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: Tutte le procedure sono state eseguite in topi in accordo con American Veterinary Medical Association (AVMA) le linee guida e approvati Istituzionale cura degli animali e Usa Comitati (IACUC) protocolli. 1. Disegno dello studio <ol><li> Utilizzare 8-10 settimana vecchio maschio C57BL / 6 topi (BW ~ 25 g) nello studio. </li><li> Randomizzare topi (n = 11) in due gruppi, il gruppo di studio selezionato per bande aortico (n = 8), e il gruppo di controllo (n = 3) sottoporsi operazione sham via toracotomia. </li><li> Preparare l&#39;animale per l&#39;imaging dalla depilazione petto con crema depilatoria che è di grado medico. </li><li> Eseguire una prima ultrasuoni (sezione 2) 24 ore prima bendaggio aortico per determinare i parametri di base al giorno -1, tra una gamma di 1% e 2,5% isoflurano (miscelato con 100% O 2 tramite nosecone) anestesia indotta. </li><li> Scegli un anestetico medicalmente approvato (cioè isoflurano) e monitorare il grado di anestesia (2-3% a induce, e 1,0% per mantenere). NOTA: anestesia corretta è fondamentale per il mantenimento della battito cardiaco a tassi fisiologici normali (circa 500 battiti / min). </li><li> Conferma la profondità di anestesia da perdita di movimento dall&#39;animale in risposta ad un riflesso pedale recesso. Utilizzare paralube pomata veterinario sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Eseguire l&#39;intervento chirurgico al Giorno 0 20,21. </li><li> Per banding aortico, legare l&#39;aorta con una sutura 7-0 seta intorno ad un rastremato 26 G ago inserito sull&#39;arco. NOTA: I dettagli riguardanti il protocollo sperimentale, comprese le procedure chirurgiche bendaggio aortiche, sono stati descritti in precedenza 20,21. </li><li> Eseguire post-chirurgia ad ultrasuoni di imaging (sezione 2) a Day (s) 2, 6 e 13. </li><li> Eutanasia i topi al giorno 14 e raccogliere i cuori per la valutazione istologica. Euthanize gli animali con una overdose di pentobarbital seguita dalla rimozione di un organo vitale come il cuore. Arrestate i cuori in diastole e fissare con formalina. Utilizzare la procedura di raccolta del cuore che è stato descritto in precedenza 22. </li><li> Fissare tutti i tessuti del cuore con tamponata formalina al 10%. Per la colorazione tricromica, incorporare tessuti in paraffina prima sezionamento. Utilizzare i dettagli di colorazione tricromica che sono stati ben illustrato in precedenza 14,23. </li><li> Analizzare i dati utilizzando il software offline (sezione 3). </li></ol> 2. Imaging Protocol <ol><li> Immagini lunghe e corte degli assi del setto coronarica (SCA) (Modalità B-) <ol><li> Utilizzando la sonda MS550D con frequenza centrale di 40 MHz collegati alla porta attiva, impostare il preset applicazione di &quot;imaging cardiaco&quot;. </li><li> Con la supina animale sulla piattaforma riscaldata, sotto anestesia e controllato tramite cono, posizionare la sonda con il sistema ferroviario avere la vista parasternale asse lungo (PSLAX) (Figura 1A). Verificare sempre che l&#39;animale è tenuto caldo sul platfor preriscaldatam e la temperatura corporea è mantenuta a livelli fisiologici. </li><li> Ruotare la sonda (con tacca rivolta caudalmente) in senso orario in modo che l&#39;angolo della sonda è di 15 ° rispetto alla linea di sinistra parasternale (lungo l&#39;asse