We thank Fred Roberts for exemplary technical support and also appreciate the help from the histology core in Beth Israel Hospital. We thank Brigham Women&#8217;s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by a Department of Medicine Sundry Fund.

NOTA: Todos os procedimentos foram realizados em ratos de acordo com a American Veterinary Medical Association (AVMA) orientações e aprovado Institutional Animal Care e usar os comités (IACUC) protocolos. 1. Projeto de Estudo <ol><li> Use 8-10 semanas de idade do sexo masculino camundongos C57BL / 6 (BW ~ 25 g) no estudo. </li><li> Aleatória a ratinhos (n = 11) em dois grupos, o grupo de estudo seleccionados para bandeamento aórtico (n = 8), e o grupo de controlo (n = 3) se submeter a operação simulada através de toracotomia. </li><li> Prepare o animal para geração de imagens através da remoção de pêlos do peito usando creme depilatório que é da classe médica. </li><li> Realizar uma primeira ultra-sonografia (seção 2) 24 horas antes da bandagem aórtica para determinar parâmetros basais no dia -1, entre um intervalo de 1% e 2,5% de isoflurano (misturado com 100% de O2 via nosecone) anestesia induzida. </li><li> Escolha um agente anestésico medicamente aprovado (ou seja, o isoflurano) e monitorar o grau de anestesia (2-3% para induce, e 1,0% para manter). NOTA: a anestesia adequada é essencial para a manutenção dos batimentos cardíacos em taxas fisiológicas normais (cerca de 500 batimentos / min). </li><li> Confirmar a profundidade de anestesia por perda de movimento a partir do animal em resposta a um reflexo de pedal retirada. Use pomada paralube veterinário sobre os olhos para evitar a secura e sob anestesia. </li><li> Realizar a cirurgia no Dia 0 20,21. </li><li> Para bandagem aórtica, ligadura da aorta, com uma sutura de seda 7-0 em torno de um cônico 26 G agulha colocada no arco. Nota: Os detalhes referentes ao protocolo experimental, incluindo os procedimentos de bandagem aórtica cirúrgicos, têm sido descritos anteriormente 20,21. </li><li> Realizar imagem de ultra-som pós-cirurgia (seção 2) pelo Dia (s) 2, 6 e 13. </li><li> Eutanásia os ratos no dia 14 e colher os corações para avaliação histológica. Eutanásia dos animais usando uma overdose de pentobarbital seguido por remoção de um órgão vital tal como o coração. Prender os corações em diastole e corrigir com formalina. Use o procedimento da retirada de coração que tem sido descrito anteriormente 22. </li><li> Corrigir todos os tecidos do coração com solução tamponada de formol a 10%. Para trichrome coloração, incorporar tecidos em parafina antes do corte. Use os detalhes de tricromo que têm sido bem ilustrado anteriormente 14,23. </li><li> Analisar os dados utilizando software off-line (seção 3). </li></ol> 2. Imagem Protocol <ol><li> Imagens eixo longo e curto da artéria coronária septal (SCA) (Modo B-) <ol><li> Usando sonda MS550D com frequência central de 40 MHz conectados à-porta ativa, defina o preset aplicação a &quot;imagem cardíaca&quot;. </li><li> Com o supina animais na plataforma aquecida, sob anestesia e controlado através de cone de nariz, posicionar a sonda utilizando o sistema ferroviário obter o paraesternal eixo longo (PSLAX) (Figura 1A). Assegure-se sempre que o animal é mantido quente no Platfor pré-aquecidom e a temperatura do corpo é mantida a níveis fisiológicos. </li><li> Gire a sonda (com entalhe apontando caudally) no sentido horário de tal forma que o ângulo da sonda é de 15 ° com a linha esquerda parasternal (long-eixo view) (Figura 1B). </li><li> Ajustar o ângulo da sonda pela inclinação ligeiramente ao longo do eixo y da sonda para se obter uma vista longitudinal de comprimento completo do SCA no centro da tela (Figura 1B). </li><li> Uma vez que as referências apropriadas (válvula aórtica e da artéria pulmonar) são vistos, loja de cine a imagem utilizando a mais alta taxa de quadros possível. </li><li> Ao utilizar o &quot;xy&quot; eixos micro-manipuladores (Figura 1D), ajuste a posição da sonda para obter a imagem mais clara da SCA. </li><li> Gire a sonda 90 ° (com entalhe apontando caudally) no sentido horário como o efeito dentada da sonda está à esquerda de linha média (eixo curto) (Figura 1C). </li></ol></li><li> Imagens eixo longo e curto de SCA (Color-Doppler Mode) <ol><li> Uma vez que uma imagem de modo B é capturada ou armazenada cine-, clique na tecla Doppler colorido no teclado para ativar a janela acústica Doppler colorido (Figura 2). NOTA: Este ajuda a isolar artéria coronária (seta branca indica SCA) ou no comprimento (Figura 2A) ou no eixo curto (Figura 2C). A cor vermelha é como pode ser visto em tempo real e é indicativa do sentido do fluxo (para longe da válvula aórtica). </li><li> Certifique-se que a profundidade do foco (indicado por uma seta amarela na direita da tela de imagem), encontra-se no centro da artéria coronária. </li><li> Certifique-se de que os dados são gravados, utilizando a tecla cine-store, na maior taxa de quadros possível (&gt; 100 frames / seg). </li></ol></li><li> PW Doppler Imaging da SCA (Pulsed-Wave ou Modo PW) <ol><li> Enquanto estiver no modo cor-Doppler, clique na tecla PW para abrir uma linha amarela-indicador sobre a artéria coronária (Figura 2, mostrada em vermelho). </li><li> Coloque a amareloPW linha no meio da artéria coronária em vista, a um ângulo que é paralela à direccionalidade do fluxo. Note-se que as medidas de velocidade são altamente dependentes do ângulo de aquisição de imagem. </li><li> Ajuste o ângulo do fluxo (tecla ângulo PW) e do volume da amostra (chave SV) de tal forma que a chave ângulo PW é de 60 ° ou menos, e volume de amostra captura fluir bem no centro da SCA. </li><li> Use loja cine para capturar as formas de onda que indicam a velocidade do fluxo coronário no pico sístole (S) e a diástole (D) (figuras 3A e 3B), utilizando 1% e 2,5% de isoflurano. </li></ol></li></ol> 3. Cálculo e Análise de Dados <ol><li> Selecione o tempo de velocidade integral (VTI) ferramenta para obter o sistólica de pico e velocidade diastólica das imagens mostradas nas Figuras 3A e 3B. </li><li> Calcule o índice de reserva de fluxo coronariano (CFR) como a razão de hiperemia (2,5% de isoflurano) pico diastólica fbaixa velocidade de linha de base (1% de isoflurano) velocidade de fluxo diastólica de pico. </li><li> Calcule a razão S / D como a velocidade do fluxo coronário sistólica velocidade do fluxo de pico / pico diastólica coronária. Determinar a proporção na linha de base (isoflurano a 1%) e a hiperemia (2,5% de isoflurano). </li><li> Para os parâmetros padrão da função cardíaca, como a FS, FAC, LVM, consulte os manuais do fabricante para realizar a análise de dados utilizando software proprietário ou consulte JoVE papel de Cheng 2. </li></ol>

