Name: a rat carotid artery pressure-controlled segmental balloon injury with periadventitial therapeutic application
Uploaded: 2020-07-09T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application at JoVE.com

N.E.B. recibió el apoyo de una beca de capacitación del Instituto Nacional de Ciencias de la Salud Ambiental (5T32ES007126-35, 2018), y una beca predoctoral de la Asociación Americana del Corazón (20PRE35120321). E.S.M.B. fue un académico de KL2 parcialmente apoyado por el Programa de Académicos clínicos y traslacionales de la UNC (KL2TR002490, 2018), y el Instituto Nacional del Corazón, los Pulmones y la Sangre (K01HL145354). Los autores agradecen al Dr. Pablo Ariel del Laboratorio de Servicios de Microscopía de la UNC por ayudar con LSFM. La microscopía de fluorescencia de lámina de luz se realizó en el Laboratorio de Servicios de Microscopía. El Laboratorio de Servicios de Microscopía, Departamento de Patología y Medicina de Laboratorio, es apoyado en parte por P30 CA016086 Cancer Center Core Support Grant al UnC Lineberger Comprehensive Cancer Center.

Todos los métodos descritos aquí han sido aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de Carolina del Norte en Chapel Hill.
1. Procedimientos preoperatorios
<ol><li>Esterilizar instrumentos quirúrgicos. Autoclave todos los instrumentos quirúrgicos antes de la cirugía. Si realiza varias cirugías el mismo día, esterilizar instrumentos entre cirugías utilizando un esterilizador de cuentas secas.</li>
	<li>Preparar terapéutico en gel Pluronic-127 al 25% (diluido en agua destilada estéril).</li>
	<li>Coloque un catéter de globo Fogarty de 2F en el insuflador y coloque el extremo del globo del catéter en una jeringa de 1 ml llena de solución salina.</li>
	<li>Induzca la anestesia colocando la rata en una cámara con un 5% de isoflurano.<ol>
<li>Retire la rata de la cámara y registre el peso de la rata. Use cortapelas para afeitarse el pelaje en la región ventral del cuello.</li>
		<li>Coloque la rata de nuevo en la cámara con un 5% de isoflurano para asegurar la inducción de anestesia.</li>
	</ol>
</li>
	<li>Coloque la rata supina en una plataforma quirúrgica, insertando la cara en el cono nasal para que la cara de la rata esté hacia el cirujano.<ol>
<li>Reduzca la anestesia inhalacional al 1,5% de isoflurano. Verifique la profundidad de la anestesia mediante un reflejo de pellizcar los dedos del pie en los cuatro pies.</li>
		<li>Tape las cuatro patas hasta la plataforma quirúrgica.</li>
	</ol>
</li>
	<li>Encienda la lámpara de calor.</li>
	<li>Inyectar Atropina (0,01 mg/kg) subcutáneamente para reducir las secreciones de las vías respiratorias.</li>
	<li>Inyectar carprofeno (5 mg/kg) por vía subcutánea para el control profiláctico del dolor. Si los antiinflamatorios no son aceptables para el experimento, consulte los pasos 3.2.2 y 3.2.3.</li>
	<li>Aplique ungüento ocular lubricante en ambos ojos con un hisopo de algodón estéril para evitar que las córneas se sequen durante la cirugía.</li>
	<li>Frotar el cuello tres veces en un movimiento circular alternando entre un 70% de alcohol etílico seguido de Betadine desde el centro de la región afeitada hacia afuera para esterilizar el sitio de la incisión.</li>
	<li>Infiltrarse en la línea de incisión s.c. con 0.25% bupivacaína.</li>
	<li>Ponte guantes quirúrgicos estériles antes de manipular instrumentos y suministros quirúrgicos estériles.</li>
	<li>Poner todos los instrumentos quirúrgicos autoclavados en una hoja quirúrgica estéril.</li>
	<li>Cortar tres hebras independientes de 1 pulgada de sutura estéril de 7-0 Prolene.</li>
	<li>Coloque hisopos de algodón y gasa en la hoja quirúrgica.</li>
	<li>Cubre a la rata con una lámina quirúrgica estéril que solo expone la región esterilizada del cuello.</li>
	<li>Corte una abertura pequeña adicional en la hoja que expone parte del cono nasal. Este será el sitio para grabar el catéter del globo durante la lesión.</li>
</ol>2. Procedimientos operativos
<ol><li>Durante todo el procedimiento quirúrgico, evalúe la profundidad de la anestesia mediante el seguimiento de la frecuencia respiratoria (la tasa debe ser consistente y considerada normal), así como pellizcar los dedos del pie cada 15 minutos. Si la frecuencia respiratoria aumenta o hay una respuesta a la pellizco del dedo del pie, entonces detenga la manipulación quirúrgica y aumente el isoflurano hasta un 2,5%.</li>
	<li>Exponer la arteria carótida común (CCA).<ol>
<li>Haga una incisión superficial, recta y longitudinal del escote entre los huesos de la mandíbula de la rata. La incisión tendrá aproximadamente 1,5-2 cm de longitud.</li>
		<li>Haga una incisión a través del tejido conectivo debajo de la piel hasta que la capa muscular esté expuesta. Desplazar las glándulas salivales debajo de la piel para acceder al tejido muscular.</li>
		<li>Separe sin rodeos el tejido conectivo del músculo insertando tijeras cerradas entre la capa muscular y el tejido conectivo y abriendo suavemente la tijera mientras tira de la piel hacia arriba.</li>
