I would like to acknowledge the Department of Research and Graduate Studies for providing funds to support my research and the Department of Comparative Medicine for their vigilance and assistance. I would also like to recognize the Department of Physiology for their support and guidance as well as the students and technicians in my lab for their help. Lastly, I would like to thank my post-doctoral mentor, Dr. Charles E. Murry, for the training opportunity during which time I learned the mouse microsurgery.

<ol>
	<li>Sterile surgical instruments (Table 1) and 3" cotton tipped applicators are placed on a sterile underpad. The bead sterilizer (Germinator 500) is turned on.</li>
	<li>Mice (age: &gt; 6 weeks; wt: &gt; 18g) are anesthetized with an i.p. injection of 20&mu;l/g BW of tribromoethanol (250mg/kg; duration - approximately 40 minutes). </li>
	<li>When the mice are unresponsive to toe-pinch, a sterile lubricant (Tears Renewed) is applied to the eyes to protect them from desiccation and the left side of the chest is coated with depilatory (e.g. Nair) to remove fur from the skin.</li>
	<li>The depilatory is washed away with warm running water and betadine/alcohol swabbing is used to disinfect the surgical area.</li>
	<li>The mouse is placed on a warm deltaphase isothermal pad which is fixed to a plexiglass table. Each limb is immobilized using tape and a thick thread is placed horizontally under the top teeth to hold the upper jaw in place.</li>
	<li>The table is positioned vertically and a fiberoptic light is shone directly onto the neck region for transesophageal illumination. This requires precise placement such that the opening of the throat is viewed as a well lit orifice, thus enabling the trachea to be visualized to facilitate insertion of the PE tubing.</li>
	<li>The tubing is then connected to the ventilator (connected to a 95% O2/5% CO2) to administer constant positive pressure ventilation (TOPO ventilator; rate 125 breaths/min; peak inspiratory pressure 10-12 cmH2O; *note: settings vary with strain and gender 1-3). Once ventilation is confirmed by synchronous chest movements, the connection is fixed to the pad with tape to avoid extubation during the surgery.</li>
	<li>Using toothed forceps to pull skin up and away from the chest, a #10 sterile scalpel blade attached to a #3 scalpel handle is used to make a 1.5cm incision in the skin parallel to the sternum.</li>
	<li>Curved Vanna microscissors are used to cut the pectoralis muscles and make a small hole in the intercostal muscle.</li>
	<li>Straight, blunt microscissors are used to cut through 3 ribs.</li>
	<li>A 9mm pediatric ophthalmic speculum is used to retract the rib cage.</li>
	<li>Using the curved forceps, pull the pericardium away from the heart and use the toothed forceps to gently tear it open.</li>
	<li>Using the Castroviejo needle holder, a 6mm tapered point 3/8 needle threads the 8-0 polyethylene suture underneath the left anterior descending coronary artery (along the long axis of the heart) perpendicular to it.
		<ol>
			<li>For a temporary ligature that can be removed for timed reperfusion, a sterile 0.5-1cm piece of PE90 is placed on the heart in parallel to the coronary artery. The suture, which has first been looped under the coronary artery, is then tied to the tubing. At the time it is to be released, the ligature is loosened. This can be repeated as desired and the time of occlusion/reocclusion can be modified 4. Depending on the length of the protocol and the type of anesthesia used, supplementation may be necessary.</li>
			<li>For a permanent occlusion, the ligature laced under the coronary artery is simply tied. Blanching and dyskinesia are apparent and the long end of the suture is cut 5-10.</li>
			<li>For intramyocardial injection(s), a sterile Hamilton syringe with a 30 gauge sterile beveled needle is introduced into the base of the heart above the area of injury on the right side of the ligature. The needle is then advanced into the area of injury and withdrawn slightly so that the bevel can be seen approximately at the border zone. Some of the solution in the syringe (2-3&mu;l) is injected into the heart and the needle is held in place. The syringe is withdrawn another 1-3mm and the rest of the solution is injected. The syringe is held in place until the bleb that is formed by the solution dissipates. The needle is then removed. If there is any bleeding, a cotton-tipped applicator is gently pressed onto the needle insertion site until the bleeding stops 5-7.</li>
		</ol>
	</li>
	<li>Once the myocardial manipulations are complete, the rib retractors are removed and the thoracic cavity is closed with 2-3 mattress sutures using 6-0 surgipro suture.</li>
	<li>Two-three mattress sutures are then made to close the pectoralis muscles, 1-3 drops of 0.25% marcaine 1:10 in sterile saline (0.1ml/25g mouse) is applied to the muscle and then 2-3 mattress sutures are made to close the skin.</li>
	<li>The mouse is removed from the ventilator. Once rhythmic, rapid, shallow breathing is verified, the mouse can be extubated.</li>
	<li>0.5ml warm sterile saline is injected into the dorsal subcutaneous space and the mouse is placed on a warming pad in a cage until it regains mobility (1 hour minimum).</li>