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The authors report no disclosures.

Neste estudo de base de ultra-som, a avaliação não-invasiva da circulação coronária foi reprodutivelmente realizado em tempo real, ao longo de dias, em ratos experimentais vivos; Além disso, o protocolo demonstraram o potencial para detectar a disfunção da artéria coronária que estava presente numa fase precoce e foi associada com deficiência de perfusão miocárdica. Este método poderia finalmente ser aproveitado como uma ferramenta clínica para estratificação de risco cardiovascular e / ou resposta ava...

Estenose aórtica clínica (AS) é bem conhecido para promover um aumento progressivo no ventrículo esquerdo (VE) da pós-carga. Para compensar essa carga hemodinâmica cronicamente crescente, hipertrofia ventricular esquerda (HVE) segue como um 1,2 resposta adaptativa. O desenvolvimento de HVE é frequentemente associada a alterações na microcirculação coronária. Pensa-se que a disfunção microvascular contribui para isquemia crónica nesses pacientes 5. Além de fluxo coronário 3,4, a reserva de fluxo coronário (CFR) representa a mudança funcional das artérias coronárias 1,3 e é definido como a razão da velocidade de fluxo máxima em hiperemia a velocidade de fluxo da linha de base ou de repouso velocidade de fluxo 4,6,7. CFR é diminuída durante a remodelação do VE 1-3,5-9 e é usado como um índice da extensão da gravidade funcional da disfunção coronária 1,10,17. Ele é conhecido por ser prejudicada em muitas formas de cardiomiopatia dilatada 10 e também s coronáriastenosis 6. CFR também é um marcador de prognóstico para desfechos clínicos 12. Remodelamento do VE no cenário de disfunção cardíaca, como isquemia ou LVH também é acompanhado por extensa fibrose, alterações na microcirculação coronariana e espessamento das artérias coronárias 1,2. Como resultado destas alterações na fisiologia coronária, é provável que exista remodelação das artérias coronárias. Isso ajuda a atenuar os efeitos da baixa difusão de oxigênio e disfunção diastólica do VE que poderia resultar em susceptibilidade a 1,2,13 isquemia miocárdica. Camundongos geneticamente modificados são agora uma ferramenta de investigação amplamente predominante para imitar condições de doenças humanas, tais como a aterosclerose coronária 5,7,10,12,17. Particularmente o modelo de sobrecarga de pressão em ratos tem sido amplamente estudada 14,17. O modelo de constrição trans-aórtico (TAC) tem demonstrado estar associada com fibrose extensiva, e coronary estenose resultante, em parte, do espessamento médio das artérias coronárias e com acompanhamento de mudanças nos padrões de fluxo coronariano 1,11,17,19 semelhantes ao que é visto no cenário de HVE em seres humanos. Embora se saiba que a sobrecarga de pressão prolongada leva à insuficiência cardíaca descompensada em cerca de 4-8 semanas, os efeitos sobre a dinâmica de fluxo coronariano e reserva de fluxo nesses modelos, no início do processo de progressão da doença, e em diferentes fases após a bandagem, são ainda ser claramente delineados. Numerosas espécies de ratos estão atualmente disponíveis para fins de pesquisa, incluindo bem caracterizado LDLR - / - ou ApoE - / - ratos 10-12, e estes têm solicitado o desenvolvimento de técnicas sensíveis para avaliar a função cardiovascular e morfologia em ratos vivos 11-15. Essas técnicas incluem ressonância magnética, PET, contraste CT, ultra-som de alta freqüência, e feixe de elétrons tomografia 2,9,17,19, todos os quais oferecem alternativas promissoras para invasivamétodos como cateterismos cardíacos e cineangiocoronariografia 12. No entanto, nos ratos com tamanho muito pequeno das artérias coronárias e os batimentos cardíacos elevados (HR), a imagem latente de circulação coronariana ainda constitui um desafio técnico para muitas técnicas disponíveis atualmente 4,12. Curiosamente, tem havido um aumento exponencial avanços técnicos na área de ecocardiografia transtorácica com Doppler (TTDE), incluindo o desenvolvimento de alta freqüência cabeças de digitalização matriz com frequências centrais de 15 a 50 MHz, permitindo resoluções axiais de aproximadamente 30-100 mm, em profundidades de 8-40 mm, e taxas de quadro superior a 400 quadros-capturado / sec. Por sua vez, as técnicas baseadas em TTDE surgiram como uma ferramenta de enorme potencial para geração de imagens navios maiores 2 ou até menores, como artérias coronárias 5,12. Outro avanço fundamental que tem permitido os investigadores a realizar estudos de diagnóstico por imagem da vasculatura em um pequenonimals é o uso cuidadosamente controlada de anestésicos que mantêm a frequência cardíaca e respiratória dos animais durante o exame 11. Manutenção da anestesia controlada é particularmente importante para os estudos relacionados à vasodilatação em camundongos, e o efeito da anestesia também precisa ser mais explorado neste contexto 10,11. Nos seres humanos, por outro lado, as medições do CFR TTDE derivados tornaram-se uma ferramenta mais comumente utilizado para avaliação das artérias coronárias epicárdicas estenosadas e não obstruídos, predominantemente na descendente anterior esquerda (LAD) artéria coronária 5,16. No entanto, o papel prognóstico do CFR e mudanças na circulação coronariana em pacientes assintomáticos ou camundongos com função sistólica preservada LV em repouso tem sido muito menos explorado 16. Portanto, o objetivo do estudo foi, primeiramente, estabelecer um protocolo passo-a-passo claro, para avaliar mudanças no fluxo coronariano, por meio TTDE em um modelo de sobrecarga de pressão do mouse; segundo, este estudo examinou o sinal prognósticoificance do CFR e alterações de fluxo coronariano em resposta à pressão estresse sobrecarga nesses camundongos. Nossa hipótese é que a avaliação com base TTDE do CFR e do fluxo coronário pode ser útil na detecção precoce de disfunção coronária que pode preceder a disfunção ventricular esquerda.

