This work was supported by Onderzoekstoelagen grant OT/13/090 of the KU Leuven and by grant G0A3114N of the FWO-Vlaanderen.

NOTE: All experimental procedures described in this section were approved by the Institutional Animal Care and Research Advisory Committee of the Katholieke Universiteit Leuven (Project: 154/2013-B De Geest).
1. Permanent Ligation of the Left Anterior Descending Coronary Artery
<ol>
	<li>Anesthetize the mouse by intraperitoneal administration of 40&#160;mg/kg to 70 mg/kg of sodium pentobarbital. Ensure the mouse reaches its proper plane of anesthesia when it no longer reacts a firm toe pinch. Always confirm proper anesthetization this way prior to any surgical procedure or intervention. Use lubricating ophthalmic ointment to prevent dryness of the cornea while under anesthesia.&#160;Provide preoperative analgesia 2-4 hr before the start of the procedure (buprenorphine 0.05 mg/kg SQ).
	<ol>
		<li>Apply consistent aseptic techniques during survival surgery. Implement procedures that inhibit to a maximally possible extent microbial contamination so that significant infection or suppuration does not occur. These procedures include use of sterile instruments and sterile materials, disinfection of the surgical area, and removal of fur/hair over the surgical site and disinfection of this site.</li>
	</ol>
	</li>
	<li>Intubate the mouse with a self-prepared blunted 20-gauge needle.
	<ol>
		<li>Put the mouse in supine position with hyperextension of the head.
		<ol>
			<li>Focus the light on the neck region. Lift the tongue with a blunted pincet. The entrance of the larynx can be clearly seen.</li>
			<li>Pass the blunted needle through the larynx into the trachea under direct vision. Assess correct intubation by connecting the mouse to the ventilator (stroke volume in &#181;l: 3 x body weight (g) + 155; frequency: 120 strokes per min).</li>
		</ol>
		</li>
		<li>Alternatively, enhance visualization of endotracheal intubation by first carefully exposing the trachea.
		<ol>
			<li>Make a 5 mm mid-neck incision and retract muscle tissue just above the trachea.</li>
			<li>Perform the intubation using a surgical stereomicroscope for direct visualization of the trachea. Lift the tongue and enter the self-prepared blunted 20 gauge needle into the trachea. Confirm correct intubation by connecting the mouse to the ventilator (stroke volume in &#181;l: 3 x body weight (g) + 155; frequency: 120 strokes per min).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Keep the mouse in supine position and fix the mouse with tape. Perform surgery on a heating pad to prevent hypothermia.
	<ol>
		<li>Shave and disinfect the skin with Betadine. Take care that the left hind limb crosses the right hind limb in order to obtain a better view on the left ventricle during surgery.</li>
	</ol>
	</li>
	<li>Make a small transverse cutaneous incision up to the sternum and separate the underlying skin and muscles.</li>
	<li>Pull aside the m. pectoralis minor and m. pectoralis major with a 5-0 silk suture.</li>
	<li>Make an incision in the third intercostal space by inserting a blunted pincet.</li>
	<li>Move the pincet under the intercostal muscles from lateral to medial till the sternum is reached. Puncture the thoracic wall by pushing the pincet from inside to the skin. Complete the thoracotomy by carefully cutting the intercostal muscle just above the pincet with a small scissor. Use this technique to prevent puncturing of the lungs.</li>
	<li>Put a sponge immersed in 0.9% NaCl into the cavity to protect the lungs. Introduce a wound spreader (chest retractor) into the intercostal space to obtain exposure of the left side of the heart. As of now, the left atrium, the left ventricle, and the left anterior descending coronary artery are visible under the stereomicroscope.</li>
	<li>Perform a ligation of the left anterior descending coronary artery with a single 6-0 prolene ligature about 1 mm under the tip of the left atrium. This is distal from the first diagonal branch. 
	NOTE: Alternatively, 7-0 (0.05 mm diameter) or 8-0 threads (0.04 mm diameter) may be used. The needle is a C-1 13 mm 3/8 circle Taper Point needle. Successful ligation of the left anterior descending coronary artery induces immediate discoloration, resulting in a pale appearing myocardium in the affected territory.</li>
	<li>Remove the wound spreader (chest retractor).
	<ol>
		<li>Place three 6-0 Ti-Cron sutures around the intercostal space. Before tightening the sutures, remove the sponge from the chest cavity and re-expand the lungs by blocking the outflow of the ventilator. By doing so, the lungs reconnect with the parietal pleura.</li>
		<li>Subsequently, pull the sutures tight and repeat re-expansion by pressing down on the chest. Confirm the successful closure of the thorax using a small amount of saline (no air bubbles must be seen when applying pressure on the chest).</li>