view) (Figura 1B). </li><li> Regolare l&#39;angolo della sonda inclinando leggermente lungo l&#39;asse y della sonda per ottenere un full-length vista longitudinale della SCA al centro dello schermo (Figura 1B). </li><li> Una volta che i punti di riferimento adeguati (valvola aortica e l&#39;arteria polmonare) sono visti, negozio cine l&#39;immagine utilizzando il più alto frame-rate possibile. </li><li> Usando il &quot;xy&quot; assi micro-manipolatori (Figura 1D), regolare la posizione della sonda per ottenere l&#39;immagine più chiara della SCA. </li><li> Ruotare la sonda di 90 ° (con tacca rivolta caudalmente) in senso orario in modo che estremità del modulo sonda è a sinistra della linea mediana (asse corto) (Figura 1C). </li></ol></li><li> Immagini lunghe e corte assi di SCA (Color-Doppler Mode) <ol><li> Una volta che l&#39;immagine modalità B viene catturata o memorizzato cine-fare clic sul tasto di colore Doppler sulla tastiera per attivare il color Doppler finestra acustica (Figura 2). NOTA: Questo aiuta ad isolare coronarica (freccia bianca indica SCA) sia a lungo (Figura 2A) o in asse corto (Figura 2C). Colore rosso è come visto in tempo reale ed è indicativo della direzione del flusso (lontano dalla valvola aortica). </li><li> Assicurarsi che il centro di profondità (indicato da una freccia gialla sulla destra dello schermo dell&#39;immagine), si trova nel centro di arteria coronaria. </li><li> Assicurarsi che i dati vengono registrati, con il tasto cine-store, al più alto frame rate possibile (&gt; 100 fotogrammi / sec). </li></ol></li><li> PW Doppler Imaging di SCA (Pulsed-Wave o modalità PW) <ol><li> In modalità color-Doppler, clicca sul tasto PW per aprire una linea gialla-indicatore coronaria (Figura 2, mostrato in rosso). </li><li> Posizionare il gialloLinea PW nel mezzo dell&#39;arteria coronaria in vista, ad un angolo che porta alla direzionalità del flusso. Si noti che le misure di velocità sono altamente dipendente dall&#39;angolo di acquisizione dell&#39;immagine. </li><li> Regolare l&#39;angolo di flusso (Rif angolo PW) e il volume del campione (Rif SV) tale che l&#39;angolo di chiave PW è di 60 ° o meno ed il volume del campione cattura fluire nel centro della SCA. </li><li> Utilizzare deposito cine per catturare le forme d&#39;onda che indicano la velocità del flusso coronarico al picco sistole (S) e diastole (D) (Figure 3A e 3B), usando 1% e 2,5% isoflurano. </li></ol></li></ol> 3. Calcolo dei dati e analisi <ol><li> Selezionare il tempo di velocità integrale (VTI) strumento per ottenere la sistolica di picco e velocità diastolica rispetto alle immagini riportate nelle Figure 3A e 3B. </li><li> Calcolare l&#39;indice di riserva di flusso coronarico (CFR) come il rapporto di iperemia (2,5% isoflurano) di picco diastolico fbassa velocità di linea di base (1% isoflurano) velocità di flusso diastolica di picco. </li><li> Calcolare il rapporto S / D, come la velocità di picco di velocità di flusso coronarico sistolica / diastolica picco flusso coronarico. Determinare il rapporto al basale (1% isoflurano) e iperemia (2,5% isoflurano). </li><li> Per i parametri standard cardiaci funzionali quali FS, FAC, LVM, consultare i manuali del costruttore per eseguire l&#39;analisi dei dati utilizzando il software proprietario o fare riferimento a Giove carta di Cheng 2. </li></ol>

