The overall goal of this surgical procedure is to reproducibly induce myocardial infarction in neonatal mice. This method can help answer key questions in cardiac regeneration such as what mechanisms support regeneration in neonates and how can adult heart regeneration be promoted after myocardial infarction. The main advantage of this technique is that the left anterior descending coronary artery, or LAD, can be visualized to ensure highly reproducible LAD ligation in the neonatal mouse heart.
The changes in cardiac function after LAD ligation can be assessed by echocardiography. Before beginning the procedure place an anesthetized pup in the right lateral decubitus position on ice under the operating microscope and confirm the appropriate level of sedation by a lack of response to toe and tail pinch. Then gently disinfect the chest with povidone iodine followed by an ethanol swab.
Next use a pair of forceps to carefully lift the skin between the xiphoid and left axilla and make an incision a few millimeters below the left fore leg. Using scissors cut through the underlying pectoral muscle layer, then open a pair of forceps along the muscles and ribs in the fourth intercostal space to perform a gentle thoracotamy. Visualize the left anterior descending coronary artery emerging from the left auricle behind the pulmonary vein and descending beyond the great cardiac vein.
Location of the coronary artery is critical for the reproducibility of the technique. To ensure visualization of the artery maintain the mouse body temperature between eight and 15 degrees Celsius during the procedure. Then use a 007 mL diameter needle to pass an 11-0 nylon suture under the artery through the mid ventricle below the left auricle and ligate the artery.
The ischemia is confirmed by the blanching of the myocardium below the suture site. Next use 0.15 mL diameter needles to place two 8-0 nylon rib sutures without tying to ensure both sutures are placed without injury to internal organs such as the lungs. Then tie the rib sutures to close the thoracic incision and move the mouse onto a sterile surgical towel over a 37 degrees Celsius heating pad.
Close the muscle and skin with more 8-0 nylon sutures. Then use an ethanol swab to gently remove the povidone iodine and blood. When the animal has fully recovered from the anesthesia wipe it with bedding from the foster mother's cage to reduce the chance of maternal rejection and cannibalization and return the pup to the foster mother.
Observe the behavior of the foster mother toward the pup every 10 to 15 minutes for two to three hours to confirm the acceptance of the pup. To measure the myocardial infarct size after four to six hours with the foster mother, the pup is euthanized by isofluorane overdose followed by decapitation. Next, grasp the lower rib cage and cut through the ribs and musculature longitudinally along the left mid axillary line from the diaphragm to the axilla.
Holding the scissors at a transverse angle carefully cut through the diaphragm from the left to the right side. Then grasp the rib cage and cut the right side of the ribs and musculature along the right mid axillary line. Use the scissors to remove all of the vascular connections to the heart.
Then grasp the base of the heart and remove it from the chest cavity. Using a surgical carbon steel razor blade section the heart along the short axis at the midpoint between the suture and the cardiac apex followed by a second cut at the suture. Then place the sections in 1%TTC at room temperature for 10 to 15 minutes, carefully watching the specimens to avoid overstaining.
24 to 48 hours after LAD surgery cardiac function is assessed by echocardiography. Secure the pup in the supine position on a heated dock with its nose in an anesthesia cone and apply pre warmed echo gel to the left thoracic area. Next obtain a peristernal long axis view of the left ventricle below the suture with a 40 Mhz ultrasound probe.
Then turn the probe 90 degrees and acquire a parasternal short axis view recording the end mode echocardiographic images. The end diastolic and end systolic left ventricular internal diameters can the be measured from the short axis end mode images. Upon histological examination at three days post myocardial infarction the typical evidence of an infarction including the infiltration of inflammatory cells is observed.
At day seven the left ventricular tissue appears normal and by 21 the cardiac tissue has completely regenerated. In a total of 13 left anterior descending coronary artery ligation surgeries, 100%of the hearts were infarcted with an average infarct size of 36%To further confirm the infarct induction echocardiography can be performed 24 and 48 hours after the procedure. In this representative experiment at 24 hours post myocardial infarction the ejection fraction and the fractional shortening were significantly reduced.
By 48 hours the ejection fraction and the fractional shortening were further reduced compared to the control animals which demonstrated no reduction at all. Once mastered this technique can be completed in 10 to 15 minutes if it is performed properly. While implementing the myocardial infarction it is important to remember to operate quickly and accurately to ensure the procedure is completed within the time window during which the LAD is visible.
Following this procedure other methods like histological analysis can be performed to answer additional questions about how different treatments affect cardiac regeneration. The development of this technique paves the way for researchers in the field of cardiac biology to explore cardiac regeneration in mice. After watching this video you should have a good understanding of how to reproducibly induce myocardial infarction in neonatal mice by visualizing and ligating the LAD.