	</ol></li>
	<li>Diseccionar los dos músculos visibles (esternohiide y esternóstoide) longitudinalmente a lo largo del lado izquierdo de la tráquea hasta que se observa un tercer músculo (omohído) que corre perpendicular a los dos músculos superficiales.</li>
	<li>Utilice fórceps para crear una ventana que separa este músculo perpendicular (omohído) del músculo longitudinal (estenohído) corriendo sobre la tráquea. Realice suavemente esta separación para evitar traumatismos contundentes en la tráquea.</li>
	<li>Alcanza fórceps debajo del músculo perpendicular y corta para separar los dos músculos longitudinales y exponer el CCA.</li>
	<li>Diseccionar el CCA.<ol>
<li>Diseccionar la CCA cerca de la bifurcación hasta que se expongan la arteria carótida interna (ICA) y la arteria carótida externa (ECA).</li>
		<li>Diseccionar la arteria tiroidea superior (STA), que se ramifica del TCE.</li>
		<li>Utilizando las suturas de proleno precortado, ligar el STA y el TCE cerca de su respectiva bifurcación. Deje la mayoría de la sutura a un lado del nudo y agarre cada sutura con una hemostat curva.</li>
		<li>Termina de diseccionar alrededor del ICA, alcanza fórceps debajo y alrededor del ICA, y usa una abrazadera vascular no aplastante para lograr el control distal. Sujete la arteria occipital junto con el ICA.</li>
		<li>Disecciona el CCA proximal a la bifurcación, asegurando separar el nervio vago de la CCA.</li>
		<li>Alcanza fórceps debajo y alrededor de la CCA y usa una abrazadera vascular no aplastante para lograr un control proximal. Coloque la abrazadera al menos 5 mm de la bifurcación.</li>
	</ol></li>
	<li>Realice lesiones en el globo.<ol>
<li>Maniobrar los hemostats curvos que sostienen cada rama de arteria ligada para exponer la bifurcación entre el TCE y la rama superior.</li>
		<li>Disecciona suavemente el tejido en la bifurcación y luego hace una incisión de arteriotomía entre el TCE y la rama superior usando tijeras de microdisección.</li>
		<li>Use un hisopo de algodón para sacar toda la sangre de la CCA y limpiar el sitio de arteriotomía.</li>
		<li>Inserte el catéter de globo sin inflar a través de la arteriotomía y avance hacia la CCA hasta que el extremo proximal del globo haya pasado la bifurcación.</li>
		<li>Cinta catéter al cono nasal para que el globo no se escape de la arteria durante la inflación.</li>
		<li>Infla lentamente el balón a 5 atmósferas de presión y deja en la CCA durante 5 minutos para inducir lesiones arteriales. Asegúrese de que la presión se mantenga constante durante todo el 5 min.</li>
		<li>Después de 5 min, desinflar el globo y retirar suavemente de la CCA a través de la arteriotomía.</li>
		<li>Enjuague el CCA apretando suavemente la abrazadera en el CCA. No retire la abrazadera.</li>
		<li>Ligar el TCE proximal a la arteriotomía y luego retirar las abrazaderas de la CCA y el ICA para restaurar el flujo sanguíneo a través de la CCA al ICA. Asegúrese de que no haya sangrado visible alrededor de la arteriotomía y de que el CCA esté pulsando.</li>
	</ol></li>
	<li>Aplicar 100 μL de vehículo de gel terapéutico o plurónico solo periaventialmente a lo largo de la CCA lesionada. Para ello, aplique 50 μL en el lado izquierdo de la CCA y, a continuación, 50 μL en el lado derecho de la CCA para garantizar el recubrimiento uniforme de la arteria lesionada.</li>
	<li>Cierre el sitio de la herida.<ol>
<li>Corte el exceso de suturas de proleno.</li>
		<li>Cierre la herida usando capas de vicryl interrumpidas 4-0 o 6-0 a lo largo del tejido conectivo.</li>
		<li>Termina de cerrar la herida usando sutura de nylon 4-0 a lo largo de la piel.</li>
	</ol></li>
</ol>3. Procedimientos postoperatorios
<ol><li>Coloque a la rata sola en una jaula limpia con la mitad de la jaula debajo de una lámpara de calefacción y monitoree hasta que la rata recupere suficiente conciencia para mantener la recumbencia severa. Mantenga a la rata en una jaula separada hasta que el animal esté completamente alerta y móvil antes de transferirla de vuelta a su jaula original.</li>
	<li>Monitoree la rata diariamente durante los próximos tres días y luego tres veces por semana hasta la eutanasia. Eutanasia usando sobredosis de isoflurano seguida de toracotomía bilateral como se describe a continuación.<ol>
<li>Utilizar el Centro Nacional para el Refinamiento de Reemplazo y Reducción de Animales en La Investigación (NC3R) escala de mueca para identificar los niveles de dolor postoperatorio. Si algún animal parece estar experimentando dolor o desarrollar algún compromiso neurológico, transfiére inmediatamente.</li>
		<li>Para animales que no reciben carprofeno, administrar paracetamol 6 mg/ml en su agua potable 24 h antes de la cirugía a través de 48 h después de la cirugía. El paracetamol proporciona analgesia con efectos antiinflamatorios mínimos.</li>
		<li>Alternativamente, se pueden utilizar otras estrategias de analgesia con efectos antiinflamatorios mínimos, como la buprenorfina o la liberación prolongada de buprenorfina. Consulte al equipo veterinario de su institución.</li>