<ol>
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The authors report no disclosures.

In questo studio basato ultrasuoni, la valutazione non invasiva del flusso coronarico è riproducibile eseguita in tempo reale, per giorni, nei topi sperimentali vivi; Inoltre, il protocollo ha dimostrato la capacità di rilevare la disfunzione coronarica arteria che era presente in una fase iniziale ed è stata associata a deficit di perfusione miocardica. Questo metodo potrebbe in definitiva essere sfruttato come strumento clinico per la stratificazione del rischio cardiovascolare e / o la risposta a valutare un inter...

Clinical stenosi aortica (AS) è ben noto per promuovere un progressivo aumento del ventricolo sinistro (LV) post-carico. Per compensare questo carico emodinamico cronicamente in aumento, ipertrofia ventricolare sinistra (IVS) ne consegue come 1,2 risposta adattativa. Lo sviluppo di IVS è spesso associata ad anomalie nel microcircolo coronarico. Si pensa che la disfunzione microvascolare contribuisce a ischemia cronica in questi pazienti 5. Oltre a flusso coronarico 3,4, riserva di flusso coronarico (CFR) rappresenta modifica funzionale delle arterie coronarie 1,3 ed è definito come il rapporto tra la velocità di flusso massima in iperemia alla velocità di flusso basale o di riposo flusso velocità 4,6,7. CFR diminuisce durante LV rimodellamento 1-3,5-9 ed è usato come indice del grado di severità funzionale della disfunzione coronarica 1,10,17. E &#39;noto per essere compromessa in molte forme di cardiomiopatia dilatativa 10 e anche s coronarichetenosis 6. CFR è anche un marker prognostico per i poveri risultati clinici 12. Rimodellamento LV nella cornice di una disfunzione cardiaca, come ischemia o LVH è accompagnato anche da una fibrosi, cambiamenti nel microcircolo coronarico e l&#39;ispessimento delle arterie coronarie 1,2. Come risultato di questi cambiamenti nella fisiologia coronarica, è probabile rimodellamento delle arterie coronarie. Questo aiuta a mitigare gli effetti della bassa diffusione di ossigeno e disfunzione diastolica ventricolare sinistra che potrebbe provocare suscettibilità alla miocardica ischemia 1,2,13. I topi geneticamente modificati sono ormai uno strumento di sperimentazione ampiamente prevalente per mimare patologie umane, come l&#39;aterosclerosi coronarica 5,7,10,12,17. Particolarmente il modello sovraccarico pressorio nei topi è stato ampiamente studiato 14,17. Il modello di costrizione trans-aortica (TAC) ha dimostrato di essere associato con estesa fibrosi, e coronary stenosi conseguente, in parte, da ispessimento mediale delle arterie coronarie e con accompagnamento di cambiamenti nei modelli di flusso coronarico 1,11,17,19 simile a ciò che si vede nel setting di IVS in esseri umani. Mentre è noto che il sovraccarico di pressione prolungata porta a insufficienza cardiaca scompensata in circa 4-8 settimane, gli effetti sulla dinamica del flusso coronarico e riserva di flusso in questi modelli, nelle prime fasi del processo di progressione della malattia, e nelle diverse fasi dopo banding, sono ancora essere chiaramente delineata. Numerosi ceppi di topi sono attualmente disponibili per scopi di ricerca, tra cui ben caratterizzato LDLR - / - o ApoE - / - mice 10-12, e questi hanno spinto lo sviluppo di tecniche sensibili per valutare la funzione cardiovascolare e la morfologia nei topi che vivono 11-15. Tali tecniche includono MRI, PET, contrasto CT, ultrasuoni ad alta frequenza e fascio elettronico tomografia 2,9,17,19, tutti che forniscono alternative promettenti invasivametodi come catheterizations cardiaco e angiografia coronarica 12. Tuttavia, in topi con dimensioni molto piccole delle arterie coronarie e frequenza cardiaca elevata (HR), l&#39;imaging di circolazione coronarica costituisce ancora una sfida tecnica per molte tecniche attualmente disponibili 4,12. È interessante notare che c&#39;è stato un aumento esponenziale progressi tecnici nel campo della transtoracica Doppler (TTDE), compreso lo sviluppo di teste di scansione gamma ad alta frequenza con frequenze centrali da 15 a 50 MHz permette risoluzioni assiali di circa 30-100 micron, a profondità di 8-40 mm, e frame rate superiore a 400 fotogrammi catturati / sec. A sua volta, tecniche basate TTDE-sono emersi come uno strumento potenzialmente potente per l&#39;imaging grandi 2 o ancora più piccoli vasi, come le arterie coronarie 5,12. Un altro progresso fondamentale che ha permesso ai ricercatori di condurre studi di diagnostica per immagini del sistema vascolare in piccolo unnimals è l&#39;uso attentamente controllata di anestetici che mantengono il cuore e la frequenza respiratoria degli animali durante l&#39;imaging 11. Manutenzione dell&#39;anestesia controllata è particolarmente importante per gli studi relativi alla vasodilatazione nei topi, e l&#39;effetto dell&#39;anestesia deve inoltre essere ulteriormente esplorato in questo contesto 10,11. Nell&#39;uomo, invece, misurazioni CFR TTDE-derivati ​​sono diventati uno strumento più comunemente usato per la valutazione delle arterie coronarie epicardiche stenotiche e non ostacolato, prevalentemente discendente anteriore sinistra (LAD) arteria coronaria 5,16. Tuttavia, il ruolo prognostico di CFR e cambiamenti flusso coronarico nei pazienti asintomatici o topi con preservata funzione sistolica LV a riposo è stato molto meno esplorato 16. Pertanto, lo scopo dello studio era quello di stabilire prima un protocollo chiaro passo-passo, per valutare le variazioni di flusso coronarico utilizzando TTDE in un modello di topo di pressione di sovraccarico; secondo, questo studio ha esaminato il segno prognosticoificance di CFR e cambiamenti del flusso coronarico in risposta alla pressione di stress da sovraccarico in questi topi. Abbiamo ipotizzato che la valutazione in base TTDE di CFR e del flusso coronarico può essere utile per la diagnosi precoce di disfunzione coronarica che può precedere disfunzione ventricolare sinistra.

