Los autores no tienen reconocimientos.

Todos los animales procedimientos fueron realizados con la aprobación del Comité de Cuidado y Uso de Animales Institucional de la Universidad de Cincinnati y de conformidad con la Guía para el Cuidado y Uso de Animales de Laboratorio de los Institutos Nacionales de Salud (NIH Publication No. 85-23, Revisado 1996). 1. Preparación del ratón <ol><li> Practicar la eutanasia mediante la exposición del animal a una dosis de anestésico supraterapéutica, isoflurano inhalado al efecto. Verifique la eutanasia primario a través pizca de pie, como un estímulo nocivo. Método secundario de la eutanasia, de corte diafragma, tendrá lugar en una próxima etapa. Los métodos alternativos pueden ser utilizados se obtiene la eutanasia sentado. </li><li> Sanitizie la superficie externa del ratón por pulverización de la piel a lo largo de la región abdominal, donde se harán los cortes iniciales, con etanol al 70% hasta el punto de humedad por lo que la piel está húmeda con etanol y los pelos sueltos / seco no entran la región. </li><li> Coloque el ratón sobre un surtablero de lógica en la posición supina con apéndices superiores e inferiores se extendió hacia el exterior. </li><li> Apéndices seguros a la junta con cinta quirúrgica. </li></ol> 2. Aislamiento del corazón y la aorta <ol><li> Después de la colocación apropiada del ratón, el uso de fórceps para localizar y aislar la piel abdominal justo por debajo de la apófisis xifoides del esternón. </li><li> Crear tensión levantando la piel hacia arriba y utilizar las tijeras para quitar la piel superficial, dejando al descubierto la parte superior del peritoneo y la parte inferior de la cavidad torácica. </li><li> Introduzca la cavidad peritoneal levantando el apéndice xifoides y la realización de incisiones laterales apenas inferiores al proceso a lo largo de los márgenes subcostal. </li><li> Diseccionar hasta la cavidad torácica, pasando a través del diafragma, asegurándose de no lacerar el corazón o cualquier principales vasos sanguíneos. </li><li> Extender las incisiones laterales hechas en el paso 2.3 cranealmente para eliminar la porción anterior de la caja torácica. Sinecesario, liberar el corazón de la pared anterior del tórax mediante disección roma. </li><li> Limpie la cavidad torácica de la sangre ajena y líquido utilizando una gasa estéril para absorber el material. Una vez que el área es más visible, retire los pulmones para exponer mejor el corazón y la aorta. </li></ol> 3. La perfusión de corazón y la aorta NOTA: Para obtener la sangre mediante punción cardíaca, hacerlo justo antes de este paso. <ol><li> Llenar una jeringa de 10 cc con 10 ml de hielo frío estéril 1x solución de tampón fosfato (PBS), y adjuntar una aguja de calibre 25 a la jeringa. </li><li> Suavemente inserte la aguja en el ventrículo izquierdo del corazón. </li><li> Cortar la aurícula derecha para aliviar la acumulación de la presión de perfusión. Perfundir el contenido de la jeringa en el ratón más de 2 - 3 minutos. </li><li> Utilice una gasa estéril en la abertura en la aurícula derecha para absorber líquido de perfusión. </li></ol> 4. El aislamiento y la escisión de la aorta <ol><li> Al término de PErfusion, utilizar una gasa estéril para absorber cualquier líquido restante enturbiamiento del campo de visión dentro de la cavidad torácica. </li><li> Exponer los contenidos gastrointestinales cortando caudalmente a través de la pared abdominal, la incisión se extiende a la zona suprapúbica. Extienda la incisión más hacia los miembros inferiores bilateralmente para crear colgajos de piel que pueden ser puestas abajo o extirpados. </li><li> Retire los lóbulos del hígado, el páncreas, el estómago, el bazo y los intestinos para visualizar mejor la aorta. Tenga cuidado cuando la disección de cerca de la región perirrenal, como la aorta es superficial en las ramas de las arterias renales. NOTA: Realice este paso en un preciso y matricularse manera como el tracto gastrointestinal es rico en bacterias que aumenta la propensión a la contaminación. </li><li> Enjuague el área con 1x PBS y retirar todo el fluido por absorción con una gasa estéril. </li><li> El uso de micro tijeras estériles y microforceps, separar la aorta dorsal de la columna vertebral y el esófago ventralmente.Lo mejor es usar una disección roma y la utilización de un microscopio de disección para esto. NOTA: En el aislamiento de la aorta, iniciar caudalmente en la bifurcación ilíaca y mover cranealmente limpieza y separación de la aorta de la superficie dorsal de la cavidad o iniciar cranealmente en las carótidas y arterias braquiocefálica de disección caudalmente. Cualquier método es apropiado y es hasta la comodidad investigadores. </li><li> Retire el tejido adiposo perivascular utilizando finas micro tijeras asegurándose de no eliminar una parte de la pared aórtica. Esto es esencial para minimizar el potencial de contaminación de fibroblastos cuando el cultivo de células del músculo liso aórtico. </li></ol>

