Name: induction of myocardial infarction in adult zebrafish using cryoinjury
Uploaded: 2012-04-18T12:00:00
Duration: 11 min 12 s
Description: Watch this Scientific Journal Video about Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury at JoVE.com

We thank V. Zimmermann for excellent technical assistance and for fish care. This work was supported by the Swiss National Science Foundation, grant number: 310000_120611.

1. Equipment Set-up
<ol>
 <li>The main tool used to perform cryoinjury is the cryoprobe, the pen-like instrument that is made of stainless steel (Figure 1A). The cylindrical handle is 40 mm long and has a diameter of 8 mm. The tip of the applicator is 6 mm long and 0.8 mm of diameter. The junction between the tip and the handle is conical 4 mm long. In addition, the handle of the cryoprobe should be isolated with a plastic tube and tape to avoid frostbite of the fingers while holding the tool during the procedure.</li>
 <li>Other materials that are needed for the cryosurgery are a stereomicroscope, sharp forceps, micro dissecting spring scissors and a plastic transfer pipette. In addition prepare a beaker for anesthetizing the fish and a plastic spoon for transferring the fish.</li>
 <li>During the surgery, the fish are placed on a moist sponge with its ventral side up (Figure 1B). To hold the animal in a stable procedure, an appropriate groove should be manually cut in the sponge.</li>
 <li>Prepare a tank with system water to transfer the fish after the surgery.</li>
 <li>Set up a double timer first with a 10 second countdown and then automatically a 24 second countdown.</li>
</ol>
2. The Cryoinjury Procedure
<ol>
 <li>Cool down the cryoprobe by immersing the tip in 3-5 cm of liquid nitrogen for at least 3 min.</li>
 <li>Anesthetize an adult zebrafish by immerging it in 0.02% tricaine until the fish turns on its back and its gills almost stop moving (approximately 90 seconds).</li>
 <li>Transfer the fish with a plastic spoon to a moist sponge that has been cut to hold a fish upside down (Figure 1B). Hold fish steady with forceps using non-dominant hand. Visually locate the posterior medial margin of the heart and use straight iridectomy scissors to puncture the skin. (Figure 1C).</li>
 <li>Make a small (approx. 2 mm) incision above the heart by cutting straight anteriorly through the skin, muscles (Figure 1D). Do not cut through the bony branchial apparatus, as this will kill the animal.</li>
 <li>Gently tear the silvery epithelial layer of the hypodermis (Figure 1E) with the tip of the scissors to have a direct access to the beating ventricle. Do not insert the scissors deeper in the body cavity, as this will puncture the heart. The beating heart should be well visible, and no extensive bleeding should occur during the thoracotomy (Figure 1F).</li>
 <li>Start the programmed timer that has been set for 10 seconds followed by 24 seconds. During 10 seconds take out the cryoprobe from the liquid nitrogen. Make sure that there is no more nitrogen on the cryoprobe by shaking it gently. Spread the incision laterally using forceps to open the chest (Figure 1F).</li>
 <li>Once the timer rings 10 seconds, touch immediately but gently the ventricle with the tip of the pre-cooled cryoprobe (Figure 1G).</li>
 <li>Once the timer rings 24 seconds, pour 2-3 mL of system water using a plastic pipette onto the chest to release the cryoprobe from tissue, and transfer the fish into the tank with system water.</li>
 <li>The fish should restart breathing after a few seconds, and then it should resume swimming. If it doesn't breathe after around 45 seconds, stimulate the animal by squirting water into the gills with a plastic pipette until it starts breathing by itself. In our experience, 95% of the fish survive surgery, and all deaths occur on the day of surgery. It will not be necessary to suture incisions.</li>
</ol>
3. Heart Collection and Fixation
<ol>
 <li>Prepare 1 mL of 2% formalin in a microcentrifuge tube for fixing the heart, two forceps, micro-dissecting scissors and the moist slotted sponge.</li>
 <li>At the selected day after cryoinjury, euthanize the fish in 0.1% tricaine for 5 minutes.</li>
 <li>Place the fish ventral side up into a moist, slotted sponge. Make a large (approx. 4 mm) incision above the heart through the branchial cartilage with the micro dissecting scissors. Open widely the incision with forceps (Figure 1H).</li>
 <li>Pinch off the bulbus arteriosus, a white structure anterior to the ventricle (Figure 1 H-I), and remove the heart from the cavity by pulling it. A whole dissected heart is shown in Figure 1J and Figure 2A.</li>
 <li>Place up to 3 hearts into a microcentrifuge tube with 1 ml of 2% formalin. Gently turn the tube several times and keep it overnight at 4 &deg;C.</li>
</ol>
4. Heart Mounting
<ol>
 <li>Rinse the hearts in PBS for 5 minutes. Transfer the hearts into 10 mL of 30% sucrose that should be pre-cooled at 4 &deg;C, and mix gently. The specimen should initially float at the surface of sucrose solution. Continue incubation for 1 hour 20 minutes at 4 &deg;C. After this time, the hearts will sink to the bottom of the tube.</li>
 <li>Prepare a box with dry ice. Take an embedding mold, and pour a 5 mm layer of O.C.T. mounting medium at the bottom of the mold.</li>
 <li>With forceps place the heart into the O. C. T mounting medium in the mold. Under the stereomicroscope, adjust the orientation of the specimen to achieve the ventricular apex on the bottom of the mold, and the bulbus arteriosus towards the top.</li>
 <li>Place the molds with specimen on dry ice. When the mounting medium begins to freeze, fill up the rest of the mold with O.C.T. medium and let it freeze completely. Keep the mold for at least 1 h at -80 &deg;C before sectioning. The frozen specimen can be stored for many months at -80 &deg;C.</li>