		<li>Look through the intercostal muscle to confirm normal expansion of the lungs. Reposition both pectoralis muscles, serving as an extra barrier for prevention of a pneumothorax.</li>
	</ol>
	</li>
	<li>Close the skin with 5-0 silk-sutures.</li>
	<li>Disconnect the mouse from the ventilator and allow recovery on the heating pad. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Consistently provide postoperative analgesia (buprenorphine 0.05 mg/kg SQ BID for at least 48 hr after surgery).&#160;</li>
</ol>
2. In vivo Invasive Hemodynamic Measurements in Mice
<ol>
	<li>Before the procedure, submerge the 1.0 French Millar pressure catheter in sterile water at 37 &#176;C for at least 30 min to minimize signal drift. Electronically calibrate the pressure sensor at 0 mm Hg and 100 mm Hg and record data at 2,000 Hz.</li>
	<li>Perform anesthesia by intraperitoneal administration of 1.4 g/kg urethane. Check that the mouse reaches its proper plane of anesthesia when it no longer reacts a firm toe pinch.</li>
	<li>Place the anesthetized mouse in supine position. Secure its limbs with tape. Maintain body temperature with a heating pad and monitor with a rectal probe. Shave the neck region and make a midline incision in the neck region to expose the thyroid gland.</li>
	<li>Fix the neck with bent needles.</li>
	<li>Pull aside the salivary gland and expose the right common carotid artery. The vagus nerve, which resembles a white thread, lies along the artery. Carefully separate the carotid artery from the vagus nerve using a curved forceps.</li>
	<li>Pass a curved forceps under the right common carotid artery to separate it from other tissues. Remove connective tissue around the artery.</li>
	<li>Pass two 6-0 silk wires under the right common carotid artery. Make a tight knot on the top wire, which is placed towards the head, close, and fix with a kocher (distal occlusive ligation). Pass the proximal wire twice from left to right and fix with 2 kochers (proximal non-occlusive wire).</li>
	<li>Keep the carotid artery moist by dropping sterile 0.9% NaCl. Dry off excess liquid with cotton buds.</li>
	<li>Make an incision in the right common carotid artery with a 26-gauge needle between the distal ligation and the proximal non-occlusive wire.</li>
	<li>Introduce the pressure sensor into the artery. Verify that there is no blood loss. Gently push the 1.0 French Millar pressure catheter forward and adjust the proximal non-occlusive wire in such a way that the catheter can pass carefully through the wire under the clavicle.
	<ol>
		<li>Minimize blood loss during adjustment of the proximal non-occlusive wire. Do not compress the pressure sensor too much while advancing since it is very fragile. Since the proximal wire must not occlude the artery, the vessel should remain filled with blood.</li>
	</ol>
	</li>
	<li>Start recording the pressure signal. An arterial pressure signal fluctuates in a healthy mouse between a diastolic pressure of 60 - 70 mm Hg and a systolic pressure of 100 - 120 mm Hg.</li>
	<li>Direct the catheter via the innominate artery and via the aorta into the left ventricle. The ventricular pressure fluctuates between 0 mm Hg and 100 - 120 mm Hg. Allow the catheter to stabilise inside the left ventricle. Record the signal for 30 min to 60 min depending on the experimental requirements.</li>
	<li>After completion of the experiment, soak the catheter in Alconox 1% for 30 min. Wash the catheter with Milli-Q water. Store the catheter in a foam block.</li>
	<li>Retrieve data from the recording software for further analysis.
	<ol>
		<li>For data analysis, consider a time interval where the pressure signal is stable. Choose at least 10 consecutive cardiac cycles of the recorded data of interest.</li>
		<li>Use LabChart software version 8.0 or similar to analyse the heart rate, the maximal systolic left ventricular pressure, the minimal diastolic left ventricular pressure, the peak rate of isovolumetric left ventricular contraction (dP/dtmax), the peak rate of isovolumetric left ventricular relaxation (dP/dtmin), the end-diastolic left ventricular pressure, and the time constant of the isovolumetric left ventricular pressure fall (tau)7. 
		NOTE: The end-diastolic pressure corresponds to the pressure at the time-point immediately before the pressure surge induced by isovolumetric contraction. The calculation of tau is based on fitting the left ventricular pressure into a monoexponential decay curve, expressed as P(t)=P0e-t/tau + b, In this formula, P(t) is the left ventricular pressure at time t after the maximal negative value of dP/dt has been reached. The parameter b corresponds to the theoretical asymptote, which in a simplified approach can be assumed to be zero. Enhanced isovolumetric relaxation results in a smaller value of tau.</li>
	</ol>
	</li>
</ol>

