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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<li>Kato, R., Foex, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myocardial+protection+by+anesthetic+agents+against+ischemia-reperfusion+injury%3A+An+update+for+anesthesiologists%5BTitle%5D)+AND+%22Canadian+Journal+of+Anaesthesia%22%5BJournal%5D)">Myocardial protection by anesthetic agents against ischemia-reperfusion injury: An update for anesthesiologists.</a> Canadian Journal of Anaesthesia. 49 (8), 777-791 (2002).</li>
</ol>

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.

<table><tbody><tr><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></tr><tr><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>&amp;nbsp;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></tbody></table>

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...

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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

6.5K Views.  Huazhong University of Science and Technology. Deze studie beschrijft een aanpasbare methode om een myocardfunctiemodel in Miro te creëren door precisieligatie van de linker voorste dalende kransslagader door middel van micromanipulatie. Dit protocol verkort de duur van de procedure, zorgt voor een beheersbare infarctgrootte en verbetert de overleving van muizen. Gebruik een rechthoekige OT met een verwarmingskussen dat tijdens de chirurgische ingreep is voorverwarmd tot 37 graden Celsius.Desinfecteer het bord vóór de procedure ...

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

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

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 ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

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 ...

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 ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

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

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