Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences. Aseptic technique is required throughout the procedure to prevent contamination of the surgical site.

1. Apical resection operation in neonatal 1-day-old Cx3cr1 ^GFP/+ mouse (C57BL/6 background)

1. Anesthesia
   1. Take all the mouse pups out from the cages and put them in a clean dry box.
   2. Embed each mouse in the ice for about 2-3 minutes. Ensure that there is a thin barrier such as latex or gauze between the ice and the mouse pup to prevent frostbite. 
   3. Identify sufficient anesthesia by observing for the following signs: pale skin, lack of limb movement, and lack of pedal reflex.
2. Thoracotomy
   1. Precool the bronze operating platform overnight at -20 °C to help maintain anesthesia.
   2. Take the anesthetized mouse out of the ice box and put it on a bronze operating platform. Anchor the mouse on the operating platform in a supine position using medical adhesive tape.
   3. Put the operating platform with the mouse on it under a stereoscope.
   4. Disinfect the mouse chest using a prep pad soaked with betadine and 70% alcohol or as consistent with institutional policy.
   5. Incise the skin with a 1 cm cut at the fourth intercostal area of the chest cavity and then separate the intercostal muscles using microsurgical scissors until the heart is accessed.
   6. Alternately press the chest and abdomen with the help of two forceps until the heart is exteriorized out of chest without any mechanical damage.
   7. Set the ribs below and above the small incision as a natural fixation to immobilize the heart.
3. Ventricular apex resection
   1. Locate the apex of the left ventricle. Cut a 1 mm diameter of ventricular apex tissue using iridectomy scissors under a stereoscope.
   2. Confirm that the left ventricular chamber is exposed, and begin oozing.
   3. Gently press the heart back to chest cavity using a cotton swab.
   4. Suture the muscles, ribs, and skin using 8-0 Prolene sutures.
   5. Clean the mouse thoroughly after the operation.
4. Post-operation care
   1. Transfer the mouse from the operating platform to a 37 °C heating blanket to warm up the body immediately after the operation.
   2. Confirm anabiosis of the mouse by observing for the following signs: spontaneous respiration restoration, skin color change from pale to pink, and movement of limbs.
   3. Take the operated mouse back to its mother once it has recovered.
   4. Mingle the operated mouse with its mother's nesting materials if necessary.
      ​NOTE: It is important to remove all the 1-day-old pups from the mother at once and then return them all at once after all the pups are recovered.

2. Preparation of the neonatal cardiac macrophage suspension

NOTE: All these experimental procedures should be carried out in a dark place and under sterile conditions.

1. Harvest the heart of a neonatal Cx3cr1^GFP/+ mouse 1-day after apical resection
   1. Euthanize the Cx3cr1^GFP/+ mouse one day after the apical resection. Use an excess of carbon dioxide and decapitate the mouse sequentially to apply euthanasia.
   2. Take the heart out of the chest and immerse it in a 10 cm dish with PBS.
   3. Cut the vessels and remaining connective tissue away from the ventricles. Cut the auricular appendix and outflow tract away from the heart.
   4. After the heart stops beating, cut the heart into 1-2 mm³ pieces in PBS buffer using microsurgical scissors (Figure 2A).
2. Neonatal mouse heart dissociation
   1. Transfer the harvested heart tissue to a tube containing 2.5 mL of preheated enzyme mix (Table of Materials), and tightly close the tube.
   2. Invert the tube and place it with the cap down (Figure 2B).
   3. Run the neonatal heart dissociation program (Table of Materials).
   4. Detach the tube from the dissociator after the program is completed.
   5. Add 7.5 mL of 1x DMEM with 10% FBS to the tube.
   6. Filter and transfer the suspension to a 15 mL centrifuge tube (Figure 2C). Centrifuge the cell suspension at 300 x g for 5 minutes.
   7. Resuspend the cell pellet in 1 mL of blood cell lysis solution and incubate for 2 minutes at room temperature.
   8. Add 5-10 mL of PBS buffer to the suspension and centrifuge at 300 x g for 5 minutes.
   9. Resuspend the cell pellet in 1 mL of DMEM with 10% FBS.
3. Neonatal cardiac macrophage isolation
   1. Sort GFP⁺ neonatal cardiac macrophages by FACS. Receive the sorted macrophages in a sterile tube containing 500 µL of DMEM with 10% FBS.
   2. Count the GFP⁺ macrophages (Figure 2D). Resuspend the macrophage in DMEM with 10% FBS at the concentration of 1 x 10⁶ macrophages per 200 µL of DMEM for later injection.
      NOTE: The number of GFP⁺ macrophages is small in one neonate (approximate 1 x 10⁵); therefore, at least 10 neonates should be used to obtain enough macrophages for each adult mouse injection.

3. Neonatal cardiac macrophage transplantation

1. Perform a myocardial infarction operation on an adult (6-8-week-old) male C57BL/6 mouse by ligation of the left anterior coronary artery⁸.
2. Put the operated mouse on a 37 °C heated blanket until it recovers.
3. Intravenously inject 200 µL of DMEM with 1 x 10⁶ GFP⁺ neonatal cardiac macrophages into the infarcted adult mouse after the operation (approximate 6 hours later) via the tail vein.
4. Send the mouse back to a clean cage.

4. Result evaluation

1. Injection efficiency validation
   1. Anesthetize the operated mouse and harvest the heart 7 days after the myocardial infarction operation.
   2. Immerse the heart in the pre-cooled PBS buffer.
   3. Fix the heart tissue with 4% polyformaldehyde for 72 hours at room temperature with shaking.
   4. Dehydrate the heart tissue in dimethylbenzene and ethanol.
   5. Embed the heart tissue in paraffin and slice it into sections with a thickness of 5 µm.
   6. Perform the standard immunofluorescence staining protocol¹. Use α-actinin to mark cardiomyocytes.
   7. Detect the GFP⁺ macrophages in the heart receiving transplantation.
   8. Perform the standard immunofluorescence staining protocol¹. Use α-actinin and pH3 to mark cardiomyocytes and cell proliferation, respectively (Figure 3A).
2. Cardiac repair evaluation after the transplantation
   1. Use echocardiography on the operated mouse one month after the operation¹.
   2. Analyze the cardiac function by comparing the left ventricular ejection fraction and fractional shortening in different groups (Figure 3B).
   3. Anesthetize the operated mouse and harvest the heart.
   4. Repeat steps 4.1.2-4.1.5.
   5. Perform a Masson's staining protocol.
   6. Analyze the infarcted area after macrophage injection (Figure 3C).