This protocol demonstrates new middle cardiac macrophages separation and the transplantation into adult mice, which might be a permissive way to promote cardiac repair. The implications of this technique can be extended to the heart regeneration field. Begin by pre-cooling the bronze operating platform overnight at 20 degrees Celsius.
Take the anesthetized mouse out of the icebox and put it on the platform. Anchor the mouse in a supine position using medical adhesive tape. Put the operating platform with the mouse under a stereoscope, and disinfect the mouse chest using a prep pad soaked with betadine and 70%alcohol.
Incise the skin with a one centimeter cut at the fourth intercostal area of the chest cavity, and separate the intercostal muscles using microsurgical scissors until the heart is accessed. Alternately press the chest and abdomen with the help of two forceps until the heart is exteriorized out of the chest without any mechanical damage. Set the ribs below and above the small incision as a natural fixation to immobilize the heart, locate the apex of the left ventricle and cut a one millimeter diameter of ventricular apex tissue using your iridectomy scissors.
Confirm that the left ventricular chamber is exposed and begins oozing. Gently press the heart back into the chest cavity using a cotton swab, suture the muscles, ribs and skin using eight by zero prolene sutures and clean the mouse thoroughly after the operation. Transfer the mouse from the operating platform to a 37 degree heating blanket to warm up the body immediately after the operation.
Confirm antibiosis of the mouse by observing for the signs of spontaneous respiration restoration, skin color change from pale to pink and movement of limbs. Take the operated mouse back to it's mother once it has recovered. Mingle the operated mouse and it's mother's nesting materials, if necessary.
Harvest the heart of a euthanized neonatal CX3-CR1-GFP mouse one day after apical resection and immerse it in a 10 centimeter dish with PBS. Cut the vessels and remaining connective tissue away from the ventricles. Cut the auricular appendix and outflow tract away from the heart.
After the heart stops beating cut the heart into one to two cubic millimeter pieces in phosphate saline buffer using microsurgical scissors. Transfer them to a tube containing 2.5 milliliters of preheated enzyme mix and tightly close the tube. Invert the tube and place it with the cap down, then run the Neonatal Heart Dissociation program.
Detach the tube from the dissociator after the program completes. Add 7.5 milliliters of 1x DMEM with 10%FBS to the tube. Filter and transfer the suspension to a 15 milliliter centrifuge tube.
Centrifuge the cell suspension at 300 times G for five minutes. Re-suspend the cell pellet in one milliliter of blood cell lysis solution and incubate for two minutes at room temperature. Add 5 to 10 milliliters of phosphate saline buffer to the suspension and centrifuge at 300 times G for five minutes.
Re-suspend the cell pellet in one milliliter of DMEM with 10%FBS, then sort GFP positive neonatal cardiac macrophages by fax. Sort macrophages into a sterile tube containing 500 milliliters of DMEM with 10%FBS. Count the GFP positive macrophages and re-suspend them in DMEM with 10%FBS at the concentration of a million macrophages per 200 milliliters of DMEM for later injection.
Neonatal cardiac macrophages were injected into the myocardial infarcted adult mouse heart and efficiency of the proliferative cardiomyocytes was confirmed by immunofluorescent staining. Statistical analysis shows that cardiomyocyte proliferation increases after macrophage transplantation. Echocardiogram images shows the cardiac function in an adult mouse one month after myocardial infarction.
Cardiac function is enhanced after macrophage transplantation. Mass in staining shows that the infarcted area in an adult mouse one month after myocardial infarction was remarkably reduced after the neonatal cardiac macrophage transplantation. Neonatal cardiac macrophage transplantation might be a promotion strategy to promote adult mouth heart regeneration.
This protocol helps researchers pursue regenerative application development and explore the heart regeneration mechanism.
