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.

Hinweis: Alle Verfahren wurden bei Mäusen nach American Veterinary Medical Association (AVMA) Richtlinien durchgeführt und genehmigt Institutional Animal Care und Verwenden Komitees (IACUC) Protokolle. 1. Studiendesign <ol><li> Verwenden Sie 8-10 Wochen alten männlichen C57BL / 6 Mäuse (BW ~ 25 g) in der Studie. </li><li> Zufälliger Mäuse (n = 11) in zwei Gruppen, die Studiengruppe für Aortenbandierung ausgewählte (n = 8) und der Kontrollgruppe (n = 3), um Scheinoperation via Thorakotomie unterzogen werden. </li><li> Bereiten Sie das Tier für die Bildgebung durch Entfernen von Haaren aus der Brust mit Enthaarungscreme, die für medizinische Zwecke ist. </li><li> Führen Sie eine erste Ultraschall (Abschnitt 2) 24 Stunden vor Aortenbandierung zum Ausgangsparameter am Tag -1 zu bestimmen, zwischen einem Bereich von 1% und 2,5% Isofluran (mit 100% O 2 über nosecone gemischt) induzierte Anästhesie. </li><li> Wählen Sie einen medizinisch zugelassenen Anästhetikum (dh Isofluran) und Überwachung der Grad der Anästhesie (2-3% auf Indu-ce und 1,0% zu halten). HINWEIS: Das richtige Anästhesie ist entscheidend bei der Aufrechterhaltung der Herzschlag bei normaler physiologischer Frequenzen (etwa 500 Schläge / min). </li><li> Bestätigen die Tiefe der Anästhesie durch Verlust der Bewegung von dem Tier als Antwort auf ein Pedal-Rückziehreflex. Verwenden paralube Tierarzt Salbe auf die Augen bis zur Trockenheit während der Narkose zu verhindern. </li><li> Führen Chirurgie am Tag 0 20,21. </li><li> Für Aortenbandierung, Ligation der Aorta mit einer 7-0 Seidenfaden um einen konischen 26 G-Nadel auf dem Bogen platziert. HINWEIS: Details zur Versuchsprotokoll, einschließlich der chirurgischen Aortenbandierung Verfahren sind bisher 20,21 beschrieben. </li><li> Führen Sie nach der Operation der Ultraschallbildgebung (Abschnitt 2) am Tag (e) 2, 6 und 13. </li><li> Euthanize die Mäuse am Tag 14 und Ernte der Herzen für die histologische Bewertung. Euthanize die Tiere mit einer Überdosis Pentobarbital, gefolgt von Entfernung ein lebenswichtiges Organ wie das Herz. Verhaften Sie die Herzen in diastole und fixieren mit Formalin. Verwenden Sie das Verfahren des Herzens der Ernte, die zuvor 22 beschrieben wurde. </li><li> Fix alle Herzgewebe mit gepufferte 10% Formalinlösung. Für Trichromfärbungen einbetten Gewebe in Paraffin vor dem Schneiden. Verwenden Sie die Details Trichromfärbungen, die gut vorher 14,23 veranschaulicht wurden. </li><li> Analysieren Sie die Daten mit Offline-Software (Abschnitt 3). </li></ol> 2. Imaging Protocol <ol><li> Lange und kurze Achse Bilder septal Koronararterie (SCA) (B-Mode) <ol><li> Mit MS550D Sonde mit Mittenfrequenz von 40 auf die aktive-Port angeschlossen MHz, stellen Sie die Anwendung voreingestellt auf &quot;Herzbildgebung&quot;. </li><li> Mit dem Tier in Rückenlage auf dem beheizten Plattform und unter Narkose über Bugnase gesteuert, positionieren Sie die Sonde mit dem Schienensystem, um die parasternalen langen Achse (PSLAX) (1A) zu erhalten. Achten Sie immer darauf, dass das Tier warm vorgewärmten platfor gehaltenm und die Körpertemperatur wird bei physiologischen Werten gehalten. </li><li> Drehen Sie die Sonde (mit Kerbe nach kaudal) im Uhrzeigersinn, so dass die Sonde Winkel 15 ° nach links Parasternallinie (Langachsenansicht) (Abbildung 