Este trabajo fue apoyado por la financiación de los NIH subvenciones HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) y un proyecto de clase mundial de la Universidad (HJ) del Ministerio de Ciencia, Tecnología y Educación de Corea del Sur.

1. La ligadura parcial de la arteria carótida izquierda <ol><li> Los ratones son utilizados en ~ 8 semanas de edad. El peso de los ratones se recomienda tener al menos 18 gramos. </li><li> Esterilizar los instrumentos durante 30 segundos en el esterilizador de instrumentos de cuentas y deje que se enfríe </li><li> Inducir la anestesia con ketamina (80mg/kg) y xilazina (10mg/kg) anestesia intraperitoneal (ip) o una alternativa aprobada por IACUC. Para la inyección ip, inyectar ratón en el cuadrante inferior izquierda del abdomen. </li><li> Coloque el ratón en una cámara de calentamiento hasta que esté completamente anestesiado. Verificar la profundidad de la anestesia con la respuesta a una pizca dedo del pie. </li><li> Lugar del ratón en una etapa quirúrgica, en posición supina (boca arriba vientre lado) posición. Tape las patas delanteras, las palmas hacia arriba y la única patas traseras hasta el escenario. </li><li> Aplicar generosa cantidad de agente depilatorio (por ejemplo, Nair) en el cuello entre la mandíbula y el esternón. Masajear suavemente hasta que todo el pelo se elimina. </li><li> Limpie el agente depilatorio y aplicar generosa cantidad de Betadine al área depilada. </li><li> Prepare una pulgada de largo secciones de sutura de seda 6-0 para ligaduras. Cada ratón se necesitan dos piezas de sutura. </li><li> Utilizando unas tijeras pequeñas afilado, haga una incisión vertical 4-6mm en la parte media del cuello. La piel y la fascia subyacente necesita ser cortado. </li><li> Mueva la glándula parótida izquierda de la línea media hacia la izquierda y comenzar la disección sin rodeos apenas a la derecha de la tráquea. Usted debe ser capaz de identificar la palpitante arteria carótida común izquierda con facilidad. </li><li> Siga la arteria carótida común caudalmente hasta encontrar la bifurcación. </li><li> Sin rodeos disecar la bifurcación para exponer los siguientes cuatro ramas distales de la arteria carótida común izquierda: arteria carótida externa, arteria carótida interna, arteria occipital y la arteria tiroidea superior. Estos vasos, tiroides, especialmente superior, puede ser frágil. Tenga mucho cuidado con su disección. </li><li> Ligar la carótida externa por encima de la tiroidea superior con sutura de seda 6-0. Para ello, pase una pinza por debajo de la arteria y agarrar un pedazo de sutura precortados con la ayuda de su pinza de otros. Tire suavemente de la sutura que pasan por debajo de la arteria. Ate firmemente evitando cualquier tejido circundante está atrapado en el nudo. Si te atan por debajo del despegue de la arteria tiroidea superior, al final del procedimiento de que efectivamente se ha realizado una ligadura completa que es un modelo muy diferente. </li><li> Ligar las arterias carótidas internas y occipital, con un nudo con la misma técnica. </li><li> Verificar la posición de las suturas y la permeabilidad de la arteria tiroidea superior. </li><li> Aproximado de la piel y la piel cerca usando una pequeña cantidad de tejido-Mend. </li><li> Los ratones en una cámara de calentamiento hasta la recuperación. Recuperación por lo general tarda de 30 a 60 minutos. </li><li> Una sola dosis de buprenorfina (0.1mg/kg) debe administrarse inmediatamente después de la cirugía si la anestesia que se utiliza no proporciona alivio del dolor prolongado (es decir, inhalación isoflurano) o si el animal está en peligro en las primeras 24 horas. En nuestra