The overall goal of this procedure is to use apical resection in neonatal mice to study early mammalian heart regeneration. This matter can help answer key questions in the heart regeneration field such as what are the molecular and cellular mechanisms that underlie early mammalian cardiac regeneration. The main advantage of this technique is that apical resection mouse model provides a relatively simple and a direct approach for studying early mammalian heart regeneration.
Before beginning the procedure, transfer all of the postnatal day one C57 black six pups from their nursing mother into a clean mouse cage with fresh bedding and nesting materials. Then for each neonate in turn, transfer the pup from an ice bed to the surgical area and immobilize the arms, legs, and tail in a supine position. Disinfect the chest with betadine followed by a gentle cleaning with a 70%alcohol prep pad.
Now, make a transfer skin incision along the fourth inner costal area of the chest cavity. Use spring scissors to make an incision in the apex of the heart. To access the heart, use spring scissors to make an incision in the apex of the heart followed by a blunt dissection of the inner costal muscles.
Use sterile 6-0 sutures to close the ribs and muscles together. Use skin glue to carefully close the skin incision and allow the neonate to recover under a heat lamp for about three minutes. Then use a 70%alcohol prep pad to clean all of the blood and glue traces from the pup and return the animal to its litter mates.
After all of the surgeries have been performed, mix the pups with the mother's bedding and excrement before returning the neonates to the mother's nest. One day after surgery, count the number of sham and surgical pups to determine the survival rate within each group. At the appropriate time point after surgery, for each animal in turn, clean the chest with 70%ethanol and incise the midline skin and muscle of the chest.
Remove the entire heart from the thoracic cavity and fix the tissue in five millimeters of four percent paraformaldehyde overnight at room temperature. The next day, transfer the samples to 70%ethanol for up to one week before paraffin embedding. After acquiring five micrometer thick paraffin sections through the entire ventricle, stain the samples with hematoxylin, and Eocene, and Masson's trichrome to examine the muscle replacement and fibrotic responses in the resected hearts respectively.
Using a slide scanner, manually obtain at least three foci per sample. Then analyze the sample images at a 40X magnification according to the manufacturer's instructions using the default parameters of the scanner. With a successful apical resection, within one day of the apical resection, a blood clot is effectively formed to seal the left ventricle.
A progressive resorption of the blood clot and early cardiac fibrosis is then observed with its gradual replacement by myocardial tissue until the complete regeneration of the myocardium by 21 days after the resection. Indeed, by three weeks after the surgery, no differences between the resected and sham operated hearts are apparent by morphological analysis. While attempting this procedure, it's important to remember to maintain sterile conditions.
Following this procedure, other measures like labeling and the linear can be performed to answer additional questions about cardiomyocyte proliferation and apical resection. After watching this video, you should have a good understanding of how to perform apical resection in neonatal mice.