1B). </li><li> Stellen Sie die Sonde Winkel durch Kippen leicht entlang der y-Achse der Sonde, um ein Volllängenlängsansicht der SCA in der Mitte des Bildschirms (1B) zu erhalten. </li><li> Wenn Sie die korrekten Sehenswürdigkeiten (Aortenklappe und Lungenarterie) betrachtet werden, cine Speicher das Bild mit der höchsten Bildrate möglich. </li><li> Bei Nutzung der &quot;xy&quot; Achsen Mikromanipulatoren (1D), passen Sie die Sondenposition, um das klarste Bild des SCA erhalten. </li><li> Drehen der Sonde 90 ° (mit Kerbe nach kaudal) im Uhrzeigersinn, so daß gekerbten Ende der Sonde ist an der linken Seite der Mittellinie (kurze Achse) (Abbildung 1C). </li></ol></li><li> Lange und kurze Achse Bildern von SCA (Farbdoppler Mode) <ol><li> Sobald ein B-Modus Bild aufgenommen oder Kino gespeichert, klicken Sie auf den Farb-Doppler-Taste auf der Tastatur, um Farb-Doppler-Schallfenster (Abbildung 2) zu drehen. ANMERKUNG: Dies hilft Koronararterie isolieren (weißer Pfeil gibt SCA) entweder in der Länge (Figur 2A) bzw. in der kurzen Achse (2C). Rote Farbe wird als in Echtzeit zu sehen und zeigt die Strömungsrichtung (weg von der Aortenklappe). </li><li> Stellen Sie sicher, dass die Tiefenschärfe (mit einem gelben Pfeilspitze auf der rechten Seite des Bildschirms angezeigt wird), liegt im Zentrum der Koronararterie. </li><li> Stellen Sie sicher, dass die Daten aufgezeichnet werden, mit dem Cine-Speichertaste, mit der höchstmöglichen Bildfrequenz (&gt; 100 Bilder / s). </li></ol></li><li> PW-Doppler-Imaging von SCA (Pulsed-Wave oder PW-Modus) <ol><li> Während in der Farbe-Doppler-Modus, klicken Sie auf den PW-Taste auf der Herzkranzgefäße (Abbildung 2, in rot dargestellt) zu bringen, ein gelb-Anzeigelinie. </li><li> Setzen Sie den gelbenPW Linie in der Mitte der Koronararterie in Ansicht, in einem Winkel, der die Richtwirkung der Strömung parallel. Beachten Sie, dass Geschwindigkeitsmessungen sind stark abhängig von dem Winkel der Bildaufnahme. </li><li> Stellen Sie den Winkel des Flusses (PW Winkel-Taste) und Probenvolumen (SV-Taste), so dass der PW Winkelschlüssel ist 60 ° oder weniger und Probenvolumen erfasst fließen direkt im Zentrum des SCA vor. </li><li> Verwenden cine Speicher, um die Wellenformen, die die Geschwindigkeit des koronaren Flusses an Spitzensystole (S) und Diastole (D) (3A und 3B) zeigen, zu erfassen, unter Verwendung von 1% und 2,5% Isofluran. </li></ol></li></ol> 3. Daten Berechnung und Analyse <ol><li> Wählen Sie die Geschwindigkeit zeitliche Integral (VTI) Werkzeug, um den Höhepunkt des systolischen und diastolischen Geschwindigkeiten von den in den 3A und 3B gezeigten Bilder zu erhalten. </li><li> Berechnen Sie die koronare Flussreserve Index (CFR) als das Verhältnis der hyperämisch (2,5% Isofluran) Spitzen diastolischen fniedriger Geschwindigkeit zum Ausgangswert (1% Isofluran) Spitzen diastolischen Strömungsgeschwindigkeit. </li><li> Berechnen Sie die S / D-Verhältnis als die maximale systolische Herzkranzströmungsgeschwindigkeit / Peak diastolischen Herzkranzströmungsgeschwindigkeit. Bestimmen Sie das Verhältnis zu Beginn der Studie (1% Isofluran) und Hyperämie (2,5% Isofluran). </li><li> Für Standard-Herzfunktionsparameter wie FS, FAC, LVM finden Sie in den Handbüchern des Herstellers, um die Datenanalyse durchführen proprietäre Software oder beziehen sich auf Chengs JoVE Papier 2. </li></ol>

