Name: isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease
Uploaded: 2014-11-24T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease at JoVE.com

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Todos os procedimentos com animais foram realizados com a aprovação do Cuidado e Uso Comitê da Universidade de Cincinnati animal Institucional e de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório do National Institutes of Health (NIH Publication No. 85-23, revista em 1996). 1. Preparar o mouse <ol><li> Eutanásia por exposição do animal a uma dose supraterapêutica de anestésico, isoflurano inalado para o efeito. Verifique eutanásia principal via toe pitada como um estímulo nocivo. Método secundário da eutanásia, o corte de diafragma, irá ocorrer em uma próxima etapa. Métodos alternativos podem ser concedidos a eutanásia utilizado é obtido. </li><li> Sanitizie a superfície externa do rato por pulverização da pele ao longo da região abdominal, onde serão feitas as reduções iniciais, com etanol a 70% para o ponto de humidade de modo a que a pele é molhado com etanol e todos os cabelos secos soltas / não entram a região. </li><li> Coloque o mouse em uma surplaca cirúrgica em decúbito dorsal, com apêndices superiores e inferiores estendidos para fora. </li><li> Apêndices seguros ao Conselho, utilizando fita cirúrgica. </li></ol> 2. Isolamento do Coração e Aorta <ol><li> Após o posicionamento adequado do mouse, utilize uma pinça para localizar e isolar a pele abdominal inferior apenas ao processo xifóide do esterno. </li><li> Criar tensão levantando a pele para cima e usar uma tesoura para remover a pele superficial, expondo a porção superior do peritônio e porção inferior da cavidade torácica. </li><li> Entrar na cavidade peritoneal, levantando o apêndice xifóide e fazer incisões laterais apenas inferior ao processo ao longo das margens subcostal. </li><li> Dissecar-se para dentro da cavidade torácica, passando pelo diafragma, sendo certo não lacerar o coração ou qualquer um dos principais vasos sanguíneos. </li><li> Estenda as incisões laterais feitas na etapa 2.3 cranial para remover a porção anterior da caixa torácica. Senecessário, libertar o coração da parede anterior do tórax usando dissecção romba. </li><li> Limpe a caixa torácica do sangue alheio e fluido, utilizando gaze estéril para absorver material. Uma vez que a área é mais visível, remoção dos pulmões para expor melhor o coração e aorta. </li></ol> 3. Perfusão de Coração e Aorta NOTA: Para obter o sangue através de punção cardíaca, fazê-lo imediatamente antes desta etapa. <ol><li> Encher uma seringa de 10 cc com 10 ml de gelo estéril solução tampão de fosfato frio 1x (PBS), e anexar uma agulha de calibre 25 para a seringa. </li><li> Inserir a agulha suavemente para o ventrículo esquerdo do coração. </li><li> Corte o átrio direito para aliviar o acúmulo de pressão de perfusão. Perfundir os conteúdos da seringa para dentro do rato de 2 - 3 minutos. </li><li> Use gaze estéril na abertura no átrio direito para absorver líquido de perfusão. </li></ol> 4. Isolamento e Excisão da Aorta <ol><li> Após a conclusão da perfusion, utilizar gaze estéril para absorver qualquer fluido remanescente turvação do campo de visão dentro da cavidade torácica. </li><li> Expor os conteúdos gastrointestinais caudalmente cortando através da parede abdominal, que se estende da incisão para a região suprapúbica. Estender a incisão mais para os membros inferiores bilateralmente para criar retalhos de pele, que pode ser fixado para baixo ou excisadas. </li><li> Retire os lobos do fígado, pâncreas, estômago, baço e intestino para visualizar melhor a aorta. Tome cuidado ao dissecar perto da região perirrenal, como a aorta é superficial com os ramos das artérias renais. NOTA: Execute esta etapa em um preciso e matricular forma como o trato gastrointestinal é rica em bactérias, o que aumenta a propensão para a contaminação. </li><li> Lavar a área com 1x PBS e remover todo o fluido por absorção com uma gaze estéril. </li><li> Usando microtesouras e micropinças estéreis, separe a aorta do dorsal da coluna e do esôfago ventrally.É melhor utilizar a dissecção romba e utilizando um microscópio de dissecção para este. NOTA: Ao isolar a aorta, iniciar caudalmente na bifurcação ilíaca e mover-se cranialmente limpeza e separação da aorta a partir da superfície dorsal da cavidade cranial ou iniciar as carótidas e braquiocefálicos dissecação caudalmente. Qualquer método é apropriado e é até o conforto pesquisadores. </li><li> Remover o tecido adiposo perivascular usando microtesouras finas sendo certo não para remover uma porção da parede da aorta. Isto é essencial para minimizar o potencial de contaminação por fibroblastos quando a cultura de células musculares lisas da aorta. </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>

