This method can help answer key questions in the field of cardiovascular research such as identifying early protective mechanisms in acute myocardia infraction. The main advantage of this technique is that an advanced hemodynamic characterization can be performed real time in mice undergoing myocardial infarction followed up by reperfusion. Endotracheal intubation and ischemia reperfusion surgery will be performed by Pia Stock, a biologist from our laboratory.
After anesthetizing the animal, remove the neck and chest hair using electric clippers. Next, clean the skin with three alternating scrubs of Betadine and 70%alcohol. Now, using small surgical scissors, make a 10-millimeter longitudinal median incision five millimeters beneath the bottom lip and towards the sternum.
Next, bluntly dissect the left and right part of the submandibular gland using forceps. Then, separate the muscle and fat tissue in the right paratracheal region to access the right commune corroded artery. Using curved forceps, carefully bluntly dissect the vessel to mobilize and separate five to 10 millimeters of it.
Always avoid moving the vagus nerve in the corroded body, as this can lead to severe hypotension and bradycardia. After the vessel mobilization, pass the two prepared 5/0 silk sutures under the vessel. Then ligate the distal vessel using a tight knot, and place a loose knot on the proximal suture where the catheter will go.
Next, place a hemostat vascular clamp on the proximal end of the exposed vessel, proximal to the suture. This will be used to reversibly block blood flow. Now, using microscissors, open the vessel with the wedge-shaped incision one millimeter proximal to the cranial knot.
A small drop of blood should be seen. Then, stretch the incision and insert the catheter 10 millimeters into the vessel. Now, start recording the data collected by the catheter probe.
Next, extract the vascular clamp, then add one or two drops of saline into the incision to help slide the catheter another 10 millimeters down the vessel. Then, tighten the knot to prevent blood reflux alongside the catheter without locking the catheter in place. Now, gently continue advancing the catheter until the pressure analysis shows an arterial blood pressure profile when the catheter has reached the aorta.
At the aortic valve, light resistance and pull synchronized movement should also be felt. When trying to advance the catheter through the aortic valve, it can help to pull it back five millimeters and advance again. Proper left ventricular catheterization will be indicated by a change in diastolic pressure.
Any change in volume is also an indicator of advancing the sensor to the left ventricle. Once certain that the catheter is in the left ventricle, fasten the proximal knot more tightly to prevent it from moving. After the catheterization, prepare the 45 minute ischemia.
First, incise the skin from the caudal sternum to the left axilla. Then proceed with the blunt preparation of the two muscle layers to expose the ribs. Next, open the thorax with an incision between the third and fourth left rib.
Make use of surgical hooks to access the pericardium, and then resect the pericardium covering the heart. Before continuing with the left main coronary artery ligation, wait 30 seconds without touching the animal to record pressure and volume data for a valid analysis. Now, identify the left main coronary artery.
It emerges under the left oracle and descends at the left side of the heart towards the apex. Then, loop a 6/0 polypropylene suture around the artery two millimeters under the left oracle. Next, place a small silicon tube under the loop and secure it with a knot.
If the distal myocardium starts to turn gray, then the left main coronary artery has been occluded. To proceed, cut the suture at one millimeter length, and then release the surgical hooks and manually close the muscle layers above the incision. Then wait 45 minutes while continuously recording pressure and volume data.
After 45 minutes, reopen the incision and remove the silicon tube to induce reperfusion. Then record data for another 20 minutes. When the heart turns back to being red, the reperfusion is successful.
After the reperfusion period, loosen the knot and extract the catheter while quickly fastening the knot again to prevent loss of blood if blood is needed for further studies. After completing the calibration steps, which are detailed in the text protocol, perform a software-guided data analysis. For the baseline data, highlight at least 10 cycles taken before the passing of the catheter into the aortic valve.
As needed, exclude any cycles with deviations due to ventilation or manipulation. Next, analyze 10 cycle blocks on five minute intervals through the ischemia and reperfusion. The various parameters calculated by this analysis are sufficient for characterization of the left ventricular function.
At the end of the experiment, after the catheter has been retracted from the left ventricle, take a final analysis of the pressure data. Following the protocol, after the left ventricular catheterization, reversible left main coronary artery ligation was performed for 45 minutes followed by 20 minutes of reperfusion. Correct placement of the volume and pressure catheter was confirmed by obtaining the characteristic left ventricular pressure-volume graph.
Successful occlusion of the left main coronary artery was visually confirmed by blanching of the distal left ventricular myocardium. After the left main coronary artery occlusion, pressure and volume data was analyzed on five minute intervals. Pressure data showed no changes in maximum left ventricular systolic pressure, indicating preserved peripheral perfusion and stable anesthesia.
Analysis of left ventricular volume revealed that during the early phase of the ischemia, there was a significant decrease in ejection fraction and in absolute stroke volume. The maximum change in pressure, a parameter of left ventricular contractility, showed a 30%reduction in mice undergoing myocardial ischemia. The minimum change in pressure significantly decreased as well, indicating impaired left ventricular compliance.
After watching this video, you should have a good understanding on how to characterize left ventricular systolic and diastolic function in mice undergoing acute myocardial infarction using pressure-volumes catheter technique.