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The authors report no disclosures.

In diesem Ultraschall basierte Studie wurde nicht invasive Beurteilung der koronaren Flusses reproduzierbar in Echtzeit durchgeführt, über Tage, in Live-Versuchsmäusen; Darüber hinaus zeigte das Protokoll die Möglichkeit, koronare Herzfunktionsstörungen, die zu einem frühen Zeitpunkt anwesend war und mit Mangel an myokardialen Perfusion assoziiert zu erkennen. Diese Methode könnte letztlich als klinischer Parameter bei kardiovaskulären Risikostratifizierung und / oder Beurteilung Reaktion auf therapeutische Int...

Clinical Aortenstenose (AS) ist gut bekannt, eine fortschreitende Zunahme der linksventrikulären (LV) Nachlast fördern. Um sich für diese chronisch steigenden hämodynamische Belastung zu kompensieren, erfolgt LV Hypertrophie (LVH) als adaptive Reaktion 1,2. Die Entwicklung der LVH ist oft mit Anomalien in koronaren Mikrozirkulation verbunden. Es wird vermutet, dass die mikrovaskuläre Dysfunktion trägt zu chronischer Ischämie in Patienten 5. Neben Koronarfluss 3,4, koronare Flussreserve (CFR) stellt Funktionsänderung der Koronararterien 1,3 und wird als das Verhältnis der maximalen Strömungsgeschwindigkeit in Hyperämie in den Ausgangsströmungsgeschwindigkeit oder ruhenden Strömungsgeschwindigkeit 4,6,7 definiert. CFR während LV Remodeling 1-3,5-9 verringert, und wird als Index für das Ausmaß der funktionalen Schwere koronare Dysfunktion 1,10,17 verwendet. Es ist bekannt, in vielen Formen der dilatierten Kardiomyopathie 10 sowie koronarer s beeinträchtigttenosis 6. CFR ist auch ein prognostischer Marker für schlechte klinische Ergebnisse 12. LV Remodeling in der Einstellung der Herzdysfunktion, wie Ischämie oder LVH wird auch durch umfangreiche Fibrose, Veränderungen bei der koronaren Mikrozirkulation und Verdickung der Koronararterien 1,2 begleitet. Als Ergebnis dieser Veränderungen in koronaren Physiologie, dürfte Umbau der Koronararterien. Dies trägt zur Milderung von niedrigen Sauerstoffdiffusion und LV diastolische Dysfunktion, die in der Anfälligkeit für myokardiale Ischämie 1,2,13 führen kann. Genetisch veränderte Mäuse, sind jetzt eine weit verbreitete Untersuchungstool für die Nachahmung menschlicher Erkrankungen Erkrankungen wie koronare Atherosklerose 5,7,10,12,17. Vor allem die Drucküberlastung Modell bei Mäusen wurde weitgehend untersucht 14,17. Das trans Aortakonstriktion Modell (TAC) wurde gezeigt, dass mit umfangreichen Fibrose und coronar zugeordnet werdeny Stenose resultierende, teilweise aus den medialen Verdickung der Koronararterien und mit begleitenden Änderungen im Koronarfluss Muster 1,11,17,19 ähnlich dem, was bei der Einstellung der LVH beim Menschen erwartet. Obwohl bekannt ist, dass eine längere Drucküberlastung führt zu dekompensierter Herzinsuffizienz in etwa 4-8 Wochen, die Auswirkungen auf die koronare Flussdynamik und Flussreserve in diesen Modellen, frühzeitig in den Prozess der Krankheitsprogression und in verschiedenen Stadien nach Streifenbildung, noch bisher klar. Zahlreiche Stämme von Mäusen sind derzeit für Forschungszwecke, auch gut charakterisierte LDLR verfügbar - / - oder ApoE - / - Mäusen 10-12, und diese haben die Entwicklung von empfindlichen Techniken zur Beurteilung der Herz-Kreislauf-Funktion und Morphologie in lebenden Mäusen 11-15 aufgefordert werden. Solche Techniken umfassen MRI, PET, Kontrast CT, hochfrequenten Ultraschall, und Elektronenstrahltomographie 2,9,17,19, die alle für versprechende Alternativen zu invasivenMethoden wie die Herzkatheter und Koronarangiographie 12. In Mäusen, die mit sehr kleinen Größe der Herzkranzgefäße und hoher Herzfrequenz (HR), Bildgebung von Herzkreislauf noch eine technische Herausforderung für viele derzeit verfügbaren Techniken 4,12. Interessanterweise gab es einen exponentiellen Anstieg in dem technischen Fortschritt auf dem Gebiet der transthorakalen Dopplerechokardiographie (TTDE), einschließlich der Entwicklung von Hochfrequenz-Array Scanköpfe mit Mittenfrequenzen von 15 bis 50 MHz eine axiale Auflösung von etwa 30 bis 100 &amp; mgr; m in einer Tiefe von 8 bis 40 mm, und Bildraten von mehr als 400 Bildern eingefangen / sec. Im Gegenzug haben TTDE-basierte Techniken als potenziell leistungsfähiges Werkzeug zur Abbildung größer 2 oder sogar kleinere Schiffe entstanden, wie Koronararterien 5,12. Ein weiterer wichtiger Fortschritt, der es ermöglicht hat Ermittler zu diagnostischen bildgebenden Untersuchungen des Gefäßsystems in kleinen a führennimals der sorgfältig kontrollierten Einsatz von Anästhetika, die das Herz und die Atmungsrate der Tiere während der Bild 11 aufrechtzuerhalten. Kontrollierter Anästhesie Wartung ist besonders wichtig für Studien zur Gefäßerweiterung bei Mäusen verwandt, und die Wirkung der Narkose muss auch in diesem Zusammenhang 10,11 weiter untersucht werden. Bei Menschen, andererseits TTDE abgeleiteten CFR Messungen zu einem häufig verwendeten Werkzeug zur Auswertung der stenosierten und nichtblockierten epikardialen Koronararterien, überwiegend in der linken vorderen absteigenden (LAD) Koronararterie 5,16. Allerdings ist die prognostische Bedeutung der CFR und koronare Flussänderungen bei asymptomatischen Patienten oder Mäuse mit gut erhaltener systolischer Funktion in Ruhe viel weniger erforscht 16 gewesen. Daher das Ziel der Studie war es, zunächst eine klare Schritt-für-Schritt-Protokoll, auf Veränderungen in Koronarfluss mit TTDE in einer Drucküberlastung Mausmodell zu bewerten; zweitens, diese Studie untersuchte die prognostisches Zeichenificance der CFR und Koronarfluss sich infolge einer Überlast Stress in diesen Mäusen unter Druck. Wir vermuten, dass TTDE fundierte Beurteilung der CFR und Koronarfluss kann bei der Früherkennung der koronaren Dysfunktion LV Dysfunktion vorausgehen kann.

