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 los procedimientos se realizaron en ratones de acuerdo con la Asociación Americana de Medicina Veterinaria (AVMA) directrices y aprobados Institucional Use Comités (IACUC) protocolos y Cuidado de Animales. 1. Diseño del estudio <ol><li> Utilice 8-10 semanas de edad macho C57BL / 6 ratones (BW ~ 25 g) en el estudio. </li><li> Selección aleatoria de los ratones (n = 11) en dos grupos, el grupo de estudio seleccionado por constricción aórtica (n = 8), y el grupo de control (n = 3) a someterse a operación simulada a través de toracotomía. </li><li> Preparar el animal para obtener imágenes por eliminar el vello del pecho usando crema depilatoria que es de grado médico. </li><li> Realizar una primera ultrasonido (sección 2) 24 horas antes de la constricción aórtica para determinar los parámetros de línea de base en el Día -1, entre una gama de 1% y 2,5% de isoflurano (mezclado con 100% de O 2 a través de ojiva) inducida por la anestesia. </li><li> Elegir un agente anestésico aprobado médicamente (es decir, isoflurano) y monitorear el grado de anestesia (2.3% a induce, y 1,0% para mantener). NOTA: la anestesia adecuada es crucial en el mantenimiento de los latidos del corazón a tasas fisiológicas normales (alrededor de 500 latidos / min). </li><li> Confirmar la profundidad de la anestesia por pérdida de movimiento del animal en respuesta a un reflejo pedal-retiro. Use ungüento veterinario paralube en los ojos para evitar la sequedad mientras que bajo anestesia. </li><li> Realizar cirugía en el día 0 20,21. </li><li> Para aórtica, ligar la aorta utilizando una sutura de seda 7-0 alrededor de una cónica 26 G aguja que se coloca en el arco. NOTA: Los detalles relativos al protocolo experimental, incluidos los procedimientos de bandas aórticas quirúrgicos, se han descrito anteriormente 20,21. </li><li> Realizar ecografía posterior a la cirugía (sección 2) en el Día (s) 2, 6 y 13. </li><li> La eutanasia a los ratones en el día 14 y la cosecha de los corazones para la evaluación histológica. La eutanasia a los animales mediante una sobredosis de pentobarbital seguido de la eliminación de un órgano vital como el corazón. Arresto los corazones en diastole y fijar con formalina. Utilice el procedimiento de la recolección de corazón que ha sido descrito previamente 22. </li><li> Fijar todos los tejidos del corazón con buffer solución de formalina al 10%. Para tricrómico tinción, incrustar tejidos en parafina antes de seccionar. Utilice los detalles de tricrómico tinción que han sido bien ilustrado anteriormente 14,23. </li><li> Analizar los datos utilizando software sin conexión (sección 3). </li></ol> 2. Protocolo Imaging <ol><li> Imágenes largas y cortas de los ejes de la arteria coronaria septal (SCA) (Modo B) <ol><li> El uso de la sonda MS550D con frecuencia central de 40 MHz conectados al puerto activo, establecer la aplicación preestablecido a &quot;imagen cardiaca&quot;. </li><li> Con la supina animal sobre la plataforma calentada, y bajo anestesia controlada a través de cono de la nariz, la posición de la sonda usando el sistema ferroviario para obtener el paraesternal eje largo (PSLAX) (Figura 1A). Asegúrese siempre de que el animal se mantiene caliente en la platfor precalentadom y la temperatura corporal se mantiene a niveles fisiológicos. </li><li> Girar la sonda (con la muesca apuntando caudalmente) en sentido horario tal que el ángulo de la sonda es 15 ° a la línea izquierda paraesternal (eje largo vista) (Figura 1B). </li><li> Ajuste el ángulo de la sonda por la inclinación ligeramente a lo largo del eje y de la sonda para obtener una vista longitudinal de longitud completa de la SCA en el centro de la pantalla (Figura 1B). </li><li> Una vez que los puntos de referencia apropiados (válvula aórtica y la arteria pulmonar) son vistos, tienda de cine la imagen utilizando la más alta frecuencia de imagen posible. </li><li> Mediante el uso de la &quot;xy&quot; ejes micromanipuladores (Figura 1D), ajustar la posición de la sonda para obtener la imagen más clara de la SCA. </li><li> Gire la sonda 90 ° (con la muesca apuntando en dirección caudal) en sentido horario tal que el extremo con muescas de la sonda está a la izquierda de la línea media (eje corto) (Figura 1 C). </li></ol></li><li> Imágenes largas y cortas de los ejes de SCA (Color-Doppler Mode) <ol><li> Una vez que una imagen de modo B es capturado o almacena cine-, haga clic en la tecla Doppler color en el teclado para activar el Doppler color de la ventana acústica (Figura 2). NOTA: Este ayuda a aislar la arteria coronaria (flecha blanca indica SCA) o bien en el largo (Figura 2A) o en el eje corto (Figura 2C). Color rojo es como se ve en tiempo real y es indicativo de la dirección del flujo (lejos de la válvula aórtica). </li><li> Asegúrese de que la profundidad de enfoque (indicado por una punta de flecha amarilla en la derecha de la pantalla de la imagen), se encuentra en el centro de la arteria coronaria. </li><li> Asegúrese de que los datos se graban utilizando la tecla cine-tienda, en la más alta velocidad de cuadro posible (&gt; 100 imágenes / seg). </li></ol></li><li> PW Doppler Imaging de SCA (Pulsada-Wave o Modo PW) <ol><li> Mientras que en el modo Doppler color, haga clic en la tecla de PW para que aparezca una línea amarilla-indicador de la arteria coronaria (Figura 2, se muestra en rojo). </li><li> Coloque el amarilloLínea de PW en el medio de la arteria coronaria a la vista, en un ángulo que es paralela a la direccionalidad del flujo. Tenga en cuenta que las mediciones de velocidad son altamente dependientes del ángulo de adquisición de imágenes. </li><li> Ajustar el ángulo de flujo (clave ángulo PW) y volumen de la muestra (clave SV) de tal manera que la tecla PW ángulo es de 60 ° o menos y el volumen de muestra fluya captura justo en el centro de la SCA. </li><li> Usar el almacén de cine para capturar las formas de onda que indican la velocidad del flujo coronario en la sístole (S) de pico y la diástole (D) (Figuras 3A y 3B), usando 1% y el 2,5% de isoflurano. </li></ol></li></ol> 3. Cálculo y Análisis de Datos <ol><li> Seleccione el tiempo de la velocidad herramienta integral (VTI) para obtener el pico sistólica y diastólica velocidades de las imágenes mostradas en las Figuras 3A y 3B. </li><li> Calcular el índice de reserva de flujo coronario (CFR) como la relación de hiperémica (2,5% de isoflurano) pico diastólica fbaja velocidad de la línea de base (1% isoflurano) flujo diastólico pico. </li><li> Calcular la relación S / D como la velocidad de flujo coronario sistólica / velocidad de flujo máximo pico diastólica coronaria. Determine la relación al inicio (1% de isoflurano) y en hiperemia (2,5% de isoflurano). </li><li> Para los parámetros estándar de función cardiaca tales como FS, FAC, LVM, consulte los manuales del fabricante para realizar análisis de datos utilizando el software propietario o se refieren a papel JoVe de Cheng 2. </li></ol>