</ol>
5. Hearts Sectioning
<ol>
 <li>Set up a cryostat with a cutting size of 16 &mu;m, a chamber temperature of -24 &deg;C and the temperature of the specimen at -22 &deg;C.</li>
 <li>Place the frozen block with the specimen in the cryostat and fix its orientation to begin cutting parallel to the bottom of the block.</li>
 <li>Prepare six Superfrost-plus slides per one block and numerate them from 1 to 6. Start cutting until the tissue is reached, and trim the block around the specimen with a razorblade.</li>
 <li>To obtain 6 replicates of one heart, take up the first section on slide 1, the second section on slide 2, the third on slide 3, etc. Once you placed the sixth section on slide 6, restart with slide 1 and continue until the whole organ is cut. Collect two rows of around 8 sections on the slide (Figure 3).</li>
 <li>Let the slides dry for 1 hour at room temperature. Store them for up to 1 year in tightly closed boxes at -20 &deg;C.</li>
</ol>
6. Representative Results
Representative cardiac injury following this protocol is shown in dissected whole hearts at 4 dpci (days post cryoinjury; Figure 2D-F). To visualize the myocardial tissue in vivo, we used transgenic fish expressing EGFP and nuclear DsRed2 under the control of cardiac-specific cmlc-2 promoter11,12. The absence of EGFP and DsRed2 fluorescent signals demarcated a disc-shaped infarct zone along the apical-lateral ventricular wall.
To perform cellular and molecular analyses of the tissue, the hearts were fixed and sectioned with a cryostat. A representative slide with a consecutive series of transversal heart sections is shown in Figure 3.
The sections can be analyzed with various methods (Figure 4). To determine the extent of heart regeneration versus scarring, we performed histological analysis using Acid Fuchsin Orange-G (AFOG) staining, which differentially labels cardiac and fibrotic tissues9. The gene expression analyses were achieved according to the in situ hybridization procedure13. To detect the distribution of marker proteins, we applied immunofluorescence assay with specific antibodies9. A combination of diverse staining procedures leads to the identification of molecular and cellular mechanisms involved in cardiac regeneration following cryoinjury.
<img alt="Figure 1" src="/files/ftp_upload/3666/3666fig1.jpg" /> 
Figure 1. Inducing cryoinjury of the ventricle in the adult zebrafish. (A) A photograph of the cryoprobe. (B) An adult zebrafish is placed in the slit of the sponge with its ventral side up. (C-F) Chest incision to access the heart. (C) A small cut through the skin and muscles is made between the two pectoral fins (red line). (D) The scissors are inserted into the incision to cut the skin following the red arrow. (E) Underneath the skin, the fine layer of silvery hypodermis (encircled by blue dashed line) is gently opened to access the heart. (F) The incision is spread with the forceps and the beating ventricle (green arrow) is accessible for cryoinjury. (G) The cold cryoprobe is gently inserted into the chest to touch the heart. (H-J) Heart collection. (H) A deep and long incision is made through the branchial arches of the chest to access the pericardial cavity. Two heart structures are visible: bulbus arteriosus (white arrow) and ventricle (green arrow). (I) The bulbus arteriosus is hold with forceps and pulled out from the body cavity. (J) The entire heart is excised from of the body.
<img alt="Figure 2" src="/files/ftp_upload/3666/3666fig2.jpg" /> 
Figure 2. Representative control and cryoinjured hearts dissected from adult transgenic zebrafish expressing EGFP and nuclear DsRed2 in cardiomyocytes. (A-C) Uninjured heart. (A) The dark field illumination shows the ventricle (V), bulbus arteriosus (Ba, white) and the valves (Va), where the atrium was linked to the ventricle. (B and C) Fluorescent images of the intact ventricle display EGFP and DsRed2 expression in cardiomyocytes. (D-F) Heart at 4 days post cryoinjury (4 dpci) (D) The dark field illumination reveals a disc-shaped infarct (yellow dashed line). (E-F) Cardiomyocyte markers EGFP and DsRed2 are not detected in the infarct area, indicating the damaged myocardium (encircled by dashed line).
<img alt="Figure 3" src="/files/ftp_upload/3666/3666fig3.jpg" /> 
Figure 3. Sectioning of the heart. (A) Whole heart with the drawing of transversal sections starting from the bottom of the heart (1 and 2, in red) towards the top of the heart (3 and 4 in green). (B) Photography of a slide with the series of consecutive transversal sections stained with AFOG (Acid Fuchsin Orange-G). The first sections (1 and 2, red squares) contain the ventricular apex, and the last sections of the heart (3 and 4, green squares) comprise bulbus arteriosus.
<img alt="Figure 4" src="/files/ftp_upload/3666/3666fig4.jpg" /> 
Figure 4. Examples of analysis performed on heart sections at 7 days post cryoinjury. (A) AFOG (Acid Fuchsin Orange-G) staining labels healthy muscle cells in orange, the scar tissue containing collagen in blue and fibrin in red. (B) In situ hybridization of ventricular myosin heavy chain (vmhc) mRNA visualizes the intact myocardium (blue staining). The injured tissue is devoid of the cardiac gene transcript (red dashed line). (C) Immunoassaying of Tropomyosin (green) labels intact cardiomyocytes and not damaged tissue (encircled with the dashed line). DAPI (blue) stains the nuclei of all the cells. (D) Genetically labeled cardiomyocytes express DsRed2 (red) in their nuclei. DAPI (blue) labels all the nuclei. The infarct zone does not contain DsRed2-positive cells (white dashed line).