Os autores não têm nada a 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.

A aorta desempenha um papel crucial na função cardiovascular fornecendo o corpo com sangue oxigenado e outros semelhantes para partes do corpo, é susceptível à doença. Doenças da aorta comuns incluem aterosclerose e aneurisma, que são o resultado de fatores genéticos e ambientais, incluindo dieta, tabagismo e sedentarismo 1. A aterosclerose é a deposição de cálcio e uma placa à base de lípidos para a parede da aorta, tipicamente encontrados em pacientes com hiperlipidemia crónica 2. Os aneurismas são caracterizadas por degradação de componentes estruturais da aorta e adelgaçamento da parede do vaso, seguido por um aumento do diâmetro que poderia levar à ruptura 3. Os modelos animais são importantes instrumentos para estudar o mecanismo da doença e da eficácia de tratamentos potenciais. Modelos animais comuns utilizados na investigação cardiovascular, pesquisar especificamente investigar distúrbios vasculares e metabólicosincluem camundongos geneticamente modificados para alterar os níveis de lipídios, como knockout gene apolipoproteína E e lipoproteína de baixa densidade camundongos knockout gene receptor 4. A maioria dos métodos para avaliar os mecanismos de doenças e a eficácia da terapia incluem o isolamento e a excisão da aorta. Uma vez que a aorta é localizado e isolado, a análise morfométrica pode ser determinada, tal como a presença de aneurisma, tipicamente definido como um aumento superior a 50% no diâmetro de 5. Depois necessário análise in situ é completa, a aorta pode ser excisado para análise posterior. Um aorta pode ser excisado flash congelados para conduzir estudos moleculares, tais como proteínas e / ou ensaios de expressão de gene ou fixo utilizando 4% de paraformaldeído e depois incorporado, seccionados e corados para análise histológica. A análise histológica da aorta pode demonstrar características comuns de doenças da aorta, como a degradação estrutural, a formação de placa, e infiltração de leucócitos &lt;sup&gt; 6,7. Além disso, uma aorta excisado pode ser utilizado para isolar as linhas de células primárias, incluindo células do músculo liso e endoteliais que podem posteriormente ser utilizados para uma variedade de estudos in vitro 7. Atualmente, não existem outros métodos para fornecer essa caracterização aprofundada das doenças da aorta, bem como fornecer aos pesquisadores ferramentas para estudar mais a doença cardiovascular. Modalidades de imagem, como ressonância magnética, tomografia computadorizada e ultra-sonografia são os métodos mais próximos para avaliar morfologicamente a aorta, no entanto isso é difícil em pequenos animais e na obtenção de um dispositivo com tecnologia adequada é caro 8. As linhas celulares imortalizadas pode ser comprado a investigar potenciais mecanismos da doença e da eficácia de terapias, no entanto, a natureza artificial destas células são limitantes no estudo dos efeitos sobre o ciclo de vida celular e apoptose 9. O objetivo geral doeste manuscrito é demonstrar a excisão e isolamento estéril da aorta de murino para a investigação de doenças cardiovasculares.

<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.

