Este trabalho foi financiado por recursos do NIH concede HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) e um World Class University Project (HJ) do Ministério da Ciência, Tecnologia e Educação de S. Korea.

1. Ligadura parcial da artéria carótida esquerda <ol><li> Mouses são utilizados em ~ 8 semanas de idade. O peso dos ratos é recomendado para ser pelo menos 18 gramas. </li><li> Esterilizar os instrumentos para 30 segundo no esterilizador instrumento talão e deixe esfriar </li><li> Induzir a anestesia com Ketamina (80mg/kg) e xilazina (10mg/kg) intraperitonial injeção (ip) ou alternativa anestesia aprovado pelo IACUC. Para injeção ip, injetar mouse no quadrante esquerdo baixo do abdômen. </li><li> Colocar o mouse em uma câmara de aquecimento até que esteja completamente anestesiado. Verifique a profundidade da anestesia com resposta a pitada dedo do pé. </li><li> Colocar o mouse em um estágio cirúrgico, em decúbito dorsal (barriga para cima lado) posição. Tape as patas fore-palmas para cima e na sola patas traseiras, até o palco. </li><li> Aplique uma quantidade liberal de agente depilatório (por exemplo, Nair) no pescoço entre a mandíbula eo esterno. Massageie suavemente até que todo o cabelo é removido. </li><li> Limpe o agente depilatórios e aplicar quantidade generosa de Betadine para a área depilada. </li><li> Prepare uma polegada de comprimento seções de 6-0 seda para ligadura. Cada rato vai precisar de dois pedaços de sutura. </li><li> Usando uma tesoura afiada pequena, faça uma incisão 4-6mm vertical no meio do pescoço. A pele e da fáscia subjacente precisam ser cortadas. </li><li> Mover a parótida esquerda da linha média para a esquerda e começar a dissecar sem rodeios logo à direita da traquéia. Você deve ser capaz de identificar a carótida esquerda pulsante comum facilmente. </li><li> Siga as carótida comum caudalmente até localizar a bifurcação. </li><li> Sem rodeios dissecar a bifurcação para expor os seguintes quatro ramos distais a artéria carótida comum esquerda: artéria carótida externa, artéria carótida interna, artéria occipital e da artéria tireóidea superior. Estas embarcações, de tireóide, especialmente superior, pode ser frágil. Tenha muito cuidado com o seu esvaziamento. </li><li> Empate fora da carótida externa ACIMA superior da tireóide 6-0 fio de seda. Para fazer isso, passe um fórceps debaixo da artéria e pegar um pedaço de sutura precut com a ajuda do seu fórceps outros. Puxe o fio para passar debaixo da artéria. Amarre firmemente evitando qualquer tecido circundante ser pego no nó. Se você amarrar abaixo da decolagem da artéria tireoidiana superior, até o final do procedimento você vai efetivamente ter realizado uma ligadura completa que é um modelo substancialmente diferente. </li><li> Amarrar as artérias carótida interna e occipital, com um nó usando a mesma técnica. </li><li> Verificar a posição de suturas e patência da artéria tireóidea superior. </li><li> A pele ea pele aproximada perto usando uma pequena quantidade de tecido-Mend. </li><li> Camundongos lugar em uma câmara de aquecimento até a recuperação. Recuperação normalmente leva 30 a 60 minutos. </li><li> Uma única dose de buprenorfina (0.1mg/Kg) deve ser administrado imediatamente após a cirurgia, se a anestesia utilizada não proporcionar alívio da dor prolongada (ou seja, isoflurano inalado) ou se o animal está em perigo nas primeiras 24 horas. Em nossa experiência, a dose de quetamina administrada para anestesia fornece analgesia suficiente quando a cirurgia é realizada por cirurgião experiente e sem analgesia adicional é necessária. </li></ol> 2. Carótida exame ultra-sonográfico <ol><li> Para validar se a ligadura parcial induzida perturbado fluxo, fluxo carotídeo devem ser examinados um dia pós-ligadura </li><li> Induzir a anestesia com isoflurano - caixa de indução da anestesia pode ser usada, se necessário </li><li> Coloque os ratos anestesiados no palco de imagem, lado de barriga para cima </li><li> Tape braços e pernas para ECG sensor de imagem no palco </li><li> Desfolhar pescoço pela aplicação liberal de Nair (esta etapa só é necessária se não tiver feito para o procedimento ligadura) </li><li> Insira o termômetro retal para registro de temperatura </li><li> Aplique gel eco no pescoço </li><li> Iniciar em modo-B. </li><li> Plano de imagem deve ser de 90 graus (botão na sonda deve apontar para o nariz do mouse) </li><li> Menor sonda para o pescoço do mouse até a imagem é obtida. </li><li> Manipular estágio esquerda e direita até traquéia (mid-line estrutura) é identificado </li><li> Identificar artéria carótida comum esquerda </li><li> Estágio de imagem inclinação, para que um ângulo é criado entre o plano de imagem e da carótida. Quanto maior o ângulo isso, o forte é o sinal Doppler será. </li><li> Lugar onda de pulso Doppler no meio da carótida comum esquerda. </li><li> Use correção do ângulo, conforme necessário. </li><li> Repetir as medições sobre a carótida comum direita. </li><li> Ligadura parcial da carótida sucesso resultará em redução global no fluxo (~ 80-90%) na