecting the stapled colorectal anastomosis. In this video, details of the manufacturing, deployment and use are shown.

O C-selo: A Drenagem biofragmentável Proteção da anastomose grampeada colo-retal de fuga

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL ...

Medicina

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Ventilação a pressão controlada para induzir lesão pulmonar aguda em ratos

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

O modelo animal CYP2D6: Como induzir hepatite auto-imune em ratos

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

A Medição e Tratamento de Supressão de ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Um ferimento Modelo Alkali queima de córnea neovascularização no Rato

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

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 ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Lesão tecido isquêmico na Skinfold Câmara do mouse Dorsal: Um Modelo retalho de pele para investigar aguda isquemia persistente

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Criação de abdominais aderências em Ratos

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

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 ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Um Modelo de Cardiologia remodela Através constrição da Aorta Abdominal em Ratos

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...