To begin, remove the hair from the precordial area of the anesthetized mouse using hair removal cream and position the mouse with its back down on the physiologic monitoring platform. Apply a small amount of conductive gel to the copper sheet on the physiological monitoring table. Secure the mouse pause onto this gel to facilitate the capture of electrocardiogram and respiration data.
Apply a small amount of ultrasound transmission gel to ensure optimal image quality. Align the platform laterally at a small angle between the probe and platform, and obtain a parasternal long axis view of the heart. Then, locate the suitable heart section in B mode.
Activate Color Doppler on the touch screen and slightly move the probe to find the right position. Now, switch to the Pulse Wave mode by pressing the corresponding button. Position the yellow indicator line on the coronary artery parallel to the direction of the flow, and press the Save Clip button to document the baseline data.
Increase the isoflurane concentration to 3%and wait for 30 seconds. Ensure that the flow velocity gradually increases with time. Frequently press the Save Clip button again to capture the blood flow at its highest velocity.
After completing the echocardiographic imaging, carefully remove the animal from the platform. Use the peak velocity function tools to measure the peak diastolic velocities and assess the heart's systolic function using the acquired images. Calculate the CFR by dividing the diastolic peak coronary flow velocity at maximal flow by the diastolic peak coronary flow velocity at baseline.
Perform a thoracotomy on the fourth intercostal space to expose the heart. Then, ligate the LAD artery using a 5-0 silk suture. Verify ischemia in mice through visual confirmation of visualizing downstream discoloration in the ischemic myocardium.
Perform an LAD artery occlusion for 30 minutes. Then, release the ligature and confirm reperfusion by monitoring the reddening of the discolored heart muscle area for one to two minutes. After coronary occlusion and reperfusion, close the chest in layers and let the mice recover for approximately half an hour.
Measure the CFR again at 1, 3, 5, 8, 24 and 48 hours after reperfusion as previously demonstrated. Before the ischemia reperfusion surgery, the CFR baseline was measured and the mice had a normal CFR value. However, after the surgery, there was a notable decrease in CFR after one hour of reperfusion.
As the reperfusion time was prolonged, the CFR values remained consistently low, suggesting hypoperfusion could persist for at least two days. No significant changes were observed in left ventricular cardiac function when the CFR was significantly reduced in the mice.