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None of the authors reports competing financial interests.

Chronic changes in myocardial structure and function, the development of left ventricular dysfunction, and progression to heart failure can be investigated in several murine models12. Cardiac remodelling and dysfunction can be induced by myocardial injury or by pressure overload secondary to transverse aortic constriction, or may be investigated in genetic models of dilated cardiomyopathy12. Obviously, the most pronounced benefit of murine models is the availability of a great number of transgenic and knock-out strains including cell-type specific and inducible transgenic models. Evaluation of cardiac remodelling in these models has been greatly facilitated by the development of technologies like ultrahigh resolution 2D- and 3D-echocardiography, micro-magnetic resonance imaging, hemodynamic evaluation using micromanometry, and micromanometer conductance technology for pressure volume loop analyses. In this report, we have focused on the model of myocardial injury induced by permanent ligation of the left anterior descending coronary artery and evaluation of left ventricular function by left ventricular micromanometry.
The first murine model of myocardial infarction was described in 1978 by Zolotareva et al13. This model of permanent occlusion should be clearly discerned from the murine model of transient occlusion of the left anterior descending coronary artery that was first described by Michael et al14. This latter model is used to investigate ischemia-reperfusion injury and myocardial salvage whereas permanent ligation is applied to study the pathophysiology of post-myocardial infarction cardiac remodelling.
The coronary artery tree of mice is different compared to humans15-17. The septal coronary artery in the mouse is variable in origin15-17. It arises classically as a branch of the right coronary artery18,19, but may also arise from a separate ostium in the right sinus of Valsalva17, or exceptionally from the left coronary artery16. The anatomy of the coronary artery tree may slightly differ between different strains20. However, ligation of the left anterior descending coronary artery as it emerges immediately under the left auricle results in reproducible large infarctions involving the anterolateral, posterior, and apical regions of the heart as demonstrated by histology and echocardiography15,17. Importantly, the septum is spared from infarction, which reflects the presence of the distinct septal coronary artery. In contrast to mice, blood supply of the septum in humans is mainly provided by branches of the left anterior descending coronary artery21. This difference in anatomy and the resulting sparing of the septum in mice will affect remodelling of the heart.
As stated in the results section, ligation of the left anterior descending coronary artery in mice induces a large myocardial infarction that comprises 50% to 60% of the left ventricle9,10. The occurrence of ventricular rupture in the first two weeks after myocardial infarction in mice is frequent22,23. This complication of myocardial infarction is strain-dependent and occurs significantly more often in male mice than in female mice22. Furthermore, ventricular rupture happens more frequently in hypercholesterolemic mice than under conditions of normocholesterolemia9. The occurrence of ventricular rupture in mice should be put in the right perspective. First of all, this complication is a manifestation of infarct expansion. This disproportionate thinning and dilatation of the infarct segment not only induces myocardial rupture but is also a trigger for cardiac remodelling and for congestive heart failure24-27. Thus, the frequency of myocardial rupture is an indicator for the degree of infarct expansion and thus for the extent of cardiac remodelling and development of cardiac dysfunction in surviving mice.
