Este proyecto está parcialmente financiado por el NIH T32 GM008440 a Espen Walker, R01 NS27713 a William L.Young, P01 NS44155 a William L.Young y Hua Su, R21 NS070153 a Hua SU y por la Asociación Americana del Corazón AHA 10GRNT3130004 a Hua Su. Dr. Ana Rodríguez-Hernández es apoyado por una beca de &quot;la Obra Social La Caixa&quot;

1. Preparación del ratón <ol><li> Inducir la anestesia general en el ratón con el gas isoflurano. Inyectar 0,15 ml de brupenorphine intraperitoneal para el tratamiento del dolor. Antes de continuar, compruebe si el nivel de anestesia es satisfactoria pinchando patas del ratón. </li><li> Ponga el mouse en decúbito dorsal con las cuatro extremidades fijados por cinta adhesiva. Quitar el pelo del cuello y la parte superior del pecho con unas tijeras. A través de inyección subcutánea, administrar 0,2-0,4 ml de 0,9% de solución salina para mantener el ratón hidratado durante el procedimiento quirúrgico. </li><li> Preparar el campo operatorio después de un estricto método estéril. El área de la incisión de la piel se debe limpiar con un 90% de alcohol. </li></ol> 2. La disección de la arteria carótida común y la vena yugular externa <ol><li> Haga una línea media incisión cervical horizontal a través de la zona del cuello inferior del ratón. Después de la profundización de la herida, elevar las glándulas salivales y el tejido blando cervical utilizando por losutura raction (Figura 1). Exponer la vena yugular externa derecha (VYE) lateral al músculo esternocleidomastoideo (ECM). Este paso se debe realizar bajo el microscopio, ya que la tracción excesiva sobre la vena podría dañarlo y provocar su trombosis. </li><li> Diseccionar cuidadosamente la VYE derecha a lo largo de su curso desde la clavícula hasta la base del cráneo. Por lo general, pinza bipolar eléctricos, se coagulan y se dividen las ramas para preparar una longitud adecuada para la posterior colocación de clip temporal y anastomosis. </li><li> Lateral a la tráquea y medial al SMC, explorar la arteria carótida común (CCA). Se debe ser cuidadosamente expuesta desde la clavícula hasta más allá de su bifurcación en las arterias carótidas externa e interna. Durante esta etapa, se debe prestar atención de nuevo para evitar la tracción excesiva a la VYE que luego pudiera comprometer la permeabilidad de la anastomosis. </li></ol> 3. Preparación de la anastomosis <ol><li> Ligar la CCA con 10-0 Nylon justo proximal a su bifurcación. A continuación, aplicar un clip temporal sobre el proximal CCA tan cerca de la clavícula como sea posible. </li><li> Una vez que el flujo ha sido interrumpido, seccionar la arteria justo debajo de la ligadura de bifurcación y regar con solución salina para lavar cualquier resto de la sangre en el interior del lumen. Evitar la coagulación bipolar en este paso, desde la lesión térmica a la pared de la arteria podría poner la anastomosis futuro en peligro. </li><li> A fin de mejorar la visibilidad de los bordes de la VYE, la pared medial está marcado con un marcador azul a lo largo del curso de la venotomía planeado. Una vez que el VYE está marcado, utilizar una sutura 10-0 para ligar el extremo distal como caudal como sea posible y aplicar un clip vascular temporal en el extremo proximal como craneal como sea posible. </li><li> Con una fina aguja 30 G y 0,5 ml de la jeringa, hacer una abertura inicial a lo largo de la zona marcada de la VYE e inmediatamente irrigar el lumen con solución salina para evitar la formación de trombos. A continuación, extender el venotomía con las microtijerashasta que la longitud es de aproximadamente 2-3 veces el diámetro de la CCA. Evite violento estiramiento y prestar atención a mantener un filo agudo y ordenada. </li><li> Aproximar el final de la CCA a la VYE. Hacer una incisión de corte lateral en el extremo donante de la CCA para ajustar el diámetro a la longitud al tamaño de venotomía (Figura 2). </li></ol> 4. De extremo a lado anastomosis <ol><li> Utilice una sutura de nylon 11-0 monofilamento para la anastomosis-CCA-a VYE de extremo a lateral. La sutura de la pared medial de la anastomosis en una dirección cráneo-caudal es el paso inicial. Cada puntada debe ser colocado desde fuera en la pared venosa primera (Figura 3) y desde dentro hacia fuera de la pared arterial (Figura 4) siguiente. Esto mantendrá el nudo en la superficie exterior de los vasos anastomóticos en todo momento (Figura 5). De cualquier interrumpidos o suturas continuas pueden ser utilizados, pero todas las suturas continuas deben ser apretados como el paso final. </li><li> Ona vez que la pared medial ha sido suturada, repita el procedimiento a partir del caudal a craneal con la pared lateral. Ahora cada puntada debe ser colocado desde fuera en la pared arterial primero y desde dentro hacia fuera de la pared venosa siguiente. La irrigación salina ayudará a mantener el lumen de la anastomosis visible en todo momento durante el procedimiento. </li><li> Después de terminar todos los pasos de la anastomosis, retire el clip temporal de la vena de primera y de la arteria siguiente. La sangre arterial fluirá en el VYE con poca o ninguna supuración de la anastomosis (ver vídeos modelo VH y el modelo VH 2). El sangrado mínimo debe dejar sin utilizar compresión de algodón sobre la anastomosis. Esto debe evitarse con el fin de prevenir la trombosis de la vena delicada. </li><li> Una vez que el flujo pulsátil a través de la anastomosis se confirma y se observa ningún sangrado aparente, irrigar el campo quirúrgico con solución salina y cerrar la incisión cervical con una sutura de nylon 6-0. Finalmente, administrar 0,15 ml más de intrapbrupenorphine eritoneal para el manejo del dolor postoperatorio y 0,2-0,4 ml de 0,9% de solución salina subcutánea para reponer cualquier pérdida de sangre durante la cirugía. </li></ol>