	</ol>
</li>
</ol>4. Cosecha de tejidos e imágenes
<ol><li>Dos semanas después de la cirugía, eutanasia a la rata por sobredosis de anestesia (5% isoflurano). Alternativamente, eutanasiar ratas en un momento anterior para analizar los diversos aspectos de la respuesta a lesiones arteriales.<ol>
<li>Una vez que la respiración se detiene realizar la toracotomía bilateral como un método secundario de eutanasia.</li>
	</ol></li>
	<li>Hacer una incisión lateral a través del abdomen, y luego cortar hacia arriba, a través del diafragma y las costillas, exponiendo la cavidad torácica.</li>
	<li>Perfundir y arreglar las arterias.<ol>
<li>Inserte una cánula de 18 G unida a un sistema gravitacional de fijación de perfusión a través del ventrículo izquierdo. Mantenga una presión equivalente entre ratas marcando la altura del sistema de perfusión en relación con la sobremesa (elevación de 120 cm, equivalente a 91 ± 3 mmHg).</li>
		<li>Sujete la cánula junto con el ventrículo con una hemostat curva.</li>
		<li>Hacer un corte en el atrio derecho, abrir el circuito vascular, y comenzar la perfusión con PBS seguido de 2-4% paraformaldehído (alrededor de 250 mL cada uno).</li>
		<li>Preparar paraformaldehído diluido en PBS el día del sacrificio, o como mucho la noche antes del sacrificio. Si se prepara el día del sacrificio, asegúrese de que el paraformaldehído se haya enfriado a temperatura ambiente antes de comenzar la perfusión. Almacene paraformaldehído a 4 °C.</li>
	</ol></li>
	<li>Después de la fijación, extraiga las arterias carótidas izquierda y derecha y guárdelas a 4 °C durante 2 h en 2-4% paraformaldehído.</li>
	<li>Transfiera las arterias al 30% de sacarosa y almacene durante la noche a 4 °C.</li>
	<li>Después de 16-24 h, incrustar las arterias en el compuesto de temperatura de corte óptima (OCT) y congelar bloques de arterias incrustadas en OCT.<ol>
<li>Acondicione las arterias en OCT a temperatura ambiente durante 10 minutos. Coloque la arteria paralela al plano del criomold lleno de OCT, marcando el lado de la criomold a la que se enfrenta la bifurcación arterial. Congelación de nitrógeno líquido.</li>
		<li>Almacene bloques congelados a largo plazo a -80 °C.</li>
	</ol></li>
	<li>Sección bloques congelados usando un criostato.<ol>
<li>Recoger seis secciones transversales arteriales de 5 μm de espesor por diapositiva, con la diapositiva 1 a partir de la bifurcación.</li>
		<li>Sección bloques congelados hasta que la hiperplasia ya no sea visible (alrededor de 100 diapositivas).</li>
	</ol></li>
	<li>La mancha de hematoxilina y eosina (H&E) se desliza18<ol>
<li>Encuentra el área de lesiones manchando uno de cada diez toboganes a lo largo de toda la arteria a partir de la bifurcación (por ejemplo, diapositivas 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 y 100).</li>
		<li>Manchar diapositivas adicionales alrededor del sitio de las lesiones para encontrar el tobogán con oclusión máxima (por ejemplo, las diapositivas 20, 30 y 40 tenían hiperplasia visible, por lo que las diapositivas de manchas 15, 25, 35 y 45).</li>
		<li>Manchar y cuantificar el tobogán con oclusión máxima y deslizamientos equidistantes antes y después de la diapositiva de oclusión pico (por ejemplo, oclusión pico que se encuentra en la diapositiva 35, luego manchar y cuantificar diapositivas 25, 45, etc.) para un total de 3-10 diapositivas por rata.</li>
	</ol></li>
	<li>Para imágenes de microscopía de fluorescencia de lámina de luz, almacene las arterias durante la noche a 4 °C después de la fijación en el paso 4.4.<ol>
<li>Arteria de sonda con dilución de 1:500 de anticuerpo primario anti-CD31 de conejo en diluyente (pH 7.4) durante 3 días. A continuación, contrarresta la arteria con 1:500 dilución del anticuerpo secundario Alexa Fluor 647 del antitulato durante 2 días19.</li>
		<li>Borre la arteria con iDISCO+20.</li>
		<li>Imagen de la arteria utilizando una lámina de luz microscopio de fluorescencia21. Renderice imágenes utilizando software (por ejemplo, Imaris)19.</li>
	</ol></li>
	<li>Cuantificar la hiperplasia neointimal. Realice la cuantificación de forma cegada si es posible.<ol>
<li>Utilice el software ImageJ para trazar el perímetro de la intima, lamina elástica interna (IEL) y lamina elástica externa (ANGUILA) de una arteria en cada una de las diapositivas 3-10 determinadas anteriormente (paso 4.8.3).</li>
		<li>Cuantifique el área de cada región trazada en ImageJ y exporte estos valores. La traza intima produce el área de lúmenes, la traza IEL produce el área IEL, y la traza EEL produce el área EEL.</li>
		<li>Promedia los valores obtenidos de las diapositivas de 3-10 para obtener la lesión promedio (% oclusión, relación intima:media (I:M), hiperplasia neointimal) por arteria carótida de rata. Hiperplasia neointimal = área IEL - Área de Lumen <img alt="Equation 1" src="/files/ftp_upload/60473/60473eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/60473/60473eq2.jpg">
</li>
	</ol></li>
</ol>