The second important consideration with regard to ventricular rupture is the necessity to take survival bias into account when functional or structural data are compared between two groups in an intervention study10. It is reasonable to assume that mice with the most pronounced infarct expansion will succumb to ventricular rupture. In case of a significant difference of survival rate between two groups, one should be aware that died mice are excluded from the functional or structural analysis. Consequently, bias is introduced in any comparison of mice surviving at a specific time-point.
As discussed above, many different technologies are available to evaluate cardiac remodelling after myocardial infarction. However, the availability of advanced and complex technologies may be limited for several investigators. Hemodynamic evaluation using a micromanometer pressure sensor introduced via the common carotid artery is relatively straightforward and significantly less expensive than other technologies. However, it should be stressed that there are inherent limitations to the interpretation of left ventricular micromanometry. In contrast, analysis of ventricular pressure-volume relationships (e.g., using conductance micromanometry) constitutes the most rigorous and comprehensive approach to evaluate intact cardiac function28. The unique advantage of pressure volume methodology to measure cardiac function is that it enables more specific measurements of the left ventricular performance independently of loading conditions and of the heart rate29.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with the 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 failure1.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. In other words, ventricular remodelling concerns an alteration of the left ventricular architecture. 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 (e.g., mitral insufficiency). Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure.
Loss of myocardial tissue following acute myocardial infarction results in a decreased systolic ejection and an increased left ventricular end-diastolic volume and pressure. The Frank-Starling mechanism, implying that an increased end-diastolic volume results in an increased pressure developed during systole, may help to restore cardiac output. However, the concomitant increased wall stress may induce regional hypertrophy in the non-infarcted segment, whereas in the infarcted area expansion and thinning may occur. Experimental animal studies show that the infarcted ventricle hypertrophies and that the degree of hypertrophy is dependent on the infarct size2. 
The loss of myocardial tissue following acute myocardial infarction results in a sudden increase in loading conditions. Post-infarct remodelling occurs in the setting of volume overload, since the stretched and dilated infarcted tissue increases the left ventricular volume. An increased ventricular volume not only implies increased preload (passive ventricular wall stress at the end of diastole) but also increased afterload (total myocardial wall stress during systolic ejection). Afterload is increased since the systolic radius is increased. Therefore, ventricular remodelling post-myocardial infarction is characterized by mixed features of volume overload and pressure overload.
The myocardium consists of 3 integrated components: cardiomyocytes, extracellular matrix, and the capillary microcirculation. All 3&#160;components are involved in the remodelling process. Matrix metalloproteinases produced by inflammatory cells induce degradation of intermyocyte collagen struts and cardiomyocyte slippage. This leads to infarct expansion characterized by the disproportionate thinning and dilatation of the infarct segment3. In later stages of remodelling, interstitial fibrosis is induced, which negatively affects the diastolic properties of the heart. 
The vascular and cardiomyocyte compartment in the myocardium should remain balanced in the process of ventricular remodelling to avoid tissue hypoxia4,5. Whether hypertrophy progresses to heart failure or not may be critically dependent on this balance between the vascular and cardiomyocyte compartment in the myocardium. 
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 reperfusion6. In contrast, the infarct area at 24 hours 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.

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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.

