Este trabajo fue apoyado la concesión SCCOR &quot;Enfermedad de AAA: Mecanismos, estratificación y tratamiento&quot; (P50 HL083800-03) de los Institutos Nacionales de Salud

<ol><li> Los ratones son anestesiados con isoflurano inhalado 2,0 3,0 ml / l. </li><li> El abdomen se rasura y limpia con alcohol y betadine. </li><li> El ratón es colocado en posición supina en el campo operatorio. </li><li> Guantes estériles son usados ​​por el cirujano, y los instrumentos utilizados han sido esterilizados con agua caliente esterilizador de cuentas. </li><li> Una larga incisión en el abdomen línea media se hace. Contenido abdominal se retraen fuera del abdomen con una gasa estéril para exponer la aorta abdominal. </li><li> La aorta abdominal es aislado del nivel de la vena renal izquierda hasta la bifurcación. </li><li> Las ramas de la aorta abdominal se exponen y se liga con 10-0. Todas las ramas de la aorta en 1,0 cm de la bifurcación se ligan. </li><li> Temporal 6,0 ligaduras de seda se colocan alrededor de las porciones proximales y distales de la aorta </li><li> Un aortotomía se crea en la bifurcación con una aguja de calibre 30. </li><li> El calor en forma de cono tubo de polietileno (PE-10) se introduce a través de la aortotomía y asegurado con un empate. </li><li> Usando una bolsa de solución salina colgado a la altura de 136 cm para calibrar 100 mmHg, la aorta se llena con la solución salina que contenía 4,5 U / ml Tipo I elastasa pancreática porcina. La aorta se dilata normalmente a 150-170% de su diámetro original durante la perfusión de la elastasa de 05.15 minutos. </li><li> El catéter se retira la perfusión y la aortotomía cierra con una sutura 10-0 para evitar la constricción. </li><li> Después de la aortotomía está cerrada, la ligadura distal se retira. </li><li> Si hay poco o ningún sangrado de la aortotomía reparado, la ligadura proximal se elimina. La aorta ampliado se llena de sangre inmediatamente. </li><li> Los intestinos se introducen en el abdomen, y la herida se cierra en dos capas con 6-0 nylon / polipropileno de sutura. </li><li> 0,25% Bupivicaime gotas (aprox una gota por cada centímetro del tamaño de la incisión) se coloca sobre la herida y una dosis de Caprofen (5mg/kg) se administra por vía subcutánea. </li><li> El ratón es colocado en un sitio caliente y se recupera en 15 minutos. </li></ol><img src="/files/ftp_upload/1280/fig1280.png" alt="Figura 1" /> Figura 1. Resultados Representante de la elastasa inducida AAA.

<ol>
	<li>Anidjar, S., Salzmann, J. L., Gentric, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-973 (1990).</li><li>Pyo, R., Lee, J. K., Shipley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+of+matrix+metalloproteinase-9+(gelatinase+B)+suppresses+development+of+experimental+abdominal+aortic+aneurysms.">Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.</a> J Clin Invest. 105 (11), 1641-1641 (2000).</li><li>Carsten, C. G., Calton, W. C., Johanning, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase+is+not+sufficient+to+induce+experimental+abdominal+aortic+aneurysms.">Elastase is not sufficient to induce experimental abdominal aortic aneurysms.</a> J Vasc Surg. 33 (6), 1255-1255 (2001).</li></ol>

The authors have nothing to disclose.

Estudios anteriores han observado importantes de lote a lote de variación en el desarrollo de diferentes preparaciones de AAA elastasa comercial 3. Por lo tanto, es fundamental para controlar los lotes de producción, así como tener grupos de control adecuados a fin de documentar la capacidad de la preparación para inducir la AAA. Curiosamente, la susceptibilidad al desarrollo de la AAA en este modelo depende de la cepa de ratón utilizada 3. El ratón utilizado en e...