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The authors report no disclosures.

En este estudio basado en la ecografía, la evaluación no invasiva del flujo coronario se realizó de forma reproducible en tiempo real, durante días, en ratones experimentales en vivo; Además, el protocolo demostró el potencial para detectar la disfunción de la arteria coronaria que estaba presente en una etapa temprana y se asoció con deficiencia en la perfusión miocárdica. Este método, en última instancia podría ser aprovechada como herramienta clínica para la estratificación del riesgo cardiovascular y ...

Estenosis aórtica Clínica (AS) es bien conocido por promover un aumento progresivo en ventricular izquierda (LV) poscarga. Para compensar este aumento de la carga hemodinámica crónica, hipertrofia ventricular izquierda (HVI) se produce como una respuesta adaptativa 1,2. El desarrollo de la HVI se asocia a menudo con alteraciones en la microcirculación coronaria. Se piensa que la disfunción microvascular contribuye a la isquemia crónica en estos pacientes 5. Además de flujo coronario 3,4, la reserva de flujo coronario (CFR) representa el cambio funcional de las arterias coronarias 1,3 y se define como la relación de la velocidad de flujo máxima en la hiperemia de la velocidad de flujo de línea de base o descansando velocidad de flujo 4,6,7. CFR se disminuyó durante LV remodelación 1-3,5-9 y se utiliza como un índice del grado de gravedad de la disfunción funcional coronaria 1,10,17. Se sabe que hay impedimento en muchas formas de miocardiopatía dilatada 10 y también s coronariastenosis 6. CFR es también un marcador pronóstico de pobres resultados clínicos 12. La remodelación del VI en el contexto de la disfunción cardiaca tales como isquemia o HVI también se acompaña de fibrosis extensa, los cambios en la microcirculación coronaria y engrosamiento de las arterias coronarias 1,2. Como resultado de estos cambios en la fisiología coronaria, es probable remodelación de las arterias coronarias. Esto ayuda a mitigar los efectos de la difusión de oxígeno baja y disfunción diastólica del VI que podría resultar en la susceptibilidad a 1,2,13 isquemia miocárdica. Ratones genéticamente modificados son ahora una herramienta de investigación muy extendido para imitar las condiciones de enfermedades humanas como la aterosclerosis coronaria 5,7,10,12,17. En particular, el modelo de sobrecarga de presión en ratones ha sido ampliamente estudiado 14,17. El modelo de constricción trans-aórtica (TAC) se ha demostrado que se asocia con fibrosis extensa, y coronary estenosis resultante, en parte, de engrosamiento de la media de las arterias coronarias y con el acompañamiento de los cambios en los patrones de flujo coronario 1,11,17,19 similares a lo que se ve en el contexto de la HVI en los seres humanos. Aunque se sabe que la sobrecarga de presión prolongada conduce a la insuficiencia cardiaca descompensada en alrededor de 4-8 semanas, los efectos sobre la dinámica de flujo y la reserva de flujo coronario en estos modelos, temprano en el proceso de progresión de la enfermedad, y en diferentes etapas después de bandas, son todavía estar claramente delineado. Numerosas cepas de ratones están disponibles actualmente para su uso en investigación, incluyendo la bien caracterizada LDLR - / - o ApoE - / - ratones 10-12, y estos han impulsado el desarrollo de técnicas sensibles para evaluar la función cardiovascular y la morfología en ratones vivos 11-15. Tales técnicas incluyen MRI, PET, contraste CT, ultrasonidos de alta frecuencia, y tomografía de haz de electrones 2,9,17,19, todos los cuales proporcionan alternativas prometedoras a invasivamétodos tales como cateterismos cardíacos y la angiografía coronaria 12. Sin embargo, en ratones con muy pequeño tamaño de las arterias coronarias y las altas tasas de corazón (HR), imágenes de la circulación coronaria sigue constituyendo un reto técnico para muchas técnicas disponibles en la actualidad 4,12. Curiosamente, se ha producido un aumento exponencial de los avances técnicos en el campo de la ecocardiografía Doppler transtorácica (EDT), incluyendo el desarrollo de alta frecuencia cabezales del escáner matriz con frecuencias centrales de 15 a 50 MHz que permite resoluciones axiales de aproximadamente 30 a 100 micras, a profundidades de 8-40 mm, y velocidades de fotogramas-capturados cuadros / seg mayor que 400. A su vez, las técnicas basadas en TTDE han surgido como una herramienta potencialmente poderosa para obtener imágenes de los vasos más grandes 2 o incluso más pequeños, tales como arterias coronarias 5,12. Otro avance importante que ha permitido a los investigadores a realizar estudios de diagnóstico por imágenes de la vasculatura en un pequeñonimals es el uso cuidadosamente controlada de anestésicos que mantienen el corazón y el ritmo respiratorio de los animales durante la formación de imágenes 11. Mantenimiento anestesia controlada es particularmente importante para los estudios relacionados con la vasodilatación en ratones, y el efecto de la anestesia también tiene que estudiar más a fondo en este contexto 10,11. En los seres humanos, por otro lado, las mediciones CFR derivados de TTDE se han convertido en una herramienta más comúnmente utilizado para la evaluación de las arterias coronarias epicárdicas estenosis y no obstruido, predominantemente en descendente anterior izquierda (LAD) de la arteria coronaria 5,16. Sin embargo, el papel pronóstico de CFR y cambios en el flujo coronario en pacientes asintomáticos o ratones con preservada la función sistólica del VI en reposo ha sido mucho menos explorado 16. Por lo tanto, el objetivo del estudio fue establecer primero un protocolo claro paso a paso, para evaluar los cambios en el flujo coronario mediante TTDE en un modelo de ratón de la sobrecarga de presión; en segundo lugar, este estudio examinó el signo de pronósticoificance de CFR y cambios en el flujo coronario en respuesta a la presión de sobrecarga de estrés en estos ratones. La hipótesis de que la evaluación basada TTDE de CFR y el flujo coronario puede ser útil en la detección precoz de la disfunción coronaria que puede preceder a la disfunción del VI.