<ol>
	<li>American Heart Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+Disease:+A+Costly+Burden+for+America,+Projections+Through+2035.">Cardiovascular Disease: A Costly Burden for America, Projections Through 2035.</a> American Heart Association CVD Burden Report. , (2017).</li><li>Singh, R. B., Mengi, S. A., Xu, Y. J., Arneja, A. S., Dhalla, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+atherosclerosis:+A+multifactorial+process.">Pathogenesis of atherosclerosis: A multifactorial process.</a> Experimental and Clinical Cardiology. 7 (1), 40-53 (2002).</li><li>Clowes, A. W., Reidy, M. A., Clowes, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+stenosis+after+arterial+injury.">Mechanisms of stenosis after arterial injury.</a> Laboratory Investigation. 49 (2), 208-215 (1983).</li><li>Clowes, A. W., Reidy, M. A., Clowes, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+cellular+proliferation+after+arterial+injury.+I.+Smooth+muscle+growth+in+the+absence+of+endothelium.">Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium.</a> Laboratory Investigation. 49 (3), 327-333 (1983).</li><li>Sartore, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+adventitial+fibroblasts+to+neointima+formation+and+vascular+remodeling:+from+innocent+bystander+to+active+participant.">Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant.</a> Circulation Research. 89 (12), 1111-1121 (2001).</li><li>Tanaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+progenitor+cells+contribute+to+neointimal+formation+in+nonirradiated+chimeric+mice.">Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice.</a> The FASEB Journal. 22 (2), 428-436 (2008).</li><li>Henry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+angioplasty+and+stenting+under+protection.+Techniques,+results+and+limitations.">Carotid angioplasty and stenting under protection. Techniques, results and limitations.</a> The Journal of Cardiovascular Surgery. 47 (5), 519-546 (2006).</li><li>Kounis, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombotic+responses+to+coronary+stents,+bioresorbable+scaffolds+and+the+Kounis+hypersensitivity-associated+acute+thrombotic+syndrome.">Thrombotic responses to coronary stents, bioresorbable scaffolds and the Kounis hypersensitivity-associated acute thrombotic syndrome.</a> Journal of Thoracic Disease. 9 (4), 1155-1164 (2017).</li><li>Jackson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+restenosis.">Animal models of restenosis.</a> Trends in Cardiovascular Medicine. 4 (3), 122-130 (1994).</li><li>Shears, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+inhibition+of+intimal+hyperplasia+by+adenovirus-mediated+inducible+nitric+oxide+synthase+gene+transfer+to+rats+and+pigs+in+vivo.">Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo.</a> Journal of the American College of Surgeons. 187 (3), 295-306 (1998).</li><li>Takayama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+arterial+restenosis:+technical+aspects+of+femoral+wire+injury.">A murine model of arterial restenosis: technical aspects of femoral wire injury.</a> Journal of Visualized Experiments. (97), (2015).</li><li>Zhang, L. N., Parkinson, J. F., Haskell, C., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+intimal+hyperplasia+learned+from+a+murine+carotid+artery+ligation+model.">Mechanisms of intimal hyperplasia learned from a murine carotid artery ligation model.</a> Current Vascular Pharmacology. 6 (1), 37-43 (2008).</li><li>Jahnke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+double-injury+restenosis+model+in+the+rat+aorta.">Characterization of a new double-injury restenosis model in the rat aorta.</a> Journal of Endovascular Therapy. 12 (3), 318-331 (2005).</li><li>Gregory, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periadventitial+atRA+citrate-based+polyester+membranes+reduce+neointimal+hyperplasia+and+restenosis+after+carotid+injury+in+rats.">Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats.</a> American Journal of Physiology-Heart and Circulatory Physiology. 307 (10), 1419-1429 (2014).</li><li>Buglak, N. E., Jiang, W., Bahnson, E. S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinnamic+aldehyde+inhibits+vascular+smooth+muscle+cell+proliferation+and+neointimal+hyperplasia+in+Zucker+Diabetic+Fatty+rats.">Cinnamic aldehyde inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in Zucker Diabetic Fatty rats.</a> Redox Biology. 19, 166-178 (2018).</li><li>Bahnson, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effect+of+PROLI/NO+on+cellular+proliferation+and+phenotype+after+arterial+injury.">Long-term effect of PROLI/NO on cellular proliferation and phenotype after arterial injury.</a> Free Radical Biology and Medicine. 90, 272-286 (2016).</li><li>Gilbert, J. C. W., Davies, M. C., Hadgraft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+Pluronic+F127+in+aqueous+solution+studied+using+fluorescent+probes.">The behaviour of Pluronic F127 in aqueous solution studied using fluorescent probes.</a> International Journal of Pharmaceutics. 40 (1-2), 93-99 (1987).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+morphometric+analyses+for+rat+carotid+balloon+injury+model.">Histological and morphometric analyses for rat carotid balloon injury model.