This work was supported by the National Natural Science Foundation of China (82070317, 81700390 to Jibin Lin, 8210021880 to Bingjie Lv and 82000428 to Boyuan Wang) and the National Key R&#38;D Program of China ( 2017YFA0208000 to Shaolin He).

Animal studies have been approved by the Animal Care and Utilization Committee of Huazhong University of Science and Technology (Wuhan, China).
NOTE: Male C57BL/6J mice (8-10 weeks) are used as models. Mice have free access to food and water and are bred in specific pathogen-free conditions. The room is maintained under controlled temperature (22 &#176;C &#177; 2 &#176;C) and humidity (45%-65%). Mice are exposed to a 12-h light/dark environment at the Animal Care Facility of Tongji Medical School (Wuhan, China) according to the guidelines set by this institution. Use sterile microsurgical instruments and surgical supplies. Surgical gloves and masks are required throughout the procedure. The experimental workflow is shown in Figure 1A.
1. Preoperative preparation
<ol>
	<li>Use a rectangular operating table (OT) with a prewarmed heating pad (37 &#176;C) throughout the surgical procedure (Figure 1B). Disinfect the board with ultraviolet light and 70% alcohol before procedure initiation.</li>
	<li>Weigh all mice accurately to calculate the dose of anesthetic drugs needed. Then, anesthetize the mice with ketamine (80 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection. Ensure appropriate depth of anesthesia by the absence of a withdrawal reflex to toe pinching and blink reflexes.</li>
	<li>Place the mouse supine on the OT with gauze under the head to avoid overheating of the eyes. Apply ophthalmic ointment to the eyes to prevent them from drying out.</li>
	<li>Shave the fur on the left precordial chest with an electric razor. Use a fur removal cream on the pre-shaved thorax and massage evenly with a sterile cotton swab for ~1 min. Wipe the excess loose fur with gauze.</li>
	<li>Use povidone-iodine, followed by 70% alcohol to clean the area. Cover the thorax with gauze.</li>
	<li>Use a 4-0 suture under the upper incisors and secure it to the anchor point (close to the edge of the OT over the nose) to keep the mouth slightly open and facilitate cannulation.</li>
	<li>Pull the tail to keep the body straight, and secure the tail to the OT using tape. Secure the four limbs and tighten them on the other anchor points. Importantly, do not over-stretch the front limbs; otherwise, respiratory compromise may occur.</li>
	<li>Use curved forceps and forceps to open the jaw and lift the tongue. Use an illuminator to clearly visualize the throat and glottis.</li>
	<li>Insert a 22-G cannula gently with a blunted and truncated needle into the trachea through the mouth ~1-cm down the throat.Use one hand to hold the tongue, move it slightly upwards with blunt forceps, and, simultaneously, use the other hand to gently insert the tube into the trachea. Be careful not to insert the tube into the esophagus.</li>
	<li>Remove the needle gently. Check the intubation by placing the tube into the water for bubbles to form before connecting to the ventilator.</li>
	<li>Connect the endotracheal tube to a ventilator set to 120/min and tidal volume adjusted to 250 &#181;L. 
	NOTE: The ventilator setting is adjusted by body weight (in general, a higher body weight requires a higher tidal volume).</li>
	<li>Verify intubation by checking bilateral symmetrical chest expansion. Then, the connection is fixed to the OT with tape to avoid the tube falling off.</li>
	<li>Place ECG electrodes on the paws and connect them to the ECG recorder. Monitor cardiac electrophysiology throughout the procedure.</li>
</ol>
2. Thoracotomy
<ol>
	<li>Remove the gauze on the thorax. Disinfect again with 70% alcohol for the incision areas using three scrub cycles. Then, cover the mouse with a sterile surgical drape with a hole over the surgical field to reduce contamination of the surgical site.</li>
	<li>Make an oblique skin incision (0.8-1.0 cm) along the left midclavicular line with a sterile scalpel.</li>
	<li>Undertake blunt dissection of subcutaneous tissues to expose the ribs underneath. Be careful not to injure vessels, ribs, and lungs. Stop the bleeding by using sterile cotton applicators.</li>
	<li>Identify and make an incision of about 6-8 mm in the third intercostal space. Then, carry out blunt dissection of tissues in the intercoastal space to open the chest cavity. Be careful not to injure the internal thoracic artery.</li>