	<li>For survival experiments, mice are placed back into their cages and returned to the vivarium until the time of sacrifice. During first 2 days, moistened food is placed on the cage floor to facilitate feeding (so they don't have to reach up which may cause pain) and buprenorphine should be administered every 6-12hr. Post-operative care also includes daily monitoring for the first week to verify adequate mobility, grooming, and eating habits.</li>
	<li>The surgical instruments are wiped clean with ethanol and inserted into the bead sterilizer before the next surgery.</li>
	<li>At the time of sacrifice, mice are anesthetized with sodium pentobarbital (65mg/ml; 55-65 mg/kg). When an adequate plane of anesthesia is achieved, the thoracic cavity is opened.</li>
	<li>While the heart is still beating, a syringe with a 23 gauge needle containing cold potassium chloride (KCl, 30mM) or 2,3-butanedione monoxime (BDM; 10mM) is used to puncture the posterior basal region of the ventricle and the solution is slowly injected into the chamber until the heart is arrested in diastole.</li>
	<li>Once the heart is removed, a syringe containing PBS is used to retrogradely perfuse rinse the heart to remove any blood that remains. For acute studies, at the end of the reperfusion period, the LAD is re-ligated at the original point of occlusion. A solution containing 1% Evan's blue is injected into the aorta. Once the heart is extracted, it is cut transversely into 3 sections of equal thickness, incubated in 1% 2,3,5-triphenyltetrazolium chloride, and imaged for morphometric analyses 11. For chronic studies, the heart is then immersed in fixative, then processed and embedded according to routine procedures. Slides can then be stained histologically and imaged for morphometric analyses (using Scion, NIH Image J, or Image Pro Plus) 9,10,12.</li>
</ol>
Representative Results:
When done correctly, the survival rates in mice (male: age 8-10 weeks, 22-28g; female: age 10-12 weeks, 20-26g) are: over 90% in acute ischemia/reperfusion and ischemic preconditioning experiments, over 85% in permanent artery ligation studies, and approximately 80% for intramyocardial injections. Since early injury is more readily visible by metabolic changes rather than structural, infarct size determination in ischemia/reperfusion and ischemic preconditioning experiments is performed by infusing 1% Evan's blue dye into the aorta which will perfuse the heart that is not supplied by the LAD (Figure 1A). Once the heart is removed and transversely cut in half, the tissue is incubated in 1% solution of 2,3,5-triphenyltetrazolium chloride to measure infarct size (Figure 1B). The areas are measured using Scion or NIH imaging software which can be calibrated using a micrometer imaged at the same magnification. These numbers are used to calculate area at risk/left ventricle and infarct size/area at risk 11. Strain differences can result in variations in body weight and heart size and so care should be taken to normalize these measures to heart weight, body weight, or tibia length for comparative purposes.
Permanent artery ligation results in gross structural changes such as necrosis, wall thinning, and chamber dilation. Comparison of the effects of treatment and/or time on infarct size and necrosis relative to the left ventricle, chamber area, septal wall and left ventricular free wall thickness in the permanent occlusion model (Figure 2A) can also be measured using Scion or NIH imaging software. Collagen staining with picrosirius red/fast green (Figure 2B) can be used to measure insterstitial fibrosis which correlates to functional indices of wall stiffening8-10. The image in Figure 3 represents the distribution of 6ul solution (Evan's blue) injected into the border zone of the heart following permanent artery ligation. Notice that it proceeds in the direction of the injury as well as toward the base and also transmurally.
<img src="/files/ftp_upload/2581/2581fig1.jpg" alt="Figure 1" /> 
Figure 1. A. Evan's Blue injected into the aorta prior to excision. This image shows the perfused regions of the heart (stained) and the occluded area (unstained). B. Evan's Blue and TTC staining following acute ischemia/reperfusion injury. This is a representative image (20x) showing the blue dye distribution which stained the unoccluded regions as well as TTC staining of metabolically viable tissue (red). Necrotic areas do not stain and so they remain pale (outlined).
<img src="/files/ftp_upload/2581/2581fig2.jpg" alt="Figure 2" /> 
Figure 2. A. Hematoxylin and eosin stain. This is a representative image (20x) of H&amp;E staining of a mouse heart cut transversely through the infarct region at 4 days post-MI (20x). The * denotes tissue necrosis, arrows point to granulation tissue, RV = right ventricle and LV = left ventricle. B. Picrosirius red and fast green stain. This is a representative image (20x) of picrosirius red/fast green staining of a cross-section of mouse heart at 4 weeks post-MI. The cytoplasm stains green and collagen fibers are red. 
<img src="/files/ftp_upload/2581/2581fig3.jpg" alt="Figure 3" /> 
Figure 3. Evan's Blue dye stain distribution following 6ul intramyocardial injection. This is a representative image showing the global and transmural distribution of Evan's Blue dye throughout the heart following 6ul intramyocardial injection at the border zone immediately following coronary artery ligation (12x).