</a> Methods in Molecular Medicine. 139, 31-66 (2007).</li><li>Buglak, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Sheet+Fluorescence+Microscopy+as+a+New+Method+for+Unbiased+Three-Dimensional+Analysis+of+Vascular+Injury.">Light Sheet Fluorescence Microscopy as a New Method for Unbiased Three-Dimensional Analysis of Vascular Injury.</a> Cardiovascular Research. , (2020).</li><li>Renier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iDISCO:+a+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Ariel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UltraMicroscope II - A User Guide. , (2018).</li><li>Touchard, A. G., Schwartz, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+restenosis+models:+challenges+and+successes.">Preclinical restenosis models: challenges and successes.</a> Toxicologic Pathology. 34 (1), 11-18 (2006).</li><li>Xiangdong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+atherosclerosis+research:+a+review.">Animal models for the atherosclerosis research: a review.</a> Protein Cell. 2 (3), 189-201 (2011).</li><li>Chen, H., Li, D., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Rat+Models+for+Atherosclerosis.">Novel Rat Models for Atherosclerosis.</a> Journal of Cardiology and Cardiovascular Sceinces. 2 (2), 29-33 (2018).</li><li>Xing, D., Nozell, S., Chen, Y. F., Hage, F., Oparil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+mechanisms+of+vascular+protection.">Estrogen and mechanisms of vascular protection.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 29 (3), 289-295 (2009).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods in Molecular Medicine. 139, 1-30 (2007).</li><li>Pellet-Many, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilins+1+and+2+mediate+neointimal+hyperplasia+and+re-endothelialization+following+arterial+injury.">Neuropilins 1 and 2 mediate neointimal hyperplasia and re-endothelialization following arterial injury.</a> Cardiovascular Research. 108 (2), 288-298 (2015).</li><li>Wu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+delivery+of+resolvin+D1+inhibits+neointimal+hyperplasia+in+a+rat+model+of+arterial+injury.">Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury.</a> Journal of Vascular Surgery. 65 (1), 207-217 (2017).</li><li>Tan, J., Yang, L., Liu, C., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-26a+targets+MAPK6+to+inhibit+smooth+muscle+cell+proliferation+and+vein+graft+neointimal+hyperplasia.">MicroRNA-26a targets MAPK6 to inhibit smooth muscle cell proliferation and vein graft neointimal hyperplasia.</a> Scientific Reports. 7, 46602 (2017).</li><li>Pearce, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effect+of+a+short-acting+NO+donor+for+the+prevention+of+neointimal+hyperplasia.">Beneficial effect of a short-acting NO donor for the prevention of neointimal hyperplasia.</a> Free Radical Biology and Medicine. 44 (1), 73-81 (2008).</li><li>Cao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100B+promotes+injury-induced+vascular+remodeling+through+modulating+smooth+muscle+phenotype.">S100B promotes injury-induced vascular remodeling through modulating smooth muscle phenotype.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1863 (11), 2772-2782 (2017).</li><li>Madigan, M., Entabi, F., Zuckerbraun, B., Loughran, P., Tzeng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+inhaled+carbon+monoxide+mediates+the+regression+of+established+neointimal+lesions.">Delayed inhaled carbon monoxide mediates the regression of established neointimal lesions.</a> Journal of Vascular Surgery. 61 (4), 1026-1033 (2015).</li><li>Khurana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis-dependent+and+independent+phases+of+intimal+hyperplasia.">Angiogenesis-dependent and independent phases of intimal hyperplasia.</a> Circulation. 110 (16), 2436-2443 (2004).</li><li>Tsihlis, N. D., Vavra, A. K., Martinez, J., Lee, V. R., Kibbe, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+is+less+effective+at+inhibiting+neointimal+hyperplasia+in+spontaneously+hypertensive+rats.">Nitric oxide is less effective at inhibiting neointimal hyperplasia in spontaneously hypertensive rats.</a> Nitric Oxide. 35, 165-174 (2013).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neointimal+hyperplasia+in+the+rat+carotid+artery+injury+model+by+a+HMGB1+inhibitor.">Inhibition of neointimal hyperplasia in the rat carotid artery injury model by a HMGB1 inhibitor.</a> Atherosclerosis. 224 (2), 332-339 (2012).</li><li>Mano, T., Luo, Z., Malendowicz, S. L., Evans, T., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+GATA-6+downregulation+promotes+smooth+muscle+differentiation+and+inhibits+intimal+hyperplasia+in+balloon-injured+rat+carotid+artery.">Reversal of GATA-6 downregulation promotes smooth muscle differentiation and inhibits intimal hyperplasia in balloon-injured rat carotid artery.</a> Circulation Research. 84 (6), 647-654 (1999).</li><li>Becher, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+Provides+Detailed+Atherosclerotic+Plaque+Morphology+and+Reveals+Angiogenesis+after+Carotid+Artery+Ligation.">Three-Dimensional Imaging Provides Detailed Atherosclerotic Plaque Morphology and Reveals Angiogenesis after Carotid Artery Ligation.</a> Circulation Research. 126 (5), 619-632 (2020).</li></ol>