	<li>Use forceps to span the intercostal space. Insert pre-sterilized homemade retractors (Figure 1C) into the rib cage and pull back to spread the incision to ~6 mm in width. Attach the retractors to the OT with rubber bands.</li>
	<li>Remove the surrounding tissues carefully to expose the heart fully. Pull off the pericardium gently with curved forceps without injuring the heart. Now a clear view of the heart is available.</li>
</ol>
3. LAD ligation
NOTE: The LAD appears as a thin red line running perpendicular from near the apex and down through the left ventricle. The LAD is bright-red color, so be careful not to mistake it for a vein. Usually, the ligation site is ~1-2 mm below the left auricle. This ligation position will produce about 40%-50% of the ischemia in the left ventricle. A higher position will create a more extensive infarct zone. A more distal site will create a smaller infarct zone.
<ol>
	<li>Use a dissecting microscope and direct a focused and appropriate light for LAD visualization. Press the site below the chosen ligation position gently to enlarge the LAD temporarily (&#8804;5 s per time). Recheck the LAD in this way.</li>
	<li>Use a tapered needle (3/8, 2.5 x 5) to pass an 8-0 silk ligature underneath the LAD under a dissecting microscope. Be careful with the needle depth: not too deep to enter the left ventricle and not too shallow to avoid damaging the LAD.</li>
	<li>Tie the ligature with a loose double-knot. The loop diameter is about 2-3 mm.</li>
	<li>Place a 2-3 mm PE-10 tubing into a loop parallel to the artery.</li>
	<li>Tighten the ligature loop gently until it is around the artery and tubing. Then, secure the loop with a slipknot. Take care not to damage the myocardial wall with excessive tightening pressure. 
	NOTE: Ligation is not carried out for the sham-operation group.</li>
	<li>Confirm cessation of blood flow in the LAD: observe a paler color in the anterior wall of the LV after ligation. In addition,significant ST-elevation within a few heartbeats also indicates occlusion16. If permanent ligation is required (e.g., MI), remove the PE-10 tubing and tie the LAD directly with a knot. Resume the remaining procedure as mentioned in step 4.3 below.</li>
	<li>Remove the retractors from the incision. Then, close the wound temporarily with a bulldog clamp. Ischemia duration is according to the experimental design. Ensure that the mouse continues to be connected to the ventilator.</li>
</ol>
4. Reperfusion
<ol>
	<li>When the period of ischemia ends, remove the bulldog clamp and insert the retractors again to open the incision and expose the heart (especially the ligation site).</li>
	<li>Untie the slipknot and remove the PE-10 tubing. Confirm the restoration of blood flow in this step by observing the color change back to pink-red within 20 s. Simultaneously, watch the ECG carefully: a potential dissolution of ST-elevation also suggests reperfusion.</li>
	<li>Leave the 8-0 ligature in situ for subsequent Evans-Blue and TTC staining. In other cases, remove the suture at this step.</li>
	<li>Remove the retractors and close the incision by suturing the third and fourth ribs with a 4-0 nylon suture. Be careful not to injure the lung. Push out the air that might be trapped in the chest cavity by pressing the chest gently while tying the suture knots.</li>
	<li>Close the muscle layers with continuous sutures. Close the skin with a 4-0 nylon suture; continuous sutures and interrupted sutures are acceptable.</li>
</ol>
5. Postoperative care
<ol>
	<li>Observe the mouse carefully for signs of recovery from anesthesia, for example, movement of the tail or whiskers. After that, the mouse usually resumes a normal breathing pattern with a respiration rate of around 150 bpm. Extubate the mouse by removing the tube slowly.</li>
	<li>Monitor the mouse for an additional 3-5 min to ensure respiratory distress is absent.</li>
	<li>Administer 100 &#181;L of buprenorphine (0.1 mg/mL, s.c.) after the mouse begins to breathe. For the next 24 h, provide an additional dose every 4-6 h. Provide ibuprofen as additional pain relief in drinking water as a 0.2 mg/mL solution for 2 days before and &#8804;7 days after surgery.</li>
	<li>Keep the mice warm and reduce mortality risk by using thermal insulation blankets as mice are prone to hypothermia after the anesthesia.</li>
</ol>