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			<td height="50" style="height:50px;width:157px;">Depilatory cream</td>
			<td style="width:108px;">Miltex, Inc.</td>
			<td style="width:96px;">Surgi-Prep</td>
			<td style="width:248px;">Apply 24 hours prior to imaging</td>
		</tr>
		<tr height="114">
			<td height="114" style="height:114px;width:157px;">Isoflurane</td>
			<td style="width:108px;">Baxter International Inc.</td>
			<td style="width:96px;">NDC 10019-773-40</td>
			<td style="width:248px;">2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td colspan="2">Table of equipments</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:157px;">Material Name</td>
			<td style="width:108px;">Company</td>
			<td style="width:96px;">Catalogue Number</td>
			<td style="width:248px;">Comments</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:157px;">High Frequency Ultrasound</td>
			<td style="width:108px;">FUJIFILM VisualSonics, Inc.</td>
			<td style="width:96px;">Vevo 2100</td>
			<td style="width:248px;"></td>
		</tr>
		<tr height="37">
			<td height="111" rowspan="3" style="height:111px;width:157px;">High-frequency Mechanical Transducer</td>
			<td rowspan="3" style="width:108px;">FUJIFILM VisualSonics, Inc.</td>
			<td rowspan="3" style="width:96px;">MS250, MS550D, MS400</td>
			<td rowspan="3" style="width:248px;"></td>
		</tr>
		<tr height="37">
		</tr>
		<tr height="37">
		</tr>
	</tbody>
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ultrasound based assessment of coronary artery flow and coronary flow reserve using the pressure overload model in mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Dos 11 ratos que foram estudados (faixas, n = 8 e sham, n = 3), imagens adequadas e reprodutíveis foram obtidos por um único observador em vários pontos de tempo: na linha de base (D-1), D2, D6 e D13 . Além disso, a velocidade de fluxo no local constritiva foi medida como 2225 ± 110,9 milímetros / s, em comparação com 277,5 ± 10,51 milímetros / s nos ratos sham no dia após a cirurgia (p &lt;0,05). O aumento da velocidade era a verificação do estabelecimento bem sucedido do modelo de sobrecarga de pressão. ...

Watch this Scientific Journal Video about Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice at JoVE.com

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Avaliação ultra-som base de Artéria Coronária Flow e Fluxo Coronariano Reserve Usando o modelo de sobrecarga de pressão no Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

ultrasound-based-assessment-coronary-artery-flow-coronary-flow

Research

JoVE Journal

Medicine

14.8K visualizações.  Brigham and Women's Hospital, Harvard Medical School. Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Vídeo: Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Uso de um Sistema de Peso de suspensão para oclusão da artéria coronária em ratos

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Medicina

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Ligadura da artéria coronária e injeção intramiocárdica em um modelo murino de infarto

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Técnica modificada para ligação da artéria coronária em ratos

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

Avaliação ultra-som de endotelial-dependente do fluxo vasodilatação mediada da artéria braquial em Pesquisa Clínica

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

A ligação permanente da artéria coronária descendente anterior esquerda em Ratos: Um Modelo de Remodelação pós-infarto do miocárdio e insuficiência cardíaca

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Re-implantação direta de Esquerda da Artéria Coronária para a aorta em adultos com Origem Anômala da Coronária Esquerda da Artéria da artéria pulmonar (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Avaliação da reserva de fluxo coronariano após reperfusão de isquemia miocárdica em ratos

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modelo de infarto do miocárdio murino usando ligadura permanente da artéria coronária descendente anterior esquerda

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Modelo In Vitro de Angiogênese Coronariana

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Caracterização ecocardiográfica da estrutura, função e fluxo coronariano do ventrículo esquerdo em camundongos recém-nascidos

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...