Los autores declaran que no hay conflictos de interés con respecto a la publicación de este manuscrito.

La lesión del globo arterial carótido de rata es uno de los modelos animales de restenosis más utilizados y estudiados. Tanto el modelo original de lesión de globo3 como la variación de lesiones segmentales modificada controlada por presión10 han informado de muchos aspectos de la respuesta a lesiones arteriales que también se produce en los seres humanos, siendo las pocas limitaciones que rara vez se desarrolla el trombo rico en fibrina y la inflamación local es m?...

Las enfermedades cardiovasculares (ECV) siguen siendo la principal causa de muerte en todo el mundo1. La aterosclerosis es la causa subyacente de la mayor parte de la morbilidad y mortalidad relacionadas con la ECV. La aterosclerosis es la acumulación de placa dentro de las arterias que resulta en un lumen estrechado, dificultando la perfusión adecuada de sangre a los órganos y tejidos distales2. Las intervenciones clínicas para el tratamiento de la aterosclerosis grave incluyen angioplastia con o sin colocación de stent. Esta intervención consiste en avanzar un catéter de globo hacia el lugar de la placa, e inflar el globo para comprimir la placa a la pared arterial, ensanchando el área luminal. Este procedimiento daña la arteria, sin embargo, iniciando la respuesta a lesiones arteriales3. La activación prolongada de esta respuesta a lesiones conduce a la restenosis arterial, o rea estrechamiento, secundaria a la hiperplasia neointimal y la remodelación de los vasos. Durante la angioplastia, la capa intimal se denuda de células endoteliales que conducen al reclutamiento inmediato de plaquetas y a la inflamación local. La señalización local induce cambios fenotípicos en las células musculares lisas vasculares (VSMC) y fibroblastos adventitiales. Esto conduce a la migración y proliferación de VSMC y fibroblastos hacia adentro hacia el lumen, lo que conduce a la hiperplasia neointimal4,5. Las células progenitoras circulantes y las células inmunes también contribuyen al volumen total de restenosis6. En su caso, los stents que eluden fármacos (DES) son el estándar actual para inhibir la restenosis7. Sin embargo, el DES inhibe la redotelización arterial, creando así un ambiente pro-trombótico que puede resultar en trombosis tardía en el stent8. Por lo tanto, los modelos animales son integrales tanto para entender la fisiopatología de la restenosis, como para desarrollar mejores estrategias terapéuticas para prolongar la eficacia de los procedimientos de revascularización.
Varios modelos animales grandes y pequeños9 se utilizan para estudiar esta patología. Estos incluyen lesión de balón3,10 o lesión de alambre11 del lado luminal de una arteria, así como ligadura parcial12 o colocación del manguito13 alrededor de la arteria. Tanto el balón como la lesión del alambre denudan la capa endotelial de la arteria, imitando lo que ocurre clínicamente después de la angioplastia. En particular, los modelos de lesiones por balón utilizan herramientas similares a las del entorno clínico (es decir, catéter de globo). La lesión del globo se realiza mejor en modelos de ratas, ya que las arterias de rata son de un tamaño adecuado para los catéteres de globo disponibles comercialmente. Aquí describimos una lesión arterial segmental controlada por presión, una versión bien establecida y modificada de la lesión del globo arterial carótido de rata. Este enfoque controlado por presión imita estrechamente el procedimiento de angioplastia clínica, y permite la formación reproducible de hiperplasia neointimal dos semanas después de la lesión14,15. Además, esta lesión arterial controlada por presión resulta en una restauración completa de la capa endotelial 2 semanas después de la cirugía16. Esto contrasta directamente con el modelo original de lesiones en globo, descrito por Clowes, donde la capa endotelial nunca vuelve a la cobertura completa3.
Después de la cirugía, las terapias pueden aplicarse o dirigirse hacia la arteria lesionada a través de varios enfoques. El método descrito aquí utiliza la aplicación periavential de una molécula pequeña incrustada en una solución de gel plurónico. En concreto, aplicamos una solución de 100 μM de aldehído cinámico en gel Pluronic-F127 al 25% inmediatamente después de una lesión para inhibir la formación de hiperplasia neointimal15. Pluronic-F127 es un gel termo reversible no tóxico capaz de suministrar medicamentos localmente de forma controlada17. Mientras tanto, la lesión arterial es local, por lo tanto la administración local permite probar un principio activo mientras minimiza los efectos fuera del objetivo. Sin embargo, la entrega efectiva de un método terapéutico con este método dependerá de la química de la molécula pequeña o biológico utilizado.

