The endogenous heart regeneration is a dynamic process that involves multiple cell types working together in concert to mount a robust regenerative response. Peering into the regenerating tissue via 3D imaging of the cleared heart in response to injury can aid in reconstruction of the cellular dynamics that guide this cardiac regenerative response. Our protocol is specifically designed to track cells during development and regeneration.
We foresee no limitations in broadly applying our clearing, staining and imaging techniques to other organ types. For LAD-induced coronary artery occlusion, first sterilize the surgical area of an anesthetized one-day-old neonatal pup with antiseptic solution. Make a transverse skin incision in the chest region.
To separate the skin from the muscle, use a pair of dressing forceps to lift the skin carefully while gently pressing the scissors against the intercostal muscles in a closed position. Locate the fourth intercostal space and use small forceps to make a small superficial puncture taking care to avoid the internal organs. Using blunt dissection and the dressing forceps, widen the area between the intercostal muscles just until a finger can be applied to the left side of the abdomen while holding the intercostal space open with dressing forceps to gently guide the heart out of the chest cavity.
Once the heart has been extracted, allow the heart to rest on the intercostal muscles and locate the LAD as the region of the heart with less pooled blood. To induce myocardial infarction, use a 6-0 suture to tie a square knot tightly around the vessel two times without severing the artery. Blanching at the apex should be observed immediately.
It's important to remain consistent on the suture placement along the LAD for a similar infarct size. This is best achieved by accessing the heart quickly while the blood is still flowing through the arteries. When the suture has been placed, allow the heart to slip back into the chest cavity.
Suture the ribs together with one surgeon and one square knot using blunt forceps to lift the upper set of ribs while passing a 6-0 suture through the upper and lower set of ribs. Apply a small volume of skin glue to the upper abdomen and use fine forceps to position the skin of the lower abdomen over the exposed chest region. Then place the pup on a heating pad and perform the ligation on the next animal.
When all of the surgeries have been performed, return the warm and mobile pups to the mother's home cage. For passive clarity of the harvested heart tissue, 21 days postsurgery after confirming a lack of response to toe pinch, place the mouse onto a clean surgical area in the supine position and secure the limbs with tape. Using tissue forceps, grasp the fur just below the xiphoid process and make an incision spanning the width of the ribcage.
Cut alongside the distal portions of the ribcage and use the forceps to lift the xiphoid process to expose the diaphragm. Detach the diaphragm with curved forceps and pull the tissue cranially until the beating heart is accessible. Use the curved forceps to grasp the heart at the base and use iridectomy scissors to cut the aorta and superior vena cava.
Place the beating heart into a Petri dish of PBS so that the heart pumps out any retained blood. The myocardial infarction can be confirmed by a correct anatomical placement of the LAD ligation. When the heart stops beating, gently squeeze the organ with forceps to expel the residual blood and transfer the specimen into a disposable 2.5 milliliter glass shell vial containing two milliliters of PBS.
Place the vial onto a shaker for at least 30 minutes at room temperature changing the PBS solution every 10 minutes until the solution remains clear of blood. When the PBS is clear, replace the PBS with two milliliters of cold 4%paraformaldehyde for a four-hour incubation at room temperature followed by three 10-minute washes on the shaker with two milliliters of fresh PBS per wash. After the last wash, fill the vial with two milliliters of 4%acrylamide supplemented with 0.5%VA-044 solution for an overnight incubation at four degrees Celsius.
The next day, place the vial in a 37 degrees Celsius heat block for three hours. At the end of the polymerization, transfer the heart into a new glass shell vial and wash the heart tissue in two milliliters of fresh PBS 10 minutes at a time refreshing the PBS at the end of every incubation. When the PBS is clear, replace the wash buffer with two milliliters of clearing solution.
Incubate the specimen at 37 degrees Celsius for several weeks until the heart is clear, refreshing the clearing solution every two to three days. When the organ is clear, wash the specimen with PBS on the shaker as demonstrated before refilling the vial with fresh PBS for a 24-hour incubation at 37 degrees Celsius. The next day, replace the PBS with RIMS for a 24-hour incubation at 37 degrees Celsius.
At the end of the incubation, the tissue is fully cleared. The tissue should become transparent after several weeks. For imaging of the cleared postnatal mouse heart after myocardial infarction induction, fill half of a chamber of a custom-made depression slide with PBS and use curved forceps to carefully transfer the cleared heart into the chamber.
Fill the remaining volume of the chamber with PBS. Then fill the chamber with PBS until the surface of the liquid forms a dome above the top of the chamber and mount the cover slide before imaging the tissue by confocal microscopy according to standard imaging protocols. If the puncture and blunt dissection are too close in proximity to the sternum, the heart may not be able to exit the chest cavity.
If the heart does not easily exit the cavity, apply pressure to the left abdomen to facilitate the process. Complications may occur from resting the heart on the intercostal muscles. Therefore, it is important to keep the blunt dissection to a minimal size in a horizontal orientation to allow for a clear visualization and accessibility of the LAD.
Note that a superficial ligation will have less room for adjustment in the final suture placement and that the suture tying around the LAD should be performed with controlled steady movements. By the end of the clearing step, the entire heart should be consistently opaque with no discoloration in the center. After a few days in RIMS solution at room temperature, the heart should be completely cleared and some tissue expansion may be observed.
As observed by tdTomato reporter protein expression, the cardiac nerves are mainly superficial with some populations residing in the epicardial layer. Importantly, reporter protein expression confirmation is preserved after undergoing the clarity protocol as demonstrated. Clearing and imaging the 3D heart provides perspective into the location and morphology of cardiac cell populations.
This allows us to identify new aspects in cell patterning and cell-cell interactions.
