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

The overall goal of this procedure is to occlude blood flow into a portion of the heart to produce ischemia reperfusion injury. For simulating a myocardial infarct, this is accomplished by first conducting a thoracotomy to expose the left anterior descending artery or LAD. In the second step, a suture is tied around the LAD to stop the blood flow.

Then after an ischemic period, the suture is removed and the artery is reperfused with blood. In the final step, the surgical incision is closed. Ultimately, the severity of myocardial infarct can be measured by the analysis of serum biomarker levels and the histology of the heart tissue.

The main advantage of our al existing method for simulating malardi infection is that it is, it is easy to find the LED and get the suture in the correct position. Well, the visual demonstration is so important because the manipulations are very difficult to learn, and the written descriptions don't convey the complexity of those manipulations Well. This is a fairly difficult procedure, and generally speaking, people are inexperienced with it Would've trouble identifying the LED or controlling bleeding if it happens to start during the procedure.

Well, one of the implications is that this approach can be used to test therapeutic compounds for their efficacy in treating myocardial infarc. Because an animal model has to be used in these approaches to be able to measure it effectively, I begin by taping a length of two hot silk, suture at least 10 centimeters long to a surgical platform. Then loop the suture around the front upper incisors of the anesthetized mouse, pull the mouse taut, and then tape the tail to the platform and the legs to the sides of the animal's body.

After cleaning the shaved surgical sites with Betadine and alcohol, place the platform with the mouse head pointing toward the end of the bench and cut a 0.5 centimeter median cervical incision into the skin. Separate the lobes of the thyroid gland at their isus to expose the Sterno OIDs muscle where the trachea can be seen under the muscle. Next, remove the inner needle of an 18 gauge trocar and then use one hand to move the mouse tongue slightly upwards with a pair of curved forceps.

Find the trachea through the cervical skin incision and then use the other hand to gently insert the trocar intubation tube until it is seen inside the trachea. Now, set an animal respirator venting 2%isoflurane in oxygen to a flow rate of 0.4 liters per minute to provide artificial ventilation for the animal, using a modified Y shape connector to attach the intubation tube to the ventilator, the correct positioning of the tracheal tube can be confirmed by judging the symmetrical chest expansion. Remove the tape on the tail and turn the mouse gently to place it in a right lateral decubitus position for the subsequent surgery.

Then secure the tail and legs to the platform with tape again to perform the thoracotomy. First, make an oblique incision approximately one centimeter long and two millimeters away from the left sternal border in the direction of where the left front leg meets the body. Approximately one to two millimeters below where the leg and body join.

The superficial thoracic vein is near this site, and the incision should be made so that the lateral end of the incision goes up to, but does not cut into the vein. Cut through the thoracic muscle to expose the ribs underneath. Taking care to avoid accidental injury of the vessel.

Then open the chest cavity with a six to eight millimeter incision in the third intercostal space, a minimum of two millimeters from the sternal border where the internal thoracic artery is located, taking care not to damage the artery. Next, insert homemade chest retractors into the incision and gently pull back to open the incision until it is eight to 10 millimeters wide. Taking care to avoid the lung, the heart should now be partly visible beneath the lungs.

Use the curved forceps to gently lift the pericardium, pull it apart, and then slide the tissue behind the retractor. Now use a dissection scope to locate the left anterior descending coronary artery, or LAD, which runs down the middle of the cardiac wall from near the apex of the heart down through the left ventricle and appears bright red and strongly pulsing on the surface of the heart. The cardiac vein is sometimes mistaken for the LAD, however proper lighting can help distinguish the two vessels.

If the lighting is too bright, it can be difficult to visualize the color differences between the vessels. Next place a one to two millimeter cotton fragment between the left atrium and left ventricle. To lift the left atrium and to help expose the LAD and clarify its position, use the pulmonary trunk to identify the left oracle to hold the heart in place and simplify tying the ligature gently apply pressure immediately below the intended ligation point about two millimeters lower than the tip of the left Oracle.

Then use a tapered needle to pass a six aught silk suture underneath the LAD taking care not to enter the left ventricle chamber or to damage the LAD. Make a loose double knot with the suture, leaving a two to three millimeter diameter loop. Then place a two to three millimeter long piece of PE 10 tubing through the loop and tighten the loop around the artery and tubing.

Secure the loop by tying one additional slip knot, taking care not to damage the ventricle wall. Confirm the occlusion of the LAD by checking for the appearance of a pale or color in the anterior wall of the left ventricle, which should appear within a few seconds of ligation. Then remove the retractor and pinch the skin together with a bulldog clamp to temporarily close the wound after the ischemia period, remove the bulldog clamp and insert the chest retractors to expose the ligature.

Then untie the knot and remove the PE 10 tubing After 15 to 20 seconds, confirm the reperfusion by observing a return of the pink red color in the anterior wall of the left ventricle. Finally, to close the chest cavity, so the incision shut in the third intercostal space with four aut silk suture. Taking care to keep the lungs clear of the suture while tying the suture knots.

Apply slight pressure to the chest with the needle holder to minimize any air that might be trapped in the chest cavity. Next, use continuous four aut silk sutures to close all of the muscle layers in the chest, and then use nylon sutures to close the skin with another continuous suture. When the suturing is complete, cease the flow of isoflurane while oxygen continues to flow.

Then after monitoring the animal carefully until it resumes a normal breathing pattern extubate the mouse, removing the tube slowly to avoid aspiration of any oral cavity. Secretions application of fallow blue dye allows the resolution of the area of the heart where occlusion of the LAD occurs, whereas hearts that are not stained with blue dye show only the area of infarct following 24 hours of reperfusion infarct. Areas in white should be distinguishable from the areas at risk in red and the areas not at risk.

In blue infarct sizes are dependent on the duration of the ischemia. Importantly, cardiac troponin eye levels are low in sham operated animals that undergo all surgical procedures except ischemia and reperfusion as compared to animals which undergo myocardial infarction. This indicates that the sham surgery does not produce significant cardiac pathology while the ischemia reperfusion injury is sufficient to produce elevation of this widely used biomarker for myocardial infarction.

After watching this video, you should have a good understanding of how to conduct a survivor surgery in the mice to simulate the malardi infect Well following this procedure, other methods like histological, staining of the heart can be done to determine infarct size and the efficacy of different therapies. It's important to remember that because this is an invasive procedure that you need to properly manage pain and distress, and it's important to manage that pain by providing analgesics in accordance with local guidelines, your institutional guidelines and national guidelines. One important thing to remember for this experiment is to design it in a way to address your specific question.

For example, if you need to have a severe infarct, you may need to extend the ischemic period during the surgery.