<table><tbody><tr><td>1 mL Syringe</td><td>Fisher</td><td>14955450</td><td></td></tr><tr><td>1 mL Syringe with needle</td><td>BD</td><td>309626</td><td></td></tr><tr><td>2 French Fogarty Balloon Embolectomy Catheter</td><td>Edwards LifeSciences</td><td>120602F</td><td></td></tr><tr><td>4-0 Ethilon (Nylon) Suture</td><td>Ethicon Inc</td><td>662H</td><td></td></tr><tr><td>4-0 Vicryl Suture</td><td>Ethicon Inc</td><td>J214H</td><td></td></tr><tr><td>7-0 Prolene Suture</td><td>Ethicon Inc</td><td>8800H</td><td></td></tr><tr><td>70% ethyl alcohol</td><td></td><td></td><td></td></tr><tr><td>Anti-Rabbit Alexa Fluor 647</td><td>Thermo Fisher Scientific</td><td>A21245</td><td></td></tr><tr><td>Atropine Sulfate</td><td>Vedco Inc</td><td></td><td>for veterinary use</td></tr><tr><td>Cotton Swabs</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Curved Hemostats</td><td>Fine Science Tools</td><td>13009-12</td><td></td></tr><tr><td>Fine Curved Forceps</td><td>Fine Science Tools</td><td>11203-25</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Gauze</td><td>Covidien</td><td>2252</td><td></td></tr><tr><td>IHC-Tek Diluent (pH 7.4)</td><td>IHC World</td><td>IW-1000</td><td></td></tr><tr><td>Insufflator</td><td>Merit Medical</td><td>IN4130</td><td></td></tr><tr><td>Iodine solution</td><td></td><td></td><td></td></tr><tr><td>Lubricating Eye Ointment</td><td>Dechra</td><td></td><td>for veterinary use</td></tr><tr><td>Mayo Scissors</td><td>Fine Science Tools</td><td>14010-15</td><td></td></tr><tr><td>Micro Serrefines</td><td>Fine Science Tools</td><td>18055-05</td><td></td></tr><tr><td>Microdissection Scissors</td><td>Fine Science Tools</td><td>15004-08</td><td></td></tr><tr><td>Micro-Serrefine Clamp Applying Forceps</td><td>Fine Science Tools</td><td>18057-14</td><td></td></tr><tr><td>Needle Holder</td><td>Fine Science Tools</td><td>12003-15</td><td></td></tr><tr><td>Pluronic-127 (diluted in sterile water)</td><td>Sigma-Aldrich</td><td>P2443</td><td>25% prepared</td></tr><tr><td>Rabbit Anti-CD31</td><td>Abcam</td><td>ab28364</td><td></td></tr><tr><td>Retractor</td><td></td><td></td><td>Bent paper clips work well</td></tr><tr><td>Rimadyl (Carprofen)</td><td>Zoetis Inc</td><td></td><td>for veterinary use</td></tr><tr><td>Saline solution</td><td></td><td></td><td></td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Sterile Drape</td><td>Dynarex</td><td>4410</td><td></td></tr><tr><td>T-Pins</td><td></td><td></td><td></td></tr></tbody></table>

a rat carotid artery pressure-controlled segmental balloon injury with periadventitial therapeutic application

Las enfermedades cardiovasculares siguen siendo la principal causa de muerte y discapacidad en todo el mundo, en parte debido a la aterosclerosis. La placa aterosclerótica estrecha la superficie luminal en las arterias, reduciendo así el flujo sanguíneo adecuado a órganos y tejidos distales. Clínicamente, los procedimientos de revascularización como la angioplastia con o sin colocación de stent tienen como objetivo restaurar el flujo sanguíneo. Aunque estos procedimientos restablecen el flujo sanguíneo reduciendo la carga de la placa, dañan la pared del vaso, lo que inicia la respuesta de curación arterial. La respuesta curativa prolongada causa restenosis arterial, o rea estrechamiento, limitando en última instancia el éxito a largo plazo de estos procedimientos de revascularización. Por lo tanto, los modelos animales preclínicos son integrales para analizar los mecanismos fisiológicos que impulsan la restenosis, y proporcionan la oportunidad de probar nuevas estrategias terapéuticas. Los modelos murinos son más baratos y fáciles de operar que los modelos animales grandes. Las lesiones por balón o alambre son las dos modalidades de lesiones comúnmente aceptadas utilizadas en modelos murinos. Los modelos de lesiones por balón en particular imitan el procedimiento de angioplastia clínica y causan daños adecuados a la arteria para el desarrollo de la restenosis. Aquí describimos los detalles quirúrgicos para realizar y analizar histológicamente el modelo modificado de lesión del globo arterial carótido de rata controlado por presión. Además, este protocolo destaca cómo la aplicación periavential local de terapias se puede utilizar para inhibir la hiperplasia neointimal. Por último, presentamos la microscopía de fluorescencia de la hoja de luz como un enfoque novedoso para la toma de imágenes y la visualización de la lesión arterial en tres dimensiones.

La Figura 1 muestra todos los materiales y herramientas quirúrgicas utilizadas para realizar esta cirugía. La tinción de hematoxilina y eosina (H&E) de secciones transversales arteriales lesionadas de dos semanas permite una visualización clara de la hiperplasia neointimal. La Figura 2 muestra imágenes representativas de las secciones transversales arteriales manchadas de H&E de una arteria sana, herida y tratada. La Figura 2 t...