carótida comum esquerda em comparação com o lado direito, com reversão do fluxo de entrada para a aorta durante a diástole. </li><li> Uma vez que as imagens foram satisfatórios obtidos, desligue a anestesia, limpe echo off gel ratos e camundongos livre de res fitatraints. Coloque os ratos na câmara de recuperação aquecido. </li></ol> 3. Isolamento de RNA íntima carótida <ol><li> Sacrifício camundongos por inalação de CO 2 de acordo com o protocolo institucional IACUC </li><li> Tape as patas a uma toalha de papel. </li><li> Cortar a pele do rato do abdômen para o topo do tórax. </li><li> Abrir a parede abdominal abaixo da caixa torácica com uma tesoura afiada. </li><li> Levantar o esterno com uma pinça e corte o diafragma e, em seguida cortar as costelas para expor o coração. </li><li> Cortar a veia cava com uma tesoura. </li><li> Perfundir pressão (120 mmHg) por 2 a 3 min com solução salina contendo 10 Unidades / mL de heparina através do ventrículo esquerdo até os pulmões e fígado tornar-se pálida. </li><li> Cortar a pele do pescoço e remover toda a gordura, músculos e tecidos conectivos até as artérias carótidas são expostos. </li><li> Posicione o mouse em um microscópio de dissecação. </li><li> Punção um furo logo abaixo os sites ligadura na carótida esquerda para a segunda perfusão. </li><li> Perfundir pressão novamente para min ~ 1 com soro fisiológico contendo 10 Unidades / mL de heparina através do ventrículo esquerdo, certificando-se a carótida esquerda é bem perused. </li><li> Usando uma pinça de ponta fina e uma tesoura pequena mola, remova cuidadosamente os tecidos peri-adventícia ao redor das carótidas. Tenha cuidado para não espremer ou esticar a carótidas durante esta etapa de limpeza. </li><li> Cortar a artéria carótida esquerda entre o arco aórtico e os pontos de ligação acima da bifurcação da carótida. </li><li> Cortar a artéria carótida direita entre o ponto de ramificação da artéria subclávia direita e na bifurcação da carótida. </li><li> Transferência das carótidas a um prato 35 milímetros cultura, contendo gelada HBSS. Se necessário, remova cuidadosamente qualquer tecido peri-adventícia restantes. </li><li> Prepare uma seringa de insulina (3 / seringa de 10 ml) com uma agulha de 29G, preencha com 150 mL de buffer QIAsol lise (Qiagen) por cada carótida. </li><li> Insira cuidadosamente a ponta da agulha em uma das extremidades da carótida. </li><li> Enquanto mantém a carótida ea ponta da agulha com uma pinça, rapidamente (~ 1 seg) liberar o buffer de lise QIAsol (150 mL) em um tubo de 1,5 ml (eluato íntima). </li><li> Lavar o restante da carótida (media + adventícia) de vez em HBSS, colocá-lo em um tubo de 1,5 ml, e encaixe-congelamento em nitrogênio líquido. </li><li> Intima eluato e os restos congelados são então utilizados para o isolamento + íntima ou mídia RNA adventícia usando o mini-kit miRNeasy (QIAGEN), de acordo com as instruções do fabricante. </li></ol> 4. Resultados representativos: Nós parcialmente ligada esquerdo do mouse artérias carótidas e ultra-sonografia foi realizada um dia após o procedimento. Conforme mostrado na Figura 1, ligadura parcial sucesso reduz a velocidade do fluxo sanguíneo e inverte o fluxo (fluxo perturbado) na artéria carótida comum esquerda durante a diástole. Ligadura parcial da ApoE knockout (KO) ratos acompanhados por uma dieta rica em gordura induz a aterosclerose robusta na artéria carótida comum esquerda (LCA), mas não na carótida direita contralateral, por duas semanas (Figura 2). RNA coletados a partir da íntima carótida contém genes marcadores endoteliais como PECAM-1, enquanto livre de medial do músculo liso genes de células marcador como um SMA (Figura 3). <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Figura 1" /> Figura 1: Esquema da anatomia carótida e ligadura: Três ramos da artéria carótida comum esquerda (LCA) [artéria carótida externa (ACE), artéria carótida interna (ACI) e artéria occipital (OA)] foram ligados, deixando a artéria tireóidea superior (STA) aberto. <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Figura 2" /> Figura 2: As imagens de ultra-som mostram perfis de velocidade de fluxo e revelam que a ligadura parcial induz inversão de fluxo (indicado por setas) na LCA durante a diástole. Fluxo em RCA permanece inalterado após a ligadura. <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Figura 3" /> Figura 3: ligadura parcial e alto teor de gordura da dieta rapidamente induz aterosclerose em camundongos ApoE LCA de camundongos ApoE KO KO foram parcialmente ligada e alimentados com a dieta rica em gordura para 1-3 semanas.. Cortes congelados de LCA foram coradas com Oil Red-O-. <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Figura 4" /> Figura 4. Método de preparação RNA íntima. RNA íntima e média + adventícia (m + a) RNA foram obtidos a partir sham-operado RCA e LCA em camundongos C57BL / 6. RNAs foram analisados ​​por semi-quantitativa de RT-PCR (A) e qPCR (B, C) ​​para PECAM-1 e α-SMA utilizando 18 anos como um controle interno. Gráficos de barras são média ± EPM, n = 3.