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Experimental research on animals has been approved by the cantonal veterinary office of Fribourg.

Cryoinjury is defined as the controlled damage of tissue by the precise application of extreme cold14. Direct thermal shock destroys the cells by protein destruction and by intracellular fluid ice crystal formation that ruptures the plasma membrane. Consequently, the injured cells die in the processes of apoptosis and necrosis14. Both of these cell death mechanisms have been shown to contribute to tissue loss after myocardial infarction15. Thus, cryoinjury represents a suitable model of inducing a heart infarct. Several studies reported this well proved method in various mammals, including mice, rats, rabbits and pigs6,7. The adaptation of the cryoinjury protocol in zebrafish enables a more comparative discussion on the infarct response between different species.
Two other groups independently developed the cryoinjury procedure for the adult zebrafish heart. The major differences are the surgery methods, the type of tools and applied freezing time. In the study by Gonz&aacute;lez-Rosa et al., the pericardium was opened by tearing the tissue instead of cutting. They used 0.3 mm copper filament linked to a polyamid tube pre-cooled in liquid nitrogen to freeze the heart for few seconds until thawing could be observed10. In the study by Schnabel et al., a conical piece of dry ice of 20 mm length with a pointed tip of 2 mm in its diameter was used to touch the heart for 10 seconds8. The two groups obtained ventricular damage through death of the myocardium. The advantage of our method is a more reproducible deposition of collagen-rich scar, which better mimics the early infarct healing responses observed in mammalian systems.
The critical step during the cryoinjury procedure in zebrafish is making an incision through the skin and the pericardial sac without puncturing the underlying heart. A correctly performed surgery causes little bleeding. Profuse bleeding indicates an unintentional puncture of the ventricle with the scissors. In such cases, the animals should be retracted from the experiment. In order to avoid puncturing the heart, the point of scissors should be aimed at a shallow angle to the skin.
Another important aspect for creating a reproducible injury is the exact positioning of the cold cryoprobe on the ventricle for the optimal precise duration. Too short freezing time will result in inflammation with little cell death. Prolonged freezing, on the other hand, may cause extensive damage of the heart, which may lead to enhanced mortality of the animals. We determined that the optimal time to hold the ventricle in the frozen state ranges between 23-26 seconds for a 2 cm long adult zebrafish, and it may differ dependently on the size of the animals.
Because our protocol is very simple and rapid, there are no experimental limitations in subjecting sufficient number of animals to obtain adequate biological replications. The cryosurgical treatment is very well tolerated by the animals. Collection of the hearts at different time points after cryoinjury allows detailed characterization of the subsequent phases during the regenerative process. The experimental analysis of the cryoinjured hearts may include 1) visualization of gene transcription by in situ hybridization, 2) detection of protein distribution by immunofluorescent staining, 3) histological imaging of different structures. Understanding the key healing processes after myocardial infarction in zebrafish will impact the field of regenerative biology. Moreover, it might be beneficial for designing novel therapeutic approaches in regenerative medicine.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Micro dissecting spring scissors</td>
 <td>Roboz Surgical Instrument Co., Inc.</td>
 <td>RS-5602</td>
 </tr>
 <tr>
 <td>Tricaine</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 </tr>
 <tr>
 <td>Formaldehyde ~36%</td>
 <td>Sigma-Aldrich</td>
 <td>47630</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>84100</td>
 </tr>
 <tr>
 <td>Tissue-Tek O.C.T. compound</td>
 <td>Sakura Finetek</td>
 <td>4583</td>
 </tr>
 <tr>
 <td>Slides Superfrost Plus</td>
 <td>Fisher Scientific</td>
 <td>12-550-15</td>
 </tr>
 <tr>
 <td>Peel-A-Way Disposable Embedding Molds - Truncated Molds T8 </td>
 <td>Polyscience, Inc.</td>
 <td>18985</td>
 </tr>
 </tbody>
</table>

induction of myocardial infarction in adult zebrafish using cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Watch this Scientific Journal Video about Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury at JoVE.com

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

induction-of-myocardial-infarction-in-adult-zebrafish-using-cryoinjury

Research

JoVE Journal

Medicine

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

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 ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...