<ol>
	<li>Go, A. 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=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129 (3), 28-292 (2014).</li><li>Ross, R., Harker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperlipidemia+and+atherosclerosis.">Hyperlipidemia and atherosclerosis.</a> Science. 193 (4258), 1094-1100 (1976).</li><li>Clouse, W. D., 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=Acute+aortic+dissection:+population-based+incidence+compared+with+degenerative+aortic+aneurysm+rupture.">Acute aortic dissection: population-based incidence compared with degenerative aortic aneurysm rupture.</a> Mayo Clin Proc. 79 (2), 176-180 (2004).</li><li>Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc Biol. 24 (3), 429-434 (2004).</li><li>Pleumeekers, H. 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=Aneurysms+of+the+abdominal+aorta+in+older+adults.+The+Rotterdam+Study.">Aneurysms of the abdominal aorta in older adults. The Rotterdam Study.</a> Am J Epidemiol. 142 (12), 1291-1299 (1995).</li><li>Brophy, C. M., Reilly, J. M., Smit, G. J., Tilson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+inflammation+in+nonspecific+abdominal+aortic+aneurysm+disease.">The role of inflammation in nonspecific abdominal aortic aneurysm disease.</a> Ann Vasc Surg. 5 (3), 229-233 (1991).</li><li>Ray, J. L., Leach, R., Herbert, J. M., Benson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+vascular+smooth+muscle+cells+from+a+single+murine+aorta.">Isolation of vascular smooth muscle cells from a single murine aorta.</a> Methods Cell Sci. 23 (4), 185-188 (2001).</li><li>Weiss, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+murine+cardiovascular+system+with+magnetic+resonance.">Imaging the murine cardiovascular system with magnetic resonance.</a> Circ Res. 88 (6), 550-551 (2001).</li><li>Graham, E. L., Balla, C., Franchino, H., Melman, Y., del Monte, F., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+culture,+and+functional+characterization+of+adult+mouse+cardiomyoctyes.">Isolation culture, and functional characterization of adult mouse cardiomyoctyes.</a> J Vis Exp. (79), 50289 (2013).</li><li>Wassef, M., Upchurch, G. R., Kuivaniemi, H., Thompson, R. W., Tilson, M. D. 3. r. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+in+abdominal+aortic+aneurysm+research.">Challenges and opportunities in abdominal aortic aneurysm research.</a> J Vasc Surg. 45 (1), 192-198 (2007).</li></ol>

Los autores no tienen nada que revelar.