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			<td height="42" style="height:42px;width:157px;">Name of the Reagent</td>
			<td style="width:108px;">Company</td>
			<td style="width:96px;">Catalogue Number</td>
			<td style="width:248px;">Comments</td>
		</tr>
		<tr height="50">
			<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>
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			<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>
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			<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>
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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.

De los 11 ratones que fueron estudiados (anillados, n = 8 y la falsa, n = 3), se obtuvieron imágenes adecuadas y reproducibles por un solo observador en varios puntos de tiempo: al inicio del estudio (D-1), D2, D6 y D13 . Además, la velocidad de flujo en el sitio constrictiva se midió como 2,225 ± 110,9 mm / s, en comparación con 277,5 ± 10,51 mm / s en los ratones sham en el día después de la cirugía (p &lt;0,05). El aumento en la velocidad era la verificación de la creación exitosa del modelo de sobrecarga ...

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.

Evaluación Ultrasonido base de la arteria coronaria de flujo y la reserva coronaria Utilizando la sobrecarga Modelo Presión en ratones

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 vistas.  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.

Video: 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.

El uso de un sistema de peso que cuelga de la oclusión coronaria en ratones

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

Investigación

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 de las arterias coronarias y la inyección intramiocárdica en un 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 la ligadura de la arteria coronaria en ratones

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.

Evaluación ecográfica de dependiente del endotelio vasodilatación mediada por flujo de la arteria braquial en Investigación 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.

La ligadura permanente de la arteria coronaria descendente anterior en ratones: un modelo de remodelación post infarto de miocardio e insuficiencia cardiaca

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.

Directa reimplantación de la arteria coronaria izquierda en la aorta en adultos con origen anómalo de la arteria coronaria izquierda desde la arteria 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.

Evaluación de la Reserva de Flujo Coronario Después de la Reperfusión de Isquemia Miocárdica en Ratas

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 de miocardio murino usando ligadura permanente de la arteria coronaria descendente anterior izquierda

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 Angiogénesis Coronaria

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.

Caracterización ecocardiográfica de la estructura, función y flujo coronario del ventrículo izquierdo en ratones neonatos

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