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	<li>Bederson, J. B., Wiestler, O. D., Br&#252;stle, O., Roth, P., Frick, R., Ya&#351;argil, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+venous+hypertension+and+the+effects+of+venous+outflow+obstruction+in+a+rat+model+of+arteriovenous+fistula.">Intracranial venous hypertension and the effects of venous outflow obstruction in a rat model of arteriovenous fistula.</a> Neurosurgery. 29, 341-350 (1991).</li><li>Gao, P., Zhu, Y., Ling, F., Shen, F., Lee, B., Gabriel, R. A., Hao, Q., Yang, G. Y., Su, H., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonischemic+cerebral+venous+hypertension+promotes+a+pro-angiogenic+stage+through+HIF-1+downstream+genes+and+leukocyte-derived+MMP-9.">Nonischemic cerebral venous hypertension promotes a pro-angiogenic stage through HIF-1 downstream genes and leukocyte-derived MMP-9.</a> J. Cereb. Blood Flow Metab. 29, 1482-1490 (2009).</li><li>Kim, H., Su, H., Weinsheimer, S., Pawlikowska, L., Brain Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=arteriovenous+malformation+pathogenesis:+a+response-to-injury+paradigm.">arteriovenous malformation pathogenesis: a response-to-injury paradigm.</a> Acta Neurochir. Suppl. 111, 83-92 (2011).</li><li>Lawton, M. T., Arnold, C. M., Kim, Y. J., Bogarin, E. A., Stewart, C. L., Wulfstat, A. A., Derugin, N., Deen, D., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+arteriopathy+in+the+transgenic+arteriovenous+fistula+model.">Radiation arteriopathy in the transgenic arteriovenous fistula model.</a> Neurosurgery. 62, 1129-1138 (2008).</li><li>Lawton, M. T., Stewart, C. L., Wulfstat, A. A., Derugin, N., Hashimoto, T., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transgenic+arteriovenous+fistula+in+the+rat:+an+experimental+model+of+gene+therapy+for+brain+arteriovenous+malformations.">The transgenic arteriovenous fistula in the rat: an experimental model of gene therapy for brain arteriovenous malformations.</a> Neurosurgery. 54, 1463-1471 (2004).</li><li>Schaller, B., Graf, R., Sanada, Y., Tolnay, M., Rosner, G., Wienhard, K., Heiss, W., D, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+changes+after+occlusion+of+the+posterior+superior+sagittal+sinus:+an+experimental+PET+study+in+cats.">Hemodynamic changes after occlusion of the posterior superior sagittal sinus: an experimental PET study in cats.</a> AJNR Am J Neuroradiol. 24, 1876-1880 (2003).</li><li>Zhu, Y., Lawton, M. T., Du, R., Shwe, Y., Chen, Y., Shen, F., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+hypoxia-inducible+factor-1+and+vascular+endothelial+growth+factor+in+response+to+venous+hypertension.">Expression of hypoxia-inducible factor-1 and vascular endothelial growth factor in response to venous hypertension.</a> Neurosurgery. 59, 687-696 (2006).