experiencia, la dosis de ketamina para anestesia administrada proporciona una analgesia suficiente cuando la cirugía se lleva a cabo por un cirujano experimentado y no se necesita más analgesia. </li></ol> 2. Ecografía carotídea <ol><li> Para validar si la ligadura parcial inducida perturbado el flujo, el flujo de la carótida debe examinar un día después de la ligadura </li><li> Inducir la anestesia con isoflurano - Caja de inducción de la anestesia podría ser utilizado si es necesario </li><li> Lugar ratones anestesiados en el escenario de imágenes, lado boca arriba </li><li> Los brazos y las piernas para cinta ECG sensor de imagen en el escenario </li><li> Defoliar cuello mediante la aplicación liberal de Nair (este paso sólo es necesario si no lo ha hecho para el procedimiento de ligadura) </li><li> Inserte el termómetro rectal para registrar la temperatura </li><li> Aplicar gel eco en el cuello </li><li> Inicio en modo-B. </li><li> Plano de la imagen debe ser de 90 grados (el mando de la sonda debe estar orientada hacia la nariz del ratón) </li><li> Inferior de la sonda en el cuello del ratón hasta que la imagen se obtiene. </li><li> Manipular la izquierda del escenario y la derecha hasta la tráquea (en la línea media la estructura) se identifica </li><li> Identificar la arteria carótida común </li><li> Etapa de formación de imágenes de inclinación de manera que un ángulo se crea entre el plano de la imagen y la carótida. Cuanto mayor sea este ángulo, mayor será la señal Doppler se. </li><li> Coloque la onda de pulso Doppler en medio de la carótida común izquierda. </li><li> Utilizar la corrección de ángulo según sea necesario. </li><li> Repetir las mediciones en la carótida común derecha. </li><li> El éxito de la ligadura de la carótida parcial resultará en una reducción general en el flujo (~ 80-90%) en la arteria carótida común izquierda en comparación con el lado derecho, con inversión del flujo de entrada hacia la aorta durante la diástole. </li><li> Una vez que las imágenes se han obtenido satisfactoria, apague la anestesia, limpie echo gel de los ratones y los ratones libres de resolución de la cintatraints. Coloque los ratones en una cámara de recuperación caliente. </li></ol> 3. La íntima de la carótida de aislamiento de ARN <ol><li> Sacrificio de los ratones por inhalación de CO 2 de acuerdo con el protocolo institucional IACUC </li><li> Tape las patas de una toalla de papel. </li><li> Cortar la piel del ratón desde el abdomen hasta la parte superior del tórax. </li><li> Abrir la pared abdominal por debajo de la caja torácica con un par de tijeras afiladas. </li><li> Levantar el esternón con una pinza y corta el diafragma, y ​​luego cortar la caja torácica para exponer el corazón. </li><li> Corte de la vena cava con unas tijeras. </li><li> La presión de perfusión (120 mmHg) de 2 a 3 minutos con solución salina normal contiene 10 unidades / mL de heparina a través del ventrículo izquierdo hasta que los pulmones y el hígado se vuelven pálidos. </li><li> Cortar la piel del cuello y retirar toda la grasa, los músculos y tejidos conectivos hasta las arterias carótidas están expuestos. </li><li> Coloque el ratón bajo un microscopio de disección. </li><li> Haga un agujero justo debajo de los sitios de ligadura de la arteria carótida izquierda para la segunda perfusión. </li><li> La presión de perfusión de nuevo ~ 1 minuto con solución salina normal contiene 10 unidades / mL de heparina a través del ventrículo izquierdo, asegurándose de la carótida izquierda y examinaba. </li><li> Utilizando una pinza de punta fina y tijeras pequeñas de primavera, retire con cuidado los tejidos peri-adventicia que rodea la