6. Validation after the procedure
<ol>
	<li>Troponin-T test
	<ol>
		<li>Collect blood samples from the retroorbital plexuses and isolate the serums by centrifugation (3,000 &#215; g, 10 min, room temperature).</li>
		<li>Dilute 20 &#181;L of serum to 100 &#181;L with saline solution for the troponin-T test. Storethe remainder of the samples at -80 &#176;C.</li>
		<li>Detect the Troponin T (cTnT using a commercial kit following the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Cardiac ultrasound 
	NOTE: Cardiac ultrasound is used to evaluate cardiac function and wall-motion abnormalities at different stages before and after surgery according to the experimental design17,18. Different parameters such as ventricular wall thickness, ventricular volume, ventricular-cavity diameter, ejection fraction, and short-axis shortening fraction are measured.
	<ol>
		<li>Anesthetize the mice with ketamine (80 mg/kg) and xylazine (10 mg/kg)via intraperitoneal injection.</li>
		<li>Shave the chest with an electric razor. Use fur removal cream and massage evenly. Wipe the excess loose fur with gauze.</li>
		<li>Place the mouse on the OT and secure the four limbs with adhesive tape.</li>
		<li>Place the ultrasound probe (30 MHz) on the anterior region of the heart at ~ 30&#176; to the sternum. The probe in this view is aligned with the long axis of the heart. Set the ultrasound in B-Mode; the left ventricle, left atrium, mitral valve, and ascending aorta can be identified clearly. Use video capture to obtain data for subsequent analysis.</li>
		<li>By rotating the transducer 90&#176;clockwise, obtain a parasternal short-axis view at the level of the papillary muscles to clearly detect the left and right ventricles. Then use B-Mode and M-Mode to assess cardiac function and morphometry.</li>
		<li>Calculate the left ventricular end-diastolic diameter (Dd), end-systolic diameter (Ds), and interventricular septal thickness by specifying the corresponding location in the ultrasound images. 
		NOTE: The machine would manually calculate left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV). Also, the machine would calculate the values for fractional shortening (FS) and ejection fraction (EF) using formulas FS = (Dd-Ds)/Dd &#215; 100% and EF= (LVEDV-LVESV)/LVEDV &#215; 100%. Choose five consecutive cardiac cycles and obtain their mean values.</li>
	</ol>
	</li>
	<li>Measurement of myocardial infarct size 
	NOTE: Evans-Blue/TTC staining is used to measure the infarct size because it can evaluate tissue viability19. It is recommended to stain within 72 h of reperfusion because the scar will shrink. This step is performed after euthanizing the animal with 200 mg/kg pentobarbital sodium via intraperitoneal injection.
	<ol>
		<li>Expose the heart again following the previous procedures from steps 2.2-2.5. Then, re-ligate the LAD at the initial site validated by the suture mentioned in step 4.3 at the end of the desired reperfusion duration.</li>
		<li>Cannulate the aorta and then perfuse the heart with 0.3 mL of 1% Evans Blue solution. The myocardium of the non-ischemic region is stained blue. After perfusion, remove the heart rapidly by cutting the aorta with scissors.</li>
		<li>Then, wash the heart in KCl solution (30 mM) to stop the heart from beating. Store at -20 &#176;C for &#8805;4 h after removing the surrounding fatty tissue.</li>
		<li>Cut the heart in the transverse direction into five slices of thickness 1 mm using a sharp scalpel. Weigh the slices and then incubate them with 2% TTC for 40 min at 37 &#176;C. 
		NOTE: After the incubation, the infarct areas are demarcated as white, whereas viable tissues in non-infarct areas remain red.</li>
		<li>Fix the slices with 4% formaldehyde overnight. 
		NOTE: This action will enhance the contrast between the infarct area and the non-infarct area. It will also shrink the slices.</li>
		<li>Photograph the slices with a digital camera. Then, calculate the area at risk (AAR), infarct area, and non-ischemic zone using graphics software. 
		NOTE: After Evans-Blue/TTC double-staining, the blue area is the &#34;normal&#34; area. The remaining areas (including white and red) are the &#34;ischemia risk&#34; areas: the white area is the myocardial infarction area (IA), and the red area is the ischemic (but not infarcted) area. Taking the inconsistency of sizes of heart slices into account, the results are adjusted for weight. 
		 