</li><li>Herman, J. M., Spetzler, R. F., Bederson, J. B., Kurbat, J. M., Zabramski, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+of+a+dural+arteriovenous+malformation+in+a+rat+model.">Genesis of a dural arteriovenous malformation in a rat model.</a> J. Neurosurg. 83, 539-545 (1995).</li><li>Terada, T., Higashida, R. T., Halbach, V. V., Dowd, C. F., Tsuura, M., Komai, N., Wilson, C. B., Hieshima, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+acquired+arteriovenous+fistulas+in+rats+due+to+venous+hypertension.">Development of acquired arteriovenous fistulas in rats due to venous hypertension.</a> J. Neurosurg. 80, 884-889 (1994).</li><li>Yassari, R., Sayama, T., Jahromi, B. S., Aihara, Y., Stoodley, M., Macdonald, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiographic,+hemodynamic+and+histological+characterization+of+an+arteriovenous+fistula+in+rats.">Angiographic, hemodynamic and histological characterization of an arteriovenous fistula in rats.</a> Acta Neurochir (Wien). 146, 495-504 (2004).</li><li>Solheim, O., Skeidsvoll, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+global+amnesia+may+be+caused+by+cerebral+vein+thrombosis.">Transient global amnesia may be caused by cerebral vein thrombosis.</a> Med. Hypotheses. 65, 1142-1149 (2005).</li><li>Yang, B., Shergill, U., Fu, A. A., Knudsen, B., Misra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+arteriovenous+fistula+model.">The mouse arteriovenous fistula model.</a> J Vasc Interv Radiol. 20, 946-950 (2009).</li><li>Choi, E. J., Choi, E. J., Walker, E. J., Shen, F., Oh, S. P., Arthur, H. M., Young, W. L., Su, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+Homozygous+Endothelial+Deletion+of+Eng+with+VEGF+Stimulation+Is+Sufficient+to+Cause+Cerebrovascular+Dysplasia+in+the+Adult+Mouse.">Minimal Homozygous Endothelial Deletion of Eng with VEGF Stimulation Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse.</a> Cerebrovascular diseases. 33, 540-547 (2012).</li><li>Hao, Q., Su, H., Marchuk, D. A., Rola, R., Wang, Y., Liu, W., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tissue+perfusion+promotes+capillary+dysplasia+in+the+ALK1-deficient+mouse+brain+following+VEGF+stimulation.">Increased tissue perfusion promotes capillary dysplasia in the ALK1-deficient mouse brain following VEGF stimulation.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H2250-H2256 (2008).</li><li>Su, H., Hao, Q., Shen, F., Zhu, Y., Lee, C. Z., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+cerebral+microvascular+dysplasia+model+in+rodents.">Development of a cerebral microvascular dysplasia model in rodents.</a> Acta Neurochir. Suppl. 105, 185-189 (2008).</li><li>Walker, E. J., Su, H., Shen, F., Degos, V., Jun, K., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bevacizumab+Attenuates+VEGF-Induced+Angiogenesis+and+Vascular+Malformations+in+the+Adult+Mouse+Brain.">Bevacizumab Attenuates VEGF-Induced Angiogenesis and Vascular Malformations in the Adult Mouse Brain.</a> Stroke; a journal of cerebral circulation. , (2012).</li></ol>

