The overall goal of this method is the visualization of the left anterior descending coronary artery function in rats using echocardiography to calculate coronary flow reserve. This method offers an important assessment tool for a diagnosis of microvascular dysfunction in diseased hearts and for evaluating for potential treatments throughout the study. Although reperfusion therapy has been shown to salvage jeopardized myocardium and to improve left ventricular function, functional abnormalities of small coronary vessels may occur after recanalization of the coronary artery.
This method can provide insight into the coronary microcirculation of rats, as well as into other models, including myocardial infarction, and pressure volume overload of the left ventricle. Before pre-ischemia reperfusion surgery ultrasound imaging, confirm a lack of response to toe pinch in an anesthetized Fischer 344 rat. Shave the hair from the chest.
Apply ointment to the animal's eyes and place the animal in the supine position on a 37 to 38 degree Celsius built-in warming platform. Connect the 13 to 24-megahertz linear probe to the ultrasound machine. Set the application preset to cardiac imaging setting and use the rail system to position the probe to obtain the parasternal short axis view.
Use the XY axis micromanipulator to move the probe in the rostral direction to locate the pulmonary artery. Move the probe, making the left ventricle more readily visible. Once the B-mode image has been captured and Cine Stored, click color Doppler and visualize the coronary artery in the short axis.
The red color, as observed in real time, is indicative of the direction of the flow. After visualizing the lower anterior descending coronary artery in color Doppler mode, change the mode to the pulse wave and look for the presence of a yellow indicator line on the coronary artery. Place the yellow pulse wave line in the middle of the coronary artery, taking care that the angle is parallel to the direction of the flow and use Cine Store to capture the velocity of the resting left anterior descending coronary flow and the peak diastole of the wave forms.
After obtaining the resting left anterior descending flow velocity, place a small strip of gauze around the base of the tail and use hemostats to grab and twist the tourniquet to apply pressure, causing the vein to enlarge. Carefully place a 25-gauge infusion butterfly needle attached to a 10-milliliter syringe loaded with dobutamine into the dilated tail vein and stabilize the needle with glue and a piece of surgical tape. When the infusion line has been secured, remove the hemostats and tourniquet to recover the flow and place the syringe into an infusion pump set to inject 20 micrograms per kilogram per minute.
During the infusion, carefully monitor the left anterior descending artery peak and heart rate, periodically recording the left anterior descending artery pulse wave peaks in the Doppler mode under the dobutamine-induced stress conditions. After the anterior descending coronary artery peaks and heart rate have plateaued during the challenge, stop the infusion, remove the tail vein infusion set, and remove the animal from the platform. Then, use the peak velocity tool to obtain the peak diastolic velocities from the images and calculate the coronary flow reserve index as the ratio of the left anterior descending artery stress peak diastolic flow velocity to the resting left anterior descending artery peak diastolic flow velocity.
Using a fiber optic light source, intubate rat with an 18-gauge IV catheter and connect to the catheter to a ventilator. Next, use an 8-0 monofilament suture to ligate the left anterior descending artery through a 15-millimeter opening at the fifth intercostal space with a plain knot for 30 minutes. At the end of the ischemia period, visually confirm the ischemia via discoloration of the heart surface and release the ligature.
Reperfusion can be verified by the reddening of the previously-discolored area of heart muscle. 72 hours after the surgery, measure the coronary flow and the coronary flow reserve as demonstrated, and compare the measurements to those obtained before the injury. In this representative experiment, before the ischemia reperfusion surgery, the resting left anterior descending coronary artery velocity was measured as approximately 423 millimeters per second to more than double the velocity after dobutamine infusion.
72 hours after ischemia reperfusion, the resting left anterior descending coronary artery velocity was significantly higher compared to the resting left anterior descending coronary artery velocity before the surgery. The stress response to a dobutamine test after the injury was significantly reduced compared to the response before the surgery responses. The coronary flow reserve is calculated as the ratio of the peak flow velocity during stress to the resting flow velocity and was approximately 2.1 in young rats before surgery, but significantly reduced 72 hours after the injury.
In addition, there were no significant changes in the systolic function of the left ventricle 72 hours after the injury. After its development, this technique will allow researchers to explore and use ultrasound imaging of coronary arteries in small animal models that accurately mimic human disease conditions. After watching this video, you should have a very good understanding of how to measure coronary blood flow and evaluate coronary microcirculation in a small animal cardiac disease model.