		Assign: 
		A1-A5 for Area of infarct zone /Area of the heart slice; 
		B1-B5 for Area of non-infarct zone/ Area of the heart slice; 
		W1-W5 for Weight of the heart slice. 
		 
		Then: 
		Total weight of infarcted myocardium: W1 &#215; A1 + W2 &#215; A2 + W3 &#215; A3 + W4 &#215; A4 + W5 &#215; A5; 
		Total weight of non-infarcted myocardium: W1 &#215; B1 + W2 &#215; B2 + W3 &#215; B3 + W4 &#215; B4+ W5 &#215; B5; 
		Total weight of AAR = (W1 + W2 +W3 + W4 + W5) - (W1 &#215; A1 + W2 &#215; A2 + W3 &#215; A3 + W4 &#215; A4 + W5 &#215; A5) 
		 
		Finally: 
		The area of myocardial ischemia is calculated as the percentage of AAR in the left ventricle: 
		<img alt="Equation 1" src="/files/ftp_upload/63257/63257eq01v2.jpg" /> 
		The area of myocardial infarction is calculated as the percentage of IA in the AAR: 
		<img alt="Equation 2" src="/files/ftp_upload/63257/63257eq02v2.jpg" /></li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

In recent years, the creation of models for MI and MIRI in clinical and scientific research has developed rapidly20,21. However, there are still some questions, such as the mechanisms of actions and how to ameliorate MI/MIRI, that must be resolved. Here, a modified protocol for establishing a murine model of MI and MIRI is described. Several key points must be considered carefully.
The first key point is endotracheal intubation. Some p...

Acute myocardial infarction (AMI) is a common cardiovascular disease worldwide and carries high mortality1. Advances in technologies make early and efficacious revascularization available for AMI patients. After these treatments in some patients, myocardial ischemia-reperfusion injury (MIRI) can occur2. Thus, it is of great significance to understand the mechanisms of actions and how to ameliorate MI/MIRI. Mice are widely used as models because of their low cost, rapid breeding time, and ease for making genetic alterations3. Scholars have developed different methods to model MIRI and MI in animal4,5,6,7,8,9. This strategy promotes research, but the different criteria and methods employed complicate the interpretation of results among research teams.
In mice, MI has been induced by isoproterenol10, cryoinjury11,12, or cauterization13. MI can be induced readily by isoproterenol, but the pathophysiological process is different from that in clinical MI. Cryoinjury-induced MI has poor consistency, elicits excessive myocardial damage around the left anterior descending coronary artery (LAD), and can easily induce arrhythmia. Cauterization-induced MI is quite different from the natural process of myocardial infarction, and the inflammatory reaction in the burning area is more intense; in addition, the surgical approach has technical difficulties. Moreover, there are some labs14 developing MI model in minipigs using balloon blocking or embolization or thrombosis method through interventional technique. All these methods can cause coronary artery occlusion directly, but needing coronary angiography devices and, above all, the too-thin mouse coronary arteries makes these operations not practical. For MIRI, the differences among different models were quite modest, such as using respirators /micromanipulation or not5,6.
Here, a simple and reliable method that can induce MI and the MIRI model, adapted from previously published methods4,5,6,7,8,9,15, is described. This method can simulate pathophysiological processes by direct blockade of the LAD through ligation. Moreover, by relieving the ligation, this model can also simulate reperfusion injury. In this protocol, a dissecting microscope is used for LAD visualization. Then, the researcher can identify the LAD readily. Subsequently, accurate ligation of the LAD leads to reproducible and predictable blood occlusion and ventricular ischemia. Moreover, electrocardiography (ECG) changes can be used to confirm ischemia and reperfusion in addition to the color changes of the LAD observed under a microscope. This strategy leads to a shorter procedure duration, lower surgical complications risk, and fewer experimental mice needed. The methods for the troponin-T test, cardiac ultrasound, and triphenyl tetrazolium chloride (TTC) staining are also described. Overall, this protocol is useful for studies of MI/MIR mechanism, as well as for drug discovery.