Los procedimientos experimentales con animales de laboratorio fueron aprobados por el Comité Institucional Cuidado de Animales y el Empleo de la Universidad de California, San Francisco (UCSF). Los autores no tienen conflictos de interés potenciales relacionados con los medicamentos y materiales utilizados en este procedimiento.

La hipertensión venosa cerebral sostenida ha estado estrechamente relacionada con las manifestaciones clínicas más graves y mal pronóstico en pacientes con FAV durales y MAV cerebrales 3. Estos efectos de la CVH han sido ampliamente estudiado en modelos de rata 1,2,8. Un modelo equivalente en el ratón permitiría el uso de animales modificados genéticamente que en última instancia permitir que el análisis de las vías moleculares implicados en la patogénesis de la hipertensión venosa y su...

Los modelos animales de hipertensión venosa cerebral han demostrado ser una herramienta clave en la comprensión de la fisiopatología de las malformaciones arteriovenosas cerebrales y fístulas arteriovenosas 1-7. El más utilizado es el modelo de rata creado a través de una fístula artificial entre la arteria carótida común (CCA) y la vena yugular externa (VYE), lo que provoca una hipertensión venosa cerebral consistente (CVH) en la rata 1,8-10. Modelos de ratones equivalentes, abriendo la posibilidad de utilizar diferentes cepas de ratones transgénicos, permitirían un estudio más en no sólo fisiopatología sino también potenciales terapias genéticas para estas enfermedades cerebrovasculares. Además, la construcción experimental también podría ser adaptado además para el estudio de otras enfermedades cerebrovasculares relacionadas con la hipertensión venosa como la migraña, amnesia global transitoria, ceguera monocular transitoria, etc. 11 Sin embargo, los intentos anteriores para construir estos modelos de ratones have demostró las dificultades con la permeabilidad de la fístula debido a la anatomía diminutivo 5,12. A continuación, describimos nuestro protocolo paso a paso para una anastomosis exitosa de la murino CCA y VYE que se traduce en una fístula patente a largo plazo y una hipertensión venosa durable en el ratón.

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>10-0 Sterile Microsuture</td>
			<td>Arosurgical Ic.</td>
			<td>VT5A010Q10</td>
		</tr>
		<tr>
			<td>11-0 Sterile Microsuture</td>
			<td>Arosurgical Ic</td>
			<td>VT4A00N07</td>
		</tr>
		<tr>
			<td>DUROTIP Scissors</td>
			<td>Aesculap</td>
			<td>BC210R</td>
		</tr>
		<tr>
			<td>Micro-Adson Tissue Forceps</td>
			<td>Aesculap</td>
			<td>BD510R</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Aesculap</td>
			<td>OC496R</td>
		</tr>
		<tr>
			<td>Micro Forceps #5 Jewelers</td>
			<td>Aesculap</td>
			<td>BD331R</td>
		</tr>
		<tr>
			<td>Angled Jewelers Forceps</td>
			<td>Aesculap</td>
			<td>BD329R</td>
		</tr>
		<tr>
			<td>Micro Suture Forceps</td>
			<td>Aesculap</td>
			<td>BD338R</td>
		</tr>
		<tr>
			<td>DUROGRIP Needle Holder</td>
			<td>Aesculap</td>
			<td>BM009R</td>
		</tr>
	</tbody>
</table>

adult mouse venous hypertension model: common carotid artery to external jugular vein anastomosis.