Watch this Scientific Journal Video about A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application at JoVE.com

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

La lesión del globo arterial carótido de rata imita el procedimiento clínico de angioplastia realizado para restaurar el flujo sanguíneo en vasos ateroscleróticos. Este modelo induce la respuesta a lesiones arteriales al desprestir la pared arterial, y desnudar la capa intimal de las células endoteliales, causando en última instancia remodelación y una respuesta hiperplástica intimal.

Una lesión de globo segmentario controlada por presión arterial carótida de rata con aplicación terapéutica periavential

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

a-rat-carotid-artery-pressure-controlled-segmental-balloon-injury

Research

JoVE Journal

Medicine

4.9K vistas.  University of North Carolina at Chapel Hill. La lesión del globo arterial carótido de rata imita el procedimiento clínico de angioplastia realizado para restaurar el flujo sanguíneo en vasos ateroscleróticos. Este modelo induce la respuesta a lesiones arteriales al desprestir la pared arterial, y desnudar la capa intimal de las células endoteliales, causando en última instancia remodelación y una respuesta hiperplástica intimal.

Video: A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

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

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Transferencia génica vascular de Stent metálico superficies utilizando Adenoviral Vectores anclada a través hidrolizables reticulantes

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Lesión Vascular Globo y Administración intraluminal en rata arteria carótida

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Cuantificación de Protección neurovascular Siguiendo repetitivo hipóxico Precondicionamiento y transitoria cerebral media oclusión de la arteria en ratones

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Una rata carótida lesión con el balón modelo para probar Terapéutica Remodelación Anti-vasculares

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Reproducible lesión arterial Denudación por pinzamiento aórtico abdominal infrarrenal en un modelo murino

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

El modelo de conejo de ateroesclerosis acelerada: una perspectiva metodológica de la lesión de globo de la arteria ilíaca

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

Transvenous Embolization of Carotid Cavernous Fistula through Inferior Petrosal Sinus with Detachable Coils and Ethylene Vinyl Alcohol Copolymer

Carotid cavernous fistula (CCF) is a rare disease caused by abnormal communications between the internal carotid artery (direct fistula) or meningeal branches of the external carotid artery (indirect fistula) and the cavernous sinus (CS). Trauma is the most common cause of CCF. The clinical presentation of CCF is closely related to the venous drainage pattern. Orbital and neuro-ophthalmological symptoms are the most common clinical presentation of CCF with drainage through the superior ophthalmic vein (SOV). Endovascular embolization by arterial or venous approaches is the most common management of CCF. Transvenous embolization using detachable coils and ethylene-vinyl alcohol copolymer (EVOH) is an alternative method for the treatment of CCF. Endovascular embolization offers different options to treat CCF by minimally invasive approach decreasing morbidity and residual fistulas. The purpose of this article is to report our treatment experiences via the inferior petrosal sinus (IPS), and immediate-term outcomes of endovascular embolization of CCF by using detachable coils and EVOH.

The purpose of the article is to present our experience in endovascular treatment via the inferior petrosal sinus of the carotid cavernous fistula with detachable coils and ethylene-vinyl alcohol copolymer.

Carotid cavernous fistula (CCF) is a rare disease caused by abnormal communications between the internal carotid artery (direct fistula) or meningeal ...

Complete and Partial Resuscitative Endovascular Balloon Occlusion of the Aorta for Hemorrhagic Shock

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for hemorrhage control. With the advent of purpose-built devices, REBOA has become increasingly common in civilian trauma and acute care settings. Currently available REBOA catheters were designed as complete aortic occlusion devices. However, the therapeutic window for complete aortic occlusion is time-limited due to ischemia-reperfusion injury. The partial procedure allows blood flow past the level of occlusion while maintaining targeted proximal pressure, which has been shown to reduce distal ischemia and adjunctive resuscitation requirements in preclinical studies with prolonged occlusion times as compared to traditional complete occlusion.
pREBOA-PRO is the first catheter designed to enable partial and complete aortic occlusion and is currently in limited market release at seven Level I trauma centers in North America. This paper will focus on procedural considerations for REBOA, including patient selection criteria and a comparison of complete and partial aortic occlusion in a simulator, along with highlighting critical steps to improve clinical outcomes. Additionally, this paper reviews a contrast-enhanced CT scan from a trauma patient that shows distal perfusion after 2 h of partial aortic occlusion using this newly designed catheter and discusses representative results from the limited market release to highlight the profound effect of technological innovation on outcomes in vascular emergencies.

A commercial catheter was designed to facilitate true partial resuscitative endovascular balloon occlusion of the aorta (REBOA) and address the complications associated with complete aortic occlusion. Initial clinical reports indicate that partial REBOA improves transition to reperfusion, reduction of distal ischemia, and extension of safe occlusion time compared to complete occlusion.

Oclusión completa y parcial con balón endovascular de reanimación de la aorta para el shock hemorrágico

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

Una lesión de globo segmentario controlada por presión arterial carótida de rata con aplicación terapéutica periavential

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