The overall goal of this procedure is to induce a myocardial infarction in a mouse by ligating the left anterior descending coronary artery. This method can help answer key questions in the cardiovascular field, such as whether overexpression or knock out of certain genes can affect the recovery of the heart following myocardial infarction. The main advantage of this technique is that the procedure is less invasive than various other myocardial infarction mirroring models, and results in a quick recovery time with fewer complications.
Generally, individuals new to this method will struggle because endotracheal intubation and identification of the LAD artery requires practice with a trained instructor. Visual demonstration of this method is critical at identifying and assess the LAD is difficult to learn without any experience or training. Prior to sedating the animal, start heating the bead sterilizer to 250 degrees Celsius which can take up to 20 minutes.
Check that the mouse is heavily sedated. The breathing rate will be approximately 32 breaths per minute. Then place the sedated mouse supine on a styrofoam board.
Position the mouse at eye level for easier visualization of the oropharynx. Before proceeding, confirm the sedation with a toe pinch. Then, using an elastic band, hold the mouth open by securing the top incisors in an open position.
Next, position a high intensity illuminator above the mouse so that the oropharynx is transilluminated. Don surgical loops to take notice of the small structures of the oropharynx. Now, using curved forceps open the jaw.
With another pair of forceps move the tongue out of the way and then observe the opening and closing of the vocal chords. When open insert a 20 gauge one inch intravenous catheter with a blunt tip needle introducer. Use the needle to guide the catheter to the tracheal opening, but avoid inserting the needle into the tracheal wall.
Then transfer the intubated mouse to an operating surface equipped with a heating device, and there, connect the mouse to a small rodent ventilator. Before proceeding, verify the mouse's plane of anesthesia using a toe pinch. Then tape down the intubation tube at the connecting site between the ventilator and the IV catheter.
Follow by taping down all of the extremities and lubricating the eyes. Now, trim the fur from the ventral left side of the thorax. Then apply a small layer of hair removal cream to the exposed skin, and let it penetrate for 30 to 45 seconds.
In the mean time, soak sterile cotton swabs in three 1.5 milliliter tubes filled with betadine. Then using distilled water gently wipe away the cream and fur. Follow this by cleaning the surgical field three times with betadine and 70%isopropanol.
Now, drape the mouse with a quarter sized hole over the surgical field. Before making the first incision, put the autoclaved surgical instruments in the pre heated hot bead sterilizer at 250 degrees Celsius for approximately 20 seconds. Now, use fine tipped forceps to gently lift the skin at a point approximately five millimeters to the left of the prominent xiphoid cartilage.
Then use a number 10 scalpel to create an upward vertical incision to the level of the manubrium. Next, gently separate the dermal layers with curved forceps. To hold open the muscle layer, pass two five zero polypropylene sutures through either side of the incision and secure them with clamps.
Now, make an incision in the third intercostal space following the natural angle of the rib cage. Next, remove the tape from the left extremities of the mouse. Then tape together the rear feet.
Then reposition the front foot so it is slightly elevated. Be sure to clean the surgical gloves after each time the mouse is handled. Proceed by using a retractor to gently spread apart the third and fourth ribs.
Then dip a small piece of gauze in sterile 0.9%saline and squeeze out the excess saline. Using forceps gently insert the wet gauze against the left lung to prevent accidental lung damage. Next, gently remove the thin pericardium with forceps.
Then, use a small cotton ball soaked with saline to clean the heart's surface, and make the arteries observable. Gently push the left oracle upwards, and locate the coronary arteries underneath. Now, pass an eight zero nylon suture under the LAD and complete two throws to secure the ligation.
If it is successful, the left ventricle distilled from the ligature will blanche. Then, remove the gauze and the retractor. Next, through the thoracotomy opening insert a six inch 25 gauge flexible tube, attached to a 25 gauge needle.
Insert it one or two inches into the chest cavity above the left lung. Then return the mouse to a supine position and clean off the surgical gloves. Now, continue to use five zero polypropylene sutures in an interrupted pattern to close the rib cage, keeping the chest tube in place.
Then remove the two sutures that hold the muscle layer open and close the muscle layer using five zero polypropylene sutures in a simple continuous pattern. Next, attach a one milliliter syringe to the needle on the chest tube. Pull back the plunger to extract air and blood.
If the syringe fills up, detach it, empty it, and reattach it. Now, extract the chest tube, and ensure that the chest is tightly sealed. Proceed by administering analgesics and a subcutaneous bolus of saline.
After five minutes remove the mouse from the intubation tube. If the chest doesn't rise bilaterally air is trapped in the chest cavity. Introduce a 25 gauge needle with a one milliliter syringe between the third and fourth ribs, into the thoracic cavity.
Then gently draw out the trapped air and the chest will start moving normally. Mice are euthanized 28 days after the described surgery using potassium chloride. Their hearts were arrested in diastole then fixed with paraformaldehyde.
This image shows the lack of Evans blue in the ischemic left ventricle. Hearts stained with trichrome dye show that there was an increase in collagen in the infarcted region, indicating fibrosis. Control hearts showed now evidence of increased collagen.
After watching this video you should have a good understanding of how to induce a myocardial infarction using a murine model. Utilizing the techniques of endotracheal intubation, a left sided thoracostomy approach to LAD ligation, and total synthesis without any additional incision in the chest wall. Once mastered this technique can be done in 30 minutes, if it is performed appropriately.
While attempting this procedure it's important to remember to provide the mangers this around vasculature by ligation the LAD artery. Following this procedure an echo cardiogram can be performed in order to answer additional questions like the extent of functional loss or recovery of the heart after a predetermined post operative period. After its development, this technique paved the way for researchers in the field of cardiovascular surgery to explore the effects of drugs and specific genes on myocardial infarction in mice.