This animal protocol was approved by and is in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee at East Carolina University.

Coronary heart disease continues to be an epidemiologically and fiscally significant public health problem. Considerable basic research is still needed to understand the mechanisms by which injury and remodeling proceed and how potential therapeutics may modulate these processes if they are to be developed for clinical use. Rodents are most commonly used and the wide range of genetically modified mice available makes this species a more attractive model.
Although there are differences between mice and other species, there are many advantages to a murine model. Use of a simple dissecting scope or magnifying glass and well lit conditions enable the vasculature to readily be seen (for detailed gross anatomy of vasculature, see Salto-Tellez et al., 200413). To reduce the risk of post-operative mortality, it is very important to avoid severing large vessels since the total blood volume of a 25g mouse is less than 2ml14. In the event that excessive bleeding occurs, gentle application of pressure or pinpoint cauterization can be used to stop the bleeding.
This procedure can also be modified in a variety of ways. For example, mice can be anesthetized using isoflurane, ketamine/xylazine, or sodium pentobarbital and appropriate selection is determined by the duration of the protocol15-18. The toe-pinch reflex is the most commonly used index of the depth of anesthesia. Further, to improve the probability for long term survival, some investigators use antiarrhythmic drugs such as lidocaine to reduce the incidence of lethal arrhythmias19,20 however, it must be taken into account that this has recently been shown to have antiapoptotic properties in an acute model21. Also, to reduce post-operative pain, analgesics such as buprenorphine can be administered for the first 48 hours after surgery3,16,17,22,23. To maintain body temperature during surgery (especially for longer protocols), a rectal probe in series with a heating pad is often used in place of the isothermal pad. For ischemia/reperfusion and/or ischemic pre- or postconditioning: the duration of the occlusion(s) and reperfusion(s) can be altered; for permanent occlusion, the size of the infarct may be modified by adjusting the location of the ligature; and for intramyocardial injections (eg. cells, proteins), there can be 1-3 injection locations and the volume per injection can be up to 15 &mu;l24. If cells are being injected, the gauge of the needle used (usually 26-30)5,25,26 should be chosen based on the size of the cells so the internal diameter of the needle is large enough to avoid sheering. To avoid confounds due to inflammatory processes triggered by the surgery, some investigators have reported using a snare that is manipulated ex vivo to occlude and reperfuse the hearts in a closed chest mouse at any point after the surgery27-29. More recently, Gao et al.30 have shown that temporary and permanent occlusion can be performed without the need for ventilation and a few laboratories have begun to use ultrasound to perform closed-chest intramyocardial injections25,31.
Since the first study demonstrating feasibility of ligating the coronary artery in mice was published by Johns and Olson in 195432, many others have adopted this model and modified it to study various aspects of myocardial injury and remodeling3,33-45. The nature of mice in terms of size, reproductive capacity, and comparatively less expense for purchase and maintenance make this species an appealing tool for a broad range of physiologic and pathophysiologic studies. As the miniaturization of technology for imaging in vivo advances46-49, as well as means to perform and analyze large scale genomics and proteomics, drug screening, efficacy of cell-based and/or protein therapies as well as biomaterials50-64, combined with the increasingly wide range of genetic manipulations afforded by ubiquitous or tissue specific transgenic or mutant/knockout mice, the murine model of myocardial infarction will undoubtedly continue to be an invaluable tool in evaluating acute cardiac injury and long term remodeling. Therefore, there is unquestionable value in being able to perform these experiments reliably and reproducibly.

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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.

Watch this Scientific Journal Video about Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction at JoVE.com

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

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 ...

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51.8K Views.  East Carolina University. 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.

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

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 ...

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.

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 ...

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 ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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 ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...