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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.

Von den 11 Mäusen, die von einem einzigen Beobachter zu verschiedenen Zeitpunkten untersucht wurden (gebändert, n = 8 und Schein, n = 3), angemessene und reproduzierbare Bilder wurden: an der Grundlinie (D-1), D2, D6 und D13 . Außerdem wurde die Strömungsgeschwindigkeit an der Verengungsstelle als 2225 ± 110,9 mm / s gemessen wird, verglichen mit 277,5 ± 10,51 mm / s in den schein-Mäusen am Tag nach der Operation (p &lt;0,05). Die Erhöhung der Geschwindigkeit war die Überprüfung der erfolgreichen Etablierung 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.

Ultraschall Basierend Beurteilung der koronaren Herzfluss und koronare Flussreserve Mit der Drucküberlastung Modell an der Maus

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 Aufrufe.  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.

Serie: 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.

Die Verwendung eines Hanging Weight System für Okklusion in Mäuse

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

Forschung

Medizin

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.

Koronare Ligation und intramyokardialen Injection in einem Mausmodell der Infarkt

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.

Geändert Technik für Coronary Artery Ligation in Mäuse

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.

Ultraschall Beurteilung der Endothelial Abhängige Fluss Vasodilatation der Arteria brachialis in der klinischen Forschung

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.

Permanent Ligation des linken vorderen absteigenden Koronararterie bei Mäusen: Ein Modell der Post-Myokardinfarkt Umbau und Herzinsuffizienz

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.

Direkter Wieder Implantation von linken Koronararterie in die Aorta bei Erwachsenen mit Anomale Ursprung der linken Koronararterie aus der Lungenarterie (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.

Bewertung des koronaren Flussreservats nach Myokard-Ischämie-Reperfusion bei Ratten

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.

Murine Myokardinfarkt Modell mit permanenten Ligation der linken Anterior absteigende Koronararterie

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.

In Vitro Modell der koronaren Angiogenese

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.

Echokardiographische Charakterisierung der linksventrikulären Struktur, Funktion und Koronarfluss bei neugeborenen Mäusen

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