Após a conclusão do processo, haverá uma aorta intacta proveniente do coração, descendo para as cavidades torácica e abdominal, com as artérias renais ainda ligado (Figura 1A). A partir daqui, a aorta pode ser visualizado in situ para quantificar as alterações morfométricas que são de diagnóstico no estudo de aneurismas da aorta abdominal (Figura 1B e 1C). Subsequentemente, a aorta pode ser removidas, fixadas e coradas para olhar para mudanças histológicas. A mancha geral e comum da aorta é a hematoxilina e eosina (Figura 2A e 2B). Além disso, a integridade estrutural da aorta pode ser qualitativamente quantificado utilizando a coloração de van Verhoff Geison olhar para as bandas elásticas (Figura 2C e 2D). Em vez de se obter uma análise histológica, o investigador pode piscar congelar a aorta para a proteína subseqüente e expre ácido ribonucléicossion. A última opção para o investigador é usar a aorta para obter o isolamento de células primárias. Estas células podem ser cultivadas em cultura (Figura 3A) e utilizados para uma variedade de estudos in vitro, incluindo estudos de viabilidade (Figura 3B) e localização de proteínas (Figura 3C). <img alt="Figura 1" src="/files/ftp_upload/52172/52172fig1.jpg" width="700" /> Figura 1. Em imagens representativas in situ de aorta murino. (A) Representação da anatomia normal de ratinho após o isolamento da aorta incluindo o coração e os rins. (B) imagem ampliada de uma aorta abdominal de murino normal intacta. (C) de uma aorta abdominal doente ampliada com um aneurisma no suprarrenal região. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig1highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior do th</a>é figura. <img alt="Figura 2" src="/files/ftp_upload/52172/52172fig2.jpg" width="700" /> Figura 2. A análise histológica da aorta murino. (A) Imagem representativa de um HE mancha normal de uma aorta supra-renal saudável com ampliação de 20x. (B) Imagem representativa de um aneurisma da aorta supra-renal com trombo intramural após HE mancha com ampliação de 20x. (C) Imagem representativa de um Verhoff van Geison mancha, com destaque para as bandas de elastina de uma aorta supra-renal normal no aumento de 10x. (D) Imagem representativa de uma mancha Verhoff van Geison destacando as bandas elásticas de uma aorta desenvolver doença supra-renal observado pelo afinamento e striating de bandas de elastina e espessamento da camada adventícia. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig2highres.jpg" target="_blank">Por favor, cli</a>ck aqui para ver uma versão maior desta figura. <img alt="Figura 3" src="/files/ftp_upload/52172/52172fig3.jpg" width="700" /> Figura 3. miócitos aórticas cultivadas de aorta primários. (A) Imagem representativa de miócitos da aorta primárias em cultura, a ampliação de 20X obtidas a partir da aorta abdominal. (B) Imagem representativa de miócitos da aorta primária durante um ensaio de exclusão com azul de tripano após um tratamento de 500 uM de H 2 O 2 a ampliação de 20X obtidas a partir de da aorta abdominal. (C) Imagem representativa de uma mancha de imuno-histoquímica para a actina alfa em células musculares lisas da aorta isolados primários da aorta abdominal. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52172/52172fig3highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta 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.

Isolamento e excisão de Murino Aorta; A técnica versátil no Estudo de Doenças Cardiovasculares

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

32.3K visualizações.  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. 

Vídeo: 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.

Transplante ortotópico de aorta em camundongos para o Estudo da Doença Vascular

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

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.

Usando a técnica de manga em um modelo do rato da transplantação da aorta - um vídeo instrutivo

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.

Murine Modelo de Transplante de Aorta Cervical usando uma técnica de manguito não 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.

Modificações do Método Langendorff para Isolamento Simultâneo de Miócitos Atrial e Ventricular de Camundongos 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.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

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

Biologia

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.

Isolamento de Murino vasculares coronários células musculares lisas

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.

Lesão por esmagamento aórtico murino: um eficiente<em&gt; In Vivo</em&gt; Modelo de proliferação de células musculares lisas e função 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.

Técnica Modificada para Constrição Aórtica Transversa em Camundongos

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.

Isolamento e análise de lesões do arco aórtico e da raiz em um modelo de camundongo 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.bdigitaluss.remotexs.co/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.

Isolamento e excisão de Murino Aorta; A técnica versátil no Estudo de Doenças Cardiovasculares

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