<table><tbody><tr><td>Type I porcine pancreatic elastase</td><td>Reagent</td><td>Sigma-Aldrich</td><td>E1250</td><td></td></tr><tr><td>polyethylene tubing (PE-10)</td><td></td><td>BD Biosciences</td><td>427400</td><td></td></tr><tr><td>CHANGE-A-TIP</td><td></td><td>Bovie Medical Corporation</td><td>18010-00 (FST)</td><td>cautery knife</td></tr><tr><td>LEICA MZ6</td><td></td><td>Leica Microsystems</td><td></td><td>stereomicroscope</td></tr><tr><td>LEICA DFC290HD</td><td></td><td>Leica Microsystems</td><td></td><td>USB camera</td></tr><tr><td>Image Pro Express ver. 6.0</td><td></td><td>Media Cybernetics Inc.</td><td></td><td>software</td></tr></tbody></table>

creation of murine experimental abdominal aortic aneurysms with elastase

Infusión intraluminal transitoria de la elastasa pancreática porcina en el segmento infrarrenal de la aorta abdominal es el modelo animal más utilizado del aneurisma de aorta abdominal (AAA) desde que fue descrita por primera vez en ratas por Anidjar y sus colegas. 1 El fundamento de su desarrollo se basó sobre la naturaleza de la elastina interrumpido observado en AAA. Este modelo de rata ha sido modificado para producir AAA en la región de la aorta infrarrenal de los ratones. 2 El modelo tiene la capacidad de agregar visión completa de la biopatología de la AAA, debido a la aparición de numerosos transgénicos y ratones knock-out de genes. Por otra parte, es una plataforma viable para probar agentes terapéuticos potenciales para la AAA. En este video, que demuestre el procedimiento de infusión elastasa AAA utilizada en nuestro laboratorio. Los ratones son anestesiados utilizando isoflurano al 2,5%, y una laparotomía se realiza en condiciones estériles. El aortais abdominal aislado con la ayuda de un microscopio estereoscópico de funcionamiento (Leica). Después de colocar las ligaduras temporales alrededor de la aorta proximal y distal, una aortotomía se crea en la bifurcación con la punta de una aguja de calibre 30. Un segmento de calor en forma de cono de la tubería de polietileno PE-10 se introduce a través de la aortotomía y seguro. La luz de la aorta es posteriormente perfundidos durante 5-15 minutos a 100 mm Hg con el tipo de solución salina con que la elastasa pancreática porcina (4,5 U / ml, Sigma Chemical Co.). Después de retirar el catéter de perfusión, el aortotomía ha sido reparada sin la constricción de la luz.

Watch this Scientific Journal Video about Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase at JoVE.com

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

Este video muestra cómo inducir a los aneurismas de aorta abdominal (AAA) en ratones a través de la infusión intraluminal transitoria de la elastasa pancreática porcina en el segmento infrarrenal de la aorta abdominal. El modelo tiene la capacidad de agregar visión completa de la biopatología de la AAA, debido a la aparición de numerosos transgénicos y ratones knock-out de genes.

Creación de murino Experimental aneurismas de aorta abdominal con elastasa

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

creation-murine-experimental-abdominal-aortic-aneurysms-with

Research

JoVE Journal

Biology

24.4K vistas.  Stanford University School of Medicine. Este video muestra cómo inducir a los aneurismas de aorta abdominal (AAA) en ratones a través de la infusión intraluminal transitoria de la elastasa pancreática porcina en el segmento infrarrenal de la aorta abdominal. El modelo tiene la capacidad de agregar visión completa de la biopatología de la AAA, debido a la aparición de numerosos transgénicos y ratones knock-out de genes.

Video: Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Un modelo murino Nueva endovascular de aneurismas aórticos

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Investigación

Medicina

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Ensayo del anillo aórtico

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Biología

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Constricción de la aorta transversa en ratones

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Aislamiento y grabaciones Kv Canal murinos en miocitos auriculares y ventriculares

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Direct Imaging de calcio ER con Targeted-esterasa inducida colorante de carga (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

Modelo Quirúrgico Murino de Aneurisma aórtico torácico descendente fortuito tópico

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

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 ...

<meta charset="utf8"></head><body>Investigación de los mecanismos moleculares que subyacen al aneurisma de aorta abdominal murino

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

<meta charset="utf8"></head><body>Autor destacado: Desarrollo y caracterización de un modelo de ratón para el aneurisma de aorta abdominal

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

Un modelo de aneurisma de aorta abdominal de ratón por infiltración periadventicial de cloruro de calcio y elastasa

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Creación de murino Experimental aneurismas de aorta abdominal con elastasa

Resumen

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Modelo Quirúrgico Murino de Aneurisma aórtico torácico descendente fortuito tópico

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Un modelo de aneurisma de aorta abdominal de ratón por infiltración periadventicial de cloruro de calcio y elastasa

Un modelo de aneurisma de aorta abdominal de ratón por infiltración periadventicial de cloruro de calcio y elastasa