carótida. Tenga cuidado de no apretar o estirar las carótidas durante este proceso de limpieza. </li><li> Corte de la arteria carótida izquierda entre el arco aórtico y los puntos de ligadura por encima de la bifurcación de la carótida. </li><li> Corte de la arteria carótida derecha entre el punto de arteria subclavia derecha y la ramificación de la bifurcación de la carótida. </li><li> Transferencia de las carótidas con un plato mm cultura 35, que contienen agua helada HBSS. Si es necesario, retire con cuidado cualquier resto de tejido peri-adventicia. </li><li> Prepare una jeringa de insulina (3 / 10 ml jeringa) con una aguja 29G, llenando con 150 l buffer de lisis QIAsol (Qiagen) por cada carótida. </li><li> Con cuidado, inserte la punta de la aguja en un extremo de la arteria carótida. </li><li> Mientras mantiene la carótida y la punta de la aguja con una pinza, de forma rápida (~ 1 segundo) a ras de la solución amortiguadora de lisis QIAsol (150 l) en un tubo de 1.5 ml (eluido íntima). </li><li> Enjuague los restos de la carótida (los medios de comunicación + adventicia) de vez en HBSS, lo puso en un tubo de 1.5 ml, y el complemento congelación en nitrógeno líquido. </li><li> Eluido íntima y las sobras congeladas se utilizan para la íntima o medios de aislamiento + adventicia de ARN utilizando el miRNeasy Mini Kit (Qiagen) de acuerdo con las instrucciones del fabricante. </li></ol> 4. Los resultados representativos: Nos parcialmente ligado izquierdo del ratón arterias carótidas y ecografía se llevó a cabo un día después del procedimiento. Como se muestra en la Figura 1, la ligadura parcial de éxito se reduce la velocidad del flujo sanguíneo y el flujo se invierte (alteración del flujo) en la arteria carótida común izquierda durante la diástole. La ligadura parcial de la ApoE knockout (KO) ratones acompañado de una dieta rica en grasas induce la aterosclerosis robusta en la arteria carótida común (LCA), pero no en la carótida derecha contralateral, en dos semanas (Figura 2). ARN recogido de la íntima carotídea contiene genes marcadores endoteliales como PECAM-1, mientras que sin medial del músculo liso genes marcadores de células tales como SMA (Figura 3). <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Figura 1" /> Figura 1: Esquema de la anatomía de la carótida y la ligadura: Tres ramas de la arteria carótida común (LCA) [arteria carótida externa (ACE), la arteria carótida interna (ICA), y la arteria occipital (OA)] se ligaron, dejando la arteria tiroidea superior (STA) abierto. <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Figura 2" /> Figura 2: Las imágenes de ultrasonido muestran los perfiles de velocidad del flujo y revelan que la ligadura parcial provoca la inversión del flujo (indicado por las flechas) en el LCA durante la diástole. Flujo de RCA se mantiene sin cambios después de la ligadura. <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Figura 3" /> Figura 3: ligadura parcial y la dieta rica en grasas induce rápidamente la aterosclerosis en la ECV de ApoE ratones ApoE KO ratones KO se ligaron parcial y alimentados con la dieta alta en grasa de 1 a 3 semanas.. Las secciones congeladas de LCA fueron teñidos con aceite rojo-O. <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Figura 4" /> Figura 4. Método de la íntima preparación de ARN. Íntima y media de ARN + adventicia (m + a) ARN se obtuvieron a partir de farsa que funciona con RCA y CLP en ratones C57BL / 6. ARN se analizaron semi-cuantitativos de RT-PCR (A) y qPCR (B, C) ​​de PECAM-1 y α-SMA con 18 años como un control interno. Los gráficos de barras son la media ± SEM, 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>