Aortic disease can lead to significant systemic pathology and possibly death in severe cases. Treatment options are currently limited therefore continued research in the field is necessary10. Murine models are ideal to further study disease progression and potentially therapeutic options4. Therefore, the successful completion of the method described in this manuscript will provide investigators with the tools to measure disease progression and drug efficacy. 
This technique can be modified in several ways in order to optimize the results. The use of surgical loupes or a dissecting microscope can aid in the visualization of the murine anatomy and vasculature details and when removing surrounding adipose tissue from the aorta. Additionally, the use of dedicated surgical utensils for specific tasks can increase the longevity of tools; for example, the use of scissors for cutting skin and separate scissors to cut through the ribs would be ideal. The use of a small gauged needle when perfusing the heart is preferred as this will allow the tissue to close around the puncture as opposed to a larger gauge needle where a hole will remain. 
After the aorta is successfully removed, it can be utilized for a variety of assays. The aorta can be used for molecular studies such as enzyme activity, protein expression and gene expression profiles. Additionally, the aorta can be fixed for histologic analysis where in situ assays can be utilized as well as characterization of the aortic morphology. Finally, one can isolate smooth muscle and/or endothelial cells from a freshly harvest aorta for in vitro studies with these primary isolated cells. 
The major limitation to isolation and excision of the aorta is that the animal must be sacrificed, especially in the cases of longitudinal studies. Currently, there is no single technique comparable to isolation and excision of the murine aorta. Small animal imaging technologies is becoming more advanced and approaching characterization capabilities of direct observation however it continues to have limitations including cost8. Immortalized cells lines can be used for in vitro studies however immortal cells are not as useful as primary isolated cells for in vitro assays especially for viability studies9. Isolation and excision of the murine aorta is currently the most versatile technique that can be used in the study of aortic disease.

La aorta juega un papel fundamental en la función cardiovascular suministrar al cuerpo con sangre oxigenada y las porciones similares a los demás del cuerpo, es susceptible a la enfermedad. Enfermedades de la aorta comunes son la aterosclerosis y el aneurisma, que son el resultado de factores genéticos y ambientales como la dieta, el tabaquismo y el sedentarismo 1. La aterosclerosis es la deposición de calcio y una placa base de lípidos a la pared aórtica típicamente encontrado en pacientes con hiperlipidemia crónica 2. Los aneurismas se caracterizan por la degradación de los componentes estructurales de la aorta y el adelgazamiento de la pared del vaso, seguido por un aumento del diámetro lo que finalmente puede conducir a la ruptura 3. Los modelos animales son herramientas importantes utilizadas para estudiar el mecanismo de la enfermedad y la eficacia de los tratamientos potenciales. Modelos animales más comunes utilizados en la investigación cardiovascular, investigar específicamente la investigación de los trastornos vasculares y metabólicasincluir ratones modificados genéticamente para alterar los niveles de lípidos, tales como golpe de gracia gen de la apolipoproteína E y lipoproteínas de densidad baja ratones knockout del gen del receptor 4. La mayoría de los métodos para evaluar los mecanismos de la enfermedad y la eficacia de la terapia incluiría el aislamiento y la escisión de la aorta. Una vez que la aorta está localizado y aislado, el análisis morfométrico se puede determinar, como la presencia de un aneurisma, típicamente definido como un incremento mayor que 50% en el diámetro 5. Después necesario el análisis in situ es completa, la aorta se puede escindir para su posterior análisis. Una aorta extirpada puede ser congelado instantáneamente para llevar a cabo estudios moleculares, tales como proteínas y / o ensayos de expresión de genes o fijo utilizando 4% de paraformaldehído y posteriormente incrustado, se seccionaron y se tiñeron para el análisis histológico. El análisis histológico de la aorta puede mostrar las características comunes de la enfermedad aórtica, como la degradación estructural, la formación de placa, y la infiltración de leucocitos &lt;sup&gt; 6,7. Además, una aorta extirpada se puede utilizar para aislar líneas celulares primarias que incluyen células endoteliales y de músculo liso que posteriormente se pueden utilizar para una variedad de estudios in vitro 7. Actualmente, no existen otros métodos para proporcionar esta caracterización en profundidad de la enfermedad de la aorta, así como brindar a los investigadores herramientas para estudiar más a fondo la enfermedad cardiovascular. Técnicas de imagen como la resonancia magnética, tomografía computarizada y la ecografía son los métodos más cercanos para evaluar morfológicamente la aorta, sin embargo esto es difícil en pequeños animales y la obtención de un dispositivo con tecnología adecuada es caro 8. Las líneas celulares inmortalizadas se pueden comprar para investigar los mecanismos potenciales de la enfermedad y la eficacia de las terapias, sin embargo, la naturaleza artificial de estas células son limitantes en el estudio de los efectos sobre el ciclo de vida celular y la apoptosis 9. El objetivo general deeste manuscrito es demostrar el aislamiento estéril y la escisión de la aorta murino en la investigación de la enfermedad cardiovascular.

