The overall goal of the following experiments is to present the technique of permanent ligation of the left anterior descending coronary artery in mice. This model is applied to investigate cardiac remodeling and the development of heart failure After myocardial infarction. In the first part of the video, the procedure of permanent ligation will be demonstrated, whereas in the second part, evaluation of cardiac function, using in vivo invasive hemodynamic measurements in mice will be shown.
The model of permanent ligation of the left anterior descending coronary artery will be demonstrated by Marlin Locks senior animal technician of the center for molecular vascular biology. Eja PhD student of micro group will present technique of invasive hemodynamic measurements in mice. Begin by focusing a light on the neck region of a sedated mouse.
Then lift the animal's tongue with a blunted pin set so that the entrance of the larynx can be clearly visualized. Pass a self prepared 20 gauge blunted needle through the larynx into the trachea, and then connect the mouse to the ventilator to assess that the intubation was placed correctly. Secure the mouse to a heating pad to prevent hypothermia.
Taking care that the left hind limb crosses the right hind limb for a better view of the left ventricle during surgery. Then make a small transverse cutaneous incision up to the sternum and separate the underlying skin and muscles. Use a five zero silk suture to pull aside the pectoralis muscles and then insert a blunted pin set to make an incision in the third intercostal space.
Move the pin set under the intercostal muscles from the lateral to the medial side until the sternum is reached. Then push the pin it from the inside through to the skin to puncture the thoracic wall. Finally, carefully cut the intercostal muscle just above the pin it to complete the thoracotomy.
Taking care to avoid puncturing the lungs, place a sponge soaked with 0.9%sodium chloride into the cavity to protect the lungs, and then introduce a chest retractor into the intercostal space to expose the left atrium, left ventricle, and left anterior descending coronary artery. Now ligate the left anterior descending coronary artery with a single six zero proline ligature about one millimeter under the tip of the left atrium and distal to the first diagonal branch. Successful ligation will induce an immediate discoloration resulting in a pale myocardium appearance within the affected territory.
Now place 3 6 0 tron sutures around the intercostal space and remove the sponge from the chest cavity. Re-expand the lungs by blocking the outflow of the ventilator, and then pull the sutures tight and press down on the chest to repeat the re expansion. Use a small amount of saline to confirm a lack of air bubbles and the successful closure of the thorax.
Then look through the intercostal muscle to confirm the normal expansion of the lungs. Reposition both pectoralis muscles to serve as an extra barrier for prevention of a pneumothorax, and use five zero silk sutures to close the skin. Then disconnect the mouse from the ventilator, warming the animal on the heating pad until it has fully recovered.
Before beginning the hemodynamic measurements submerge a 1.0 French Miller pressure catheter in sterile water at 37 degrees Celsius to minimize the signal drift After at least 30 minutes, electronically, calibrate the pressure sensor at zero and 100 millimeters of mercury and record the baseline data at 2000 hertz. Then secure the anesthetize mouse in the supine position on a heating pad, monitoring the animal's temperature with a rectal probe. Shave the neck region and then make a midline incision to expose the thyroid gland.
Then fix the neck with bent needles and pull aside the salivary gland to expose the right common carotid artery. Now identify the vagus nerve, which resembles a white thread and lies along the artery, and use a curved forceps to carefully separate the artery from the nerve. Pass the curved forceps under the right common carotid artery to separate it from the other tissues, removing any connective tissue from around the artery.
Then pass 2 6 0 silk wires under the right common carotid artery, and make a tight knot on the top wire. Place the wire towards the head, close it and fix it with a distal ligation. Next, pass the proximal wire twice from left to right and fix it with two proximal non occlusive wires at this time as well.
Keep the carotid artery moist by dripping sterile, 0.9%sodium chloride onto the artery. Then use a 26 gauge needle to make an incision in the right common carotid artery between the distal ligation and the proximal non occlusive wire. Introduce the pressure sensor into the artery.
Then gently push the pressure catheter forward and adjust the proximal non occlusive wire in such a way that the catheter can pass carefully through the wire, under the clavicle, and so that the vessel remains filled with blood. Now start recording the pressure signal. In a healthy mouse, the arterial pressure signal fluctuates between a diastolic pressure of 60 to 70 millimeters of mercury and a systolic pressure of 100 to 120 millimeters of mercury.
Direct the catheter via the innominate artery and the aorta into the left ventricle. The ventricular pressure will fluctuate between zero and 100 to 120 millimeters of mercury. Finally, after allowing the catheter to stabilize inside the left ventricle, record the signal for 30 to 60 minutes.
Depending on the experimental requirements, the extent of the myocardial infarction can be assessed by Evan's Blue, TTC double staining. Here, representative sections of the heart at 24 hours after ligation of the left anterior descending coronary artery are shown. The blue stained areas indicate the non-ischemic normal regions.
The deep red stained areas indicate the ischemic but viable regions. That is the non infarcted areas at risk. Whereas the pale red, negatively stained areas indicate the infarcted regions, which typically comprise between 50 to 60%of the total left ventricular wall area.
The infarct expansion can be quantified by evaluating the time course of the infarct length and thickness. Here, a representative, serious red stained cross-section of a heart at day 28 after permanent ligation of the left anterior descending coronary artery is shown in this cross section. The expansion of the substantially stretched infarct corresponds to an increase of the absolute infarct length and a decrease of the infarct thickness.
In these graphs, a representative arterial and ventricular pressure register is shown. The obvious difference between the arterial signals and the ventricular signals is that the ladder signal drops to approximately zero millimeters of mercury during diastole. Using this model, it's important to note that cardiac function will depend not only on the loss of myocardial tissue, but also in the process of infarct expansion, in fart healing, and scar formation, and on the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodeling.