En este procedimiento, la identificación adecuada de todas las ramas de la carótida común, incluyendo la carótida externa, la carótida interna, arteria occipital y la arteria tiroidea superior es esencial. También es importante verificar el patrón de flujo con ultrasonido como se ha demostrado. En nuestras manos experimentadas, nuestra tasa de éxito de las cirugías de ligadura parcial basado en los estudios de ultrasonido de más de 500 ratones es superior al 90%. De la carótida íntima preparaciones de ARN, e...

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<td>Normal Saline (0.9% sodium chloride)</td>
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<td>Baxter</td>
<td>2B1323</td>
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<td>Insulin syringe (3/10 ml 29g syringe)</td>
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<td>Tyco Health care</td>
<td>8881600145</td>
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<td>HBSS</td>
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<td>Vevo 770 High-Resolution Micro-Imaging System</td>
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<td>6-0 Silk Suture, sterile</td>
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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

A pesar de la estrecha asociación conocida, evidencia la vinculación directa alteración del flujo de la aterogénesis ha estado ausente. Recientemente, hemos utilizado una versión modificada de la carótida métodos de ligadura parcial [1,2] para demostrar que forma aguda induce condiciones de flujo bajo y oscilante, dos características fundamentales de la alteración del flujo, en el ratón de la arteria carótida común. El uso de este modelo, hemos proporcionado evidencia directa de que el flujo alterado de hecho conduce a un desarrollo rápido y robusto aterosclerosis en ratones apolipoproteína E nocaut [3]. También hemos desarrollado un método de preparación de ARN endoteliales de alta pureza a partir de la íntima carotídea ratón [3]. El uso de este modelo de ratón y el método, se encontró que la ligadura parcial provoca la disfunción endotelial en una semana, seguido por la formación de ateroma robusto y rápido de dos semanas en un modelo de ratón hiperlipidémicos junto con las características de la formación de lesiones complejas, como la neovascularización intraplaca por cuatro semanas. Esta rápida<em&gt; In vivo</emModelo&gt; y el endotelio método de preparación de ARN podría ser utilizado para determinar los mecanismos moleculares subyacentes de flujo dependen de la regulación de la biología y las enfermedades vasculares. Además, podría ser utilizado para poner a prueba diversas intervenciones terapéuticas dirigidas a la disfunción endotelial y aterosclerosis en la duración del estudio reducido considerablemente.

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

Esto describe una cirugía de ligadura de la carótida parcial, lo que provoca trastornos condiciones de flujo y posterior desarrollo de la aterosclerosis (en dos semanas) con intraplaca neovascularización (en cuatro semanas) en el ratón de la arteria carótida común. También describe un nuevo método de aislamiento de ARN de la carótida íntima, proporcionando ARN de alta pureza endoteliales.

Un modelo de Disturbed inducida por el flujo de Aterosclerosis en la arteria carótida del ratón por la ligadura parcial y un método sencillo para el aislamiento de ARN del endotelio carótida

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 vistas.  Emory University. Esto describe una cirugía de ligadura de la carótida parcial, lo que provoca trastornos condiciones de flujo y posterior desarrollo de la aterosclerosis (en dos semanas) con intraplaca neovascularización (en cuatro semanas) en el ratón de la arteria carótida común. También describe un nuevo método de aislamiento de ARN de la carótida íntima, proporcionando ARN de alta pureza endoteliales.

Video: 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.

Implantación de un manguito de la carótida para provocar esfuerzo cortante aterosclerosis inducida en ratones

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

Investigación

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.

La formación de una sofisticada técnica de microcirugía: La interposición de injerto de la vena yugular externa en la arteria carótida común en ratas

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.

Un modelo murino de la implantación de un stent en la arteria carótida para el Estudio de la reestenosis

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.

Un sencillo método de intubación ratón de pulmón

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.

Adultos Mouse hipertensión venosa: Modelo: arteria carótida común a la vena yugular externa anastomosis.

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.

La cateterización de la arteria carótida y la vena yugular para realizar mediciones hemodinámicas, Infusiones y muestras de sangre en un modelo de rata Consciente

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.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

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.

Trombosis modelo de ratón de la arteria carótida y Mesenterio Embarcaciones inducida por cloruro férrico-

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.

En Vivo Transferencia génica en el endotelio de la arteria carótida común conejo

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.

Un modelo de conejo de expresión del transgen Durable en la vena yugular a los injertos de interposición de la arteria carótida común

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