<table><tbody><tr><td>Isoflurane</td><td>Med-Vet International</td><td>#RXISO-250</td><td></td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>07-678-001</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>Sterile gauze</td><td>Dukal Corporations</td><td>1312</td><td></td></tr><tr><td>25 gauge needle</td><td>BD</td><td>305122</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td></td></tr></tbody></table>

isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Al finalizar el procedimiento, habrá una aorta intacta procedente del corazón, descendiendo en las cavidades torácica y abdominal con las arterias renales todavía conectados (Figura 1A). Desde aquí, la aorta se pueden obtener imágenes in situ para cuantificar los cambios morfométricos que son de diagnóstico en el estudio de los aneurismas aórticos abdominales (Figura 1B y 1C). Posteriormente, la aorta se puede quitar, fijadas y teñidas para observar los cambios histológicos. Una mancha general y común de la aorta es la hematoxilina y eosina (Figura 2A y 2B). Además, la integridad estructural de la aorta se puede cuantificar cualitativamente mediante tinción Verhoff van Geison mirar las bandas elásticas (Figura 2C y 2D). En lugar de obtener un análisis histológico, el investigador puede parpadear congelar la aorta para su posterior proteínas y ácido ribonucleico expresión. La opción final para el investigador es utilizar la aorta para obtener el aislamiento de células primarias. Estas células se pueden cultivar en cultivo (Figura 3A) y se utilizan para una variedad de estudios in vitro, incluyendo estudios de viabilidad (Figura 3B) y localización de la proteína (Figura 3C). <img alt="Figura 1" src="/files/ftp_upload/52172/52172fig1.jpg" width="700" /> La Figura 1. En imágenes representativas situ de aorta murino. (A) Representación de la anatomía normal de ratón después del aislamiento de la aorta incluyendo el corazón y los riñones. (B) imagen de una aorta abdominal murina normal intacta ampliada. (C) Imagen de un aórtico abdominal enferma ampliada con un aneurisma en la suprarrenal región. <a href="https://www.jove.com/files/ftp_upload/52172/52172fig1highres.jpg" target="_blank">Haga clic aquí para ver una versión más grande de l</a>es figura. <img alt="Figura 2" src="/files/ftp_upload/52172/52172fig2.jpg" width="700" /> Figura 2. El análisis histológico de aorta murino. (A) Imagen Representante de hematoxilina y eosina normal de una aorta suprarrenal sana de 20 aumentos. (B) Imagen Representante de una aorta suprarrenal aneurismática con trombo intramural después de hematoxilina y eosina de 20 aumentos. (C) Imagen de un Representante mancha Verhoff van Geison, destacando las bandas elásticas de una aorta suprarrenal normal a 10 aumentos. (D) imagen representativa de una mancha Verhoff van Geison destacando las bandas elásticas de una aorta desarrollar enfermedad suprarrenal señalado por el adelgazamiento y que perfila de bandas de elastina y engrosamiento de la capa adventicia. <a href="https://www.jove.com/files/ftp_upload/52172/52172fig2highres.jpg" target="_blank">Por favor cli</a>ck aquí para ver una versión más grande de esta figura. <img alt="Figura 3" src="/files/ftp_upload/52172/52172fig3.jpg" width="700" /> Figura 3. miocitos aórticas en cultivo primario de aorta. (A) imagen representativa de los miocitos aórticas primarias en cultivo a 20X de aumento obtenido de la aorta abdominal. (B) imagen representativa de los miocitos aórticas primarias durante un ensayo de exclusión con azul de tripano después de un tratamiento 500 M de H 2 O 2 de 20 aumentos obtenido a partir de la aorta abdominal. (C) imagen representativa de una mancha de inmunohistoquímica para la actina alfa en células aórticas aislado primario del músculo liso de la aorta abdominal. <a href="https://www.jove.com/files/ftp_upload/52172/52172fig3highres.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease at JoVE.com