The extent of myocardial infarction can be assessed by Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) double staining. TTC is a redox indicator, which is converted to deep-red 1,3,5-triphenylformazan in living tissues due to the activity of various dehydrogenases in the presence of NADH8. Figure 1 shows a representative section of the heart at 24 hr after ligation of the left anterior descending coronary artery. Blue-stained areas indicate non-ischemic/normal regions. The myocardial area at risk is defined as the myocardial tissue within the perfusion bed distally from the ligation of the left anterior descending coronary artery. Deep-red-stained areas indicate ischemic but viable regions (non-infarcted area at risk) regions whereas negatively stained areas (pale red) indicate infarcted regions. In this model, the non-infarcted area at risk is negligible and not visible in this image, which reflects the permanent character of the ligation of the left anterior descending coronary artery. The infarcted area is typically between 50 - 60% of the total left ventricular wall area9,10. Two components in the evolution of the infarct zone should be discerned: 1) the three-dimensional geometry of the infarct may alter as a consequence of infarct expansion leading to a thinner but more extended myocardial infarction, and 2) the total volume of the infarct may decrease, which reflects the healing process with wound contraction and scar formation.
Infarct expansion can be quantified by evaluating the time-course of infarct length and infarct thickness10. A representative Sirius Red stained cross-section of a heart at day 28 after permanent ligation of the left anterior descending coronary artery is shown in Figure 2. The image shows an infarct that has been substantially stretched. In a cross-section, this expansion corresponds to an increase of the absolute infarct length and a decrease of infarct thickness.
Besides considerations on the three-dimensional geometry and the volume of the infarct, another caveat with regard to interpretation of infarct parameters should be considered. Since the viable myocardial tissue will also undergo hypertrophy, it is clear that the ratio of infarct area versus total left ventricular wall area will decrease as a function of time. Assessment of longitudinal changes of the infarct zone requires therefore a clear insight in the difference between absolute parameters and relative parameters.
An arterial and ventricular pressure register is shown in Figure 3. After stabilization of the catheter, heart rate, maximal systolic left ventricular pressure, minimal diastolic left ventricular pressure, the peak rate of isovolumetric left ventricular contraction (dP/dtmax), the end-diastolic left ventricular pressure, and the peak rate of isovolumetric left ventricular relaxation (dP/dtmin) are determined. The time constant of isovolumetric left ventricular pressure fall (tau) is quantified using the method of Weiss et al.7.
<img alt="Figure 1" src="/files/ftp_upload/52206/52206fig1highres.jpg" width="700" /> 
Figure 1. Assessment of area at risk and infarct size 1 day after myocardial infarction. The images from left to right go from the cardiac apex to the base of the heart. 24 hr after permanent ligation of the left anterior descending coronary artery, 2 ml of Evans blue dye was injected as a bolus into the aorta to quantify the volume of the perfused myocardium. Hearts were subsequently arrested in diastole by injection of CdCl (100 &#181;l; 0.1 N), were flushed with physiological saline to wash out excess blue dye, and were embedded in 5% low gelling temperature agarose. Afterwards, 500 &#181;m thick cross-sections were made using a HM 650 V Vibration Microtome and slices were then incubated in a 1.5% 2,3,5-triphenyltetrazolium chloride containing isotonic phosphate buffer (pH 7.4) for 30 min at 37 &#176;C. Images were made using a SteREO Lumar V.12 microscope. The negatively stained area of infarcted tissue, the red-stained non-infarcted area at risk, and the blue-stained non-ischemic healthy ventricular wall area were quantified using Image J software. Scale bar represents 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52206/52206fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52206/52206fig2display.jpg" width="700" /> 
Figure 2. Representative Sirius Red stained cross-section of a heart at day 28 after ligation of the left anterior descending coronary artery. The images from left to right go from the cardiac apex to the base of the heart. The standard protocol of Junqueira et al.11 was applied for Sirius Red staining. This histochemical method stains fibrotic infarct tissue dark red and healthy tissue orange. Scale bar represents 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52206/52206fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52206/52206fig3highres.jpg" /> 
Figure 3. Arterial (A) and left ventricular (B) pressure registers obtained after catheterization of normal mice. The obvious difference between an arterial signal and a ventricular signal is that the latter signal drops to approximately 0 mm Hg during diastole. LVP: left ventricular pressure.

Watch this Scientific Journal Video about Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure at JoVE.com

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

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.

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 ...

permanent-ligation-left-anterior-descending-coronary-artery-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

27.6K Views.  Catholic University of Leuven. 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.

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

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.

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 Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort 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.

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 ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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.

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.

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

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

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.

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 ...

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.

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

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...