La comprensión de la fisiopatología de las malformaciones arteriovenosas cerebrales y fístulas arteriovenosas ha mejorado gracias a los modelos animales. Un modelo de rata creación de una fístula artificial entre la arteria carótida común (CCA) y la vena yugular externa (VYE) ha sido ampliamente descrito y demostrado técnicamente factible. Esta construcción provoca una hipertensión venosa cerebral consistente (CVH), y por lo tanto ha ayudado a estudiar la contribución de la hipertensión venosa a la formación, los síntomas clínicos y el pronóstico de MAV cerebrales y FAV durales. Modelos de ratones equivalentes sólo se han descrito y apenas han mostrado problemas con estenosis de la fístula. Un modelo murino establecido permitiría el estudio de la fisiopatología no sólo, sino también las terapias genéticas potenciales para estas enfermedades cerebrovasculares. Se presenta un modelo de la fístula arteriovenosa que produce una hipertensión venosa intracraneal duradera en el ratón. Microquirúrgica anastomosis of murino CCA y VYE puede ser difícil debido a la anatomía diminuta y con frecuencia resultará en una fístula no patente. En este protocolo paso a paso nos dirigimos a todos los importantes desafíos encontrados durante este procedimiento. Evitar la retracción excesiva de la vena durante la exposición, usando 11-0 suturas en lugar de 10-0, y hacer una anastomosis término-lateral cuidadosamente planificada son algunos de los pasos críticos. Aunque este método requiere conocimientos avanzados de microcirugía y una curva de aprendizaje ya que el equivalente en la rata, que se puede desarrollar de forma coherente. Este nuevo modelo ha sido diseñado para integrar las técnicas de ratones transgénicos con un sistema experimental previamente establecida que ha demostrado ser útil para estudiar las malformaciones arteriovenosas cerebrales y FAV durales. Al abrir la posibilidad de usar ratones transgénicos, un espectro más amplio de modelos válidos se puede lograr y tratamientos genéticos también puede ser probado. El constructo experimental también podría ser adaptado además para el estudio de otsus enfermedades cerebrovasculares relacionados con la hipertensión venosa como la migraña, la amnesia global transitoria, ceguera monocular transitoria, etc.

Un resultado positivo de la modelo es una fístula arteriovenosa patente que induce hipertensión venosa en el cerebro murino. Para validar el modelo que inicialmente midió la presión venosa intracraneal en el seno sagital de los ratones a los 2, 3 y 4 semanas después de la cirugía. 6 ratones diferentes fueron asignados a cada grupo de tiempo. La presión en los senos fue de 8,8 ± 1,2 mmHg en el grupo midieron dos semanas después de la cirugía. En los 6 ratones medidos 3 semanas después de la cirugía, la presi?...

Watch this Scientific Journal Video about Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis. at JoVE.com

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

Se describe un método para crear un modelo fiable de la hipertensión venosa cerebral en el ratón adulto. Este modelo ha sido ampliamente descrito y probado en la rata. Este nuevo homólogo en los ratones se abre la posibilidad de utilizar animales modificados genéticos y por lo tanto amplía las aplicaciones del modelo.

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 ...

adult-mouse-venous-hypertension-model-common-carotid-artery-to

Research

JoVE Journal

Medicine

14.0K vistas.  University of California, San Francisco.  Se describe un método para crear un modelo fiable de la hipertensión venosa cerebral en el ratón adulto. Este modelo ha sido ampliamente descrito y probado en la rata. Este nuevo homólogo en los ratones se abre la posibilidad de utilizar animales modificados genéticos y por lo tanto amplía las aplicaciones del modelo. 

Video: Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

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 ...

Investigación

Medicina

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

Bilateral Común oclusión de la arteria carótida como un estímulo de preacondicionamiento adecuado para inducir la tolerancia isquémica precoz de isquemia cerebral focal

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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. ...

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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Realización Permanente distal cerebral media carótida común con oclusión de la arteria en ratas de edad avanzada para el Estudio de la isquemia cortical con sostenida de la discapacidad

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Fase contraste resonancia magnética en la arteria carótida común de rata

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

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

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Oclusión de la arteria Cerebral Media permitiendo la reperfusión mediante la reparación de la arteria carótida común en ratones

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...