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li></ol>

Neste procedimento, a identificação adequada de todos os ramos da carótida comum, incluindo a carótida externa, carótida interna, artéria occipital e da artéria tireóidea superior é essencial. Também é importante verificar o padrão de fluxo com ultra-som, como demonstrado. Em nossas mãos experientes, nossa taxa de sucesso de cirurgias ligadura parcial com base nos estudos de ultra-som de mais de 500 ratos é melhor do que 90%. Para a carótida preparações íntima RNA, é importante prestar atenção à ap...

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a model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of rna isolation from carotid endothelium

Apesar da conhecida associação perto, evidências diretas ligando fluxo perturbado a aterogênese tem faltado. Recentemente, usou uma versão modificada do carótida métodos de ligadura parcial [1,2] para mostrar que induz agudamente condições de baixo fluxo e oscilatório, duas características-chave do fluxo perturbado, no rato artéria carótida comum. Usando esse modelo, nós fornecemos evidências diretas de que o fluxo perturbado realmente leva ao desenvolvimento de aterosclerose rápido e robusto em apolipoproteína rato knockout E [3]. Também desenvolveu um método de preparação de RNA endotelial com elevado grau de pureza da carótida íntima do mouse [3]. Usando este modelo de mouse e método, encontramos que a ligadura parcial causa disfunção endotelial em uma semana, seguido pela formação de ateromas robusta e rápida em duas semanas em um modelo de camundongo hiperlipidêmicos, juntamente com recursos de formação de lesão complexa como a neovascularização intraplaca por quatro semanas. Essa rápida<em&gt; In vivo</em&gt; Modelo eo método de preparação endotelial RNA poderia ser usado para determinar os mecanismos moleculares subjacentes fluxo de regulação dependente da biologia vascular e doenças. Além disso, ele poderia ser usado para testar várias intervenções terapêuticas visando a disfunção endotelial e aterosclerose na duração do estudo consideravelmente reduzido.

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

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

Esta descreve uma cirurgia de ligadura parcial da carótida, o que causa as condições de fluxo perturbado e desenvolvimento da aterosclerose subsequente (em duas semanas) com intraplaca neo-vascularização (em quatro semanas) em ratos artéria carótida comum. Descrevemos também um novo método de isolamento de RNA a íntima carótida, proporcionando RNA de alta pureza endoteliais.

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

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

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K visualizações.  Emory University. Esta descreve uma cirurgia de ligadura parcial da carótida, o que causa as condições de fluxo perturbado e desenvolvimento da aterosclerose subsequente (em duas semanas) com intraplaca neo-vascularização (em quatro semanas) em ratos artéria carótida comum. Descrevemos também um novo método de isolamento de RNA a íntima carótida, proporcionando RNA de alta pureza endoteliais.

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Implantação de um Cuff Carotid para Triggering Shear-estresse aterosclerose induzida em camundongos

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Medicina

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 ...

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

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

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

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

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

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

Cateterismo da artéria carótida e jugular para executar as medidas hemodinâmicas, infusões e Sangue de amostragem em um Rat Consciente Modelo

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

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

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

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

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

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

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

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

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

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

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

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Na Vivo Transferência de genes para o endotélio da artéria carótida comum de coelho

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

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, ...