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			<td>0.9 % sodium chloride solution</td>
			<td>Kelun Industry Group,China</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde fixing solution</td>
			<td>Servicebio,China</td>
			<td>G1101</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-0 silk suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products,China</td>
			<td>C412</td>
			<td>-</td>
		</tr>
		<tr>
			<td>8-0 suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products,China</td>
			<td>H801</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>IsoReag,China</td>
			<td>IR-11190</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Canon,Japan</td>
			<td>EOS 80D</td>
			<td>-</td>
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			<td>Depilatory cream</td>
			<td>Veet,French</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>Elecsys Troponin T hs STAT</td>
			<td>Roche,Germany</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>Electrochemical luminescence immunoanalyzer</td>
			<td>Roche,Germany</td>
			<td>Elecsys 2010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma,America</td>
			<td>E2129</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eye scissors</td>
			<td>Shanghai Medical Instruments,China</td>
			<td>JC2303</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Haemostatic forceps</td>
			<td>Shanghai Medical Instruments,China</td>
			<td>J31020</td>
			<td>-</td>
		</tr>
		<tr>
			<td>High frequency in vivo imaging systems</td>
			<td>Visualsonics,Canada</td>
			<td>Vevo2100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ibuprofen</td>
			<td>PerFeMiKer,China</td>
			<td>CLS-12921</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Intravenous catheter</td>
			<td>Introcan,Germany</td>
			<td>4254090B</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Sigma-Aldrich,America</td>
			<td>&#160;K2753</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Medical alcohol</td>
			<td>Huichang ,China</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microneedle holders</td>
			<td>Shanghai Medical Instruments,China</td>
			<td>WA2040</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microscopic shears</td>
			<td>Shanghai Medical Instruments,China</td>
			<td>WA1040</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microsurgical forceps</td>
			<td>Shanghai Medical Instruments,China</td>
			<td>WA3020</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mouse electrocardiograph</td>
			<td>Techman,China</td>
			<td>BL-420F</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Needle holders</td>
			<td>Shanghai Medical Instruments,China</td>
			<td>JC3202</td>
			<td>-</td>
		</tr>
		<tr>
			<td>operating floor</td>
			<td>Chico,China</td>
			<td>ZK-HJPT</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PE-10 tube</td>
			<td>Huamei,China</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Merck,America</td>
			<td>1030001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Rodent Ventilator</td>
			<td>Shanghai Alcott Biotech,China</td>
			<td>ALC-V8S-P</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Aomei Industry,China</td>
			<td>SZM0745-STL3-T3</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical thermostatic heating pad</td>
			<td>Globalebio, China</td>
			<td>GE0-20W</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triphenyltetrazolium chloride</td>
			<td>Servicebio,China</td>
			<td>G1017</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Huamaike Biochemicals and Life Science Research Prouducts,China</td>
			<td>323004</td>
			<td>-</td>
		</tr>
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induction of myocardial infarction and myocardial ischemia-reperfusion injury in mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

The experimental workflow is shown in Figure 1A. The researcher can schedule the time nodes according to the experimental design upon study initiation. The duration of LAD ligation is according to the research purpose. For MI, the research can ignore the reperfusion step. Cardiac ultrasound is available at different stages of the study because it is non-invasive, while Evans-Blue/TTC staining can be performed only when the mouse is sacrificed. For research that focuses on fibrosis and ventri...

Watch this Scientific Journal Video about Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice at JoVE.com

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

induction-myocardial-infarction-myocardial-ischemia-reperfusion

Research

JoVE Journal

Medicine

6.3K Views.  Huazhong University of Science and Technology. Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Video: Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

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 ...

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.

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 ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

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 ...

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 ...

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 ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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 ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...