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			<td height="42" style="height:42px;width:157px;">Name of the Reagent</td>
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			<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 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>
</table>

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.

Delle 11 topi che sono stati studiati (hanno legato, n = 8 e fittizie, n = 3), sono stati ottenuti immagini adeguate e riproducibili da un singolo osservatore in diversi punti temporali: al basale (D-1), D2, D6 e D13 . Inoltre, la velocità di flusso nel sito costrittiva è stata misurata come 2225 ± 110,9 millimetri / s, rispetto a 277,5 ± 10,51 millimetri / s nei topi sham il giorno dopo l&#39;intervento (p &lt;0.05). L&#39;aumento della velocità è la verifica dello stabilimento successo del modello sovraccarico d...

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.

Valutazione Ecografia base di coronaropatia Flow e flusso coronarico Reserve Utilizzando la pressione Sovraccarico modello nei topi

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 Views.  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.

L&#39;uso di un sistema di peso per appendere l&#39;occlusione dell&#39;arteria coronarica nei topi

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

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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.

Legatura dell&#39;arteria coronarica e di iniezione intramiocardico in un modello murino di 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.

Tecnica modificata di legatura dell&#39;arteria coronarica in Mouse

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.

Ecografia Valutazione della endoteliali-dipendente flusso-mediata vasodilatazione dell&#39;arteria brachiale in Ricerca Clinica

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.

Legatura permanente dell&#39;arteria discendente anteriore sinistra coronarica nei topi: un modello di post-infarto rimodellante miocardico e insufficienza 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.

Diretto Re-impianto di sinistra coronarica nell&#39;aorta in Adulti con Anomala origine della coronaria sinistra dall&#39;arteria polmonare (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.

Valutazione della riserva del flusso coronario dopo la miaocardia Ischemia Reperfusione nei ratti

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.

Modello di infarto miocardico Murine utilizzando la legatura permanente dell'arteria coronaria discendente anteriore sinistra

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.

Modello in vitro dell'angiogenesi coronarica

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.

Caratterizzazione ecocardiografica della struttura, della funzione e del flusso coronarico del ventricolo sinistro in topi neonati

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