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Aislamiento y Escisión de murino Aorta; Una técnica versátil en el Estudio de la Enfermedad Cardiovascular

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

isolation-excision-murine-aorta-versatile-technique-study

Nanyang Technological University

Research

JoVE Journal

Medicine

32.3K vistas.  University of Cincinnati College of Medicine. Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease. 

Video: Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

El trasplante ortotópico aórtica en los ratones para el Estudio de la Enfermedad Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Investigación

Medicina

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Utilizando la técnica de la manga en un modelo murino de trasplante aórtico - un Video instructivo

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Modelo de trasplante de aórtica cervical murino utilizando una técnica de manguito no sutura modificada

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

Modificaciones del método de Langendorff para el aislamiento simultáneo de miocitos auriculares y ventriculares de ratones adultos

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Biología

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Aislamiento de murino coronaria células musculares lisas vasculares

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Lesión por aplastamiento aórtico murino: una eficacia<em&gt; In Vivo</em&gt; Modelo de proliferación de células musculares lisas y función endotelial

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Una técnica modificada para la constricción aórtica transversa en ratones

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study introduces a comprehensive protocol for the isolation and histological analysis of aortic arch and root lesions in a widely used atherosclerotic mouse model, low-density lipoprotein receptor knock-out (Ldlr-/-) mice. The aortic arch and root are key sites for atherosclerotic lesions, and their examination is critical for assessing the onset, progression, or regression of atherosclerosis, predicting cardiovascular event risks, and identifying potential therapeutic targets. This protocol outlines methods for quantifying atherosclerotic burden in the aortic arch and root, including tissue isolation, fixation, Oil Red O staining, aortic root sectioning, Hematoxylin and Eosin (HE) staining, Verhoeff-Van Gieson (VVG) staining, and image analysis. Oil Red O staining measures plaque area in the aortic arch, evaluating the severity of atherosclerosis, while HE staining of the aortic root reveals plaque components such as the lipid core and fibrous cap, facilitating the assessment of plaque stability and rupture risk. VVG staining can stain collagen fibers within tissues, providing further insights into plaque composition and related information. This thorough analysis offers valuable insights into the mechanisms of lesion development and may guide the creation of novel therapeutic strategies for preventing and treating atherosclerosis.

This protocol provides a comprehensive methodology for the quantitative analysis of atherosclerotic plaques to facilitate more in-depth research into the mechanisms and progression of atherosclerosis.

Aislamiento y análisis de lesiones del arco aórtico y la raíz en un modelo de ratón aterosclerótico

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study ...

Surgical Aortic Debanding: A Procedure to Study Left Ventricular Reverse Remodeling in Murine Model of Aortic Constriction

Source: Goncalves-Rodrigues, P. et al. <a href="https://www.jove.com/video/60036" target="_blank">Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents</a>. J. Vis. Exp. (2021)
This video demonstrates the technique of aortic debanding, which can lead to reverse remodeling to limit the progression towards heart failure in a murine model. Post debanding, a significant reduction of ventricular afterload triggers regression of ventricular hypertrophy and fibrosis, causing a recovery in cardiac function.

Extirpación quirúrgica de la aorta: un procedimiento para estudiar la remodelación inversa del ventrículo izquierdo en el modelo murino de constricción aórtica