Cardiac injury model has significant implication for elucidating the mechanism of cardiac regeneration. This protocol established an apical resection model in adult X.Tropicalis, which can be employed to elucidate the mechanism of adult heart regeneration. Previous studies have stated that heart can regenerate after apical resection in adult X.Tropicalis.
And protocols of apex resection have been published in zebrafish and mouse models, but there is no published protocol for apical resection in X.tropicalis. Losing the regenerative capacity in adult mammals makes heart failure one of the leading causes of death worldwide. Illustrating the mechanisms of cardiac regeneration using this technique will have significant implications for therapies for ischemic heart diseases.
It is better to practice the technique well before performing the surgery, as it should not affect the operating process time. And it is highly crucial to keep the frog anesthetized. To begin, place the anesthetized Xenopus tropicalis abdomen up, and cover the abdomen with distilled water-soaked gauze to avoid drying of the animal's skin during the operation.
Gently press the chest with forceps and find the center of the chest parallel to the lower forelimb. Lift the skin with ophthalmic scissors, and gently make a small incision of approximately 1 centimeter. Using ophthalmic scissors, pick up the muscle layer under the skin, and create a wound in the central chest muscle.
As the heart is located in the upper position of the wound site, gently press the chest with the ophthalmic forceps to squeeze the heart out from the wound. Gently, clamp the pericardium with forceps and softly break it using ophthalmic scissors using the apex of the heart. Wait for the pericardium to come off due to systolic blood pumping.
Hold the tip of the heart with forceps in the non-dominant hand, and lift the heart slightly according to the cardiac contraction rhythm. When the heart contracts to recirculate blood through the blood vessels, quickly cut off the apex of the heart, approximately 14%of the ventricle. Place the heart back into the chest using forceps and an absorbent ball.
Suture the skin with a 4-0 non-absorbent surgical thread, ensuring no suture into the muscle layer to prevent postoperative mortality. In the sham group, perform the thoracotomy and open the pericardium, as demonstrated earlier, but suture without performing the apex resection. Place the postoperative animal with its abdomen facing up in a Petri dish containing a small quantity of deionized water.
Wait about 10 minutes for the animal to regain consciousness, and observe its mobility and wound suture during activity. Transfer the frogs to a container filled with pure water for cultivation and replace it with pure water daily to avoid wound infection. After apical resection of the Xenopus tropicalis heart, a blood clot formed and sealed the wound in the ventricle, as observed by hematoxylin and eosin staining, as well as through Masson's trichrome staining.
Morphological analysis of the heart at 0, 7, 14 and 30 days of post-resection revealed that the blood clot caused by the heart injury disappeared at day 30. At the same time, on day 14, significant regeneration of the apex was seen, and by day 30, the resected heart apex had almost entirely regenerated. Histological analysis showed a fibrin mass at the resection site on day 14, and the heart regenerated completely without scars by day 30.
Myocardial fibrosis analysis of the heart by Masson's staining showed that within the 14th day of apical resection, the clot disappeared and was replaced by fibrin. From day 14 to 30, the myocardium incrementally replaced the fibrin and repaired the damaged ventricle apex. This technique will allow researchers to explore the cellular mechanism of heart regeneration in Xenopus tropicalis and know the cells that are related to cardiac regeneration in heart of frogs using lineage-tracing analysis after apex resection.
