The overall aim of this procedure is to isolate functional cardiac immune cells. This is accomplished by first ventilating the rat and exposing the pericardial sac surrounding the heart. Next Hank's balance salt solution or HBSS is injected into the pericardial space.
Then the HBSS is aspirated, which now contains immune cells from the pericardial space. Finally, the number of immune cells obtained from the isolation procedure accounted. Ultimately, chemical degranulation or co-culture experiments, flow cytometry, immunofluorescent analysis, or pharmacological treatment of the cells can be performed to evaluate the functionality and biological activity of the cardiac immune cells.
There will be three individuals from my laboratory who will be demonstrating this procedure. Jennifer McLarty, a graduate student, Giselle Melendez, a postdoctoral fellow, and my chief technician will Spencer. The main advantage of this technique over existing methods of immune cell isolation, for example, collagenous digestion, is that it prevents unwanted immune cell activation and damage, and it preserves the immune cell responsiveness to experimental treatment Before getting started, incline a cutting board to a 30 degree angle.
Then use a Dremmel power tool to trim off half to three quarters of the end of an 18 gauge bevel needle. Making a shorter tip for uses an endotracheal tube once the rat has been monitored. To ensure adequate anesthesia, shave the abdomen, chest, and throat of the animal, then make a skin incision from the mid abdomen to below the chin.
Next, dissect away the muscles of the anterior triangle of the neck to expose the trachea once exposed, place a three zero black braided suture underneath it. Now make an incision in the trachea between the cartilaginous rings and insert the trachea tube. Tie the three zero suture tightly around the trachea to secure the tracheal tube.
Next, connect the endotracheal tube to a small animal ventilator and induce breathing. To ensure adequate oxygenation of the heart and lungs, confirm equal chest rise and fall in the rat during inspiration and expiration of the ventilator respectively. Before beginning the immune cell isolation, fill a 10 cc syringe with HBSS.
Then trim off half to three quarters of the end of the soft Teflon tip of a 24 gauge Baxter intravascular over the needle. Quick cath to make a shorter tip for the isolation procedure. Place the 24 gauge Baxter intravascular over the needle.
Quick cath tip on the syringe containing the HBSS. Then place the HBSS filled syringe, all buffers and an empty 50 milliliter conical tube on ice. Now make a midline incision in the abdominal wall from the linear alba to the xiphoid process.
Then starting just lateral to the xiphoid process, make bilateral incisions through the rib cage. Continuing at an angle towards the axilla while being careful not to lacerate the lungs heart or pericardium. Then dissect the diaphragm away from the lower ribs cutting lateral to medial.
Next, use the falciform ligament as a landmark denote where the pericardium is attached on the opposite side of the diaphragm. Gently lift one side of the rib cage to expose the thoracic cavity and the heart Performing. The pericardial puncture aspirating the HBSS and cells and reinserting.
The syringe are the trickiest parts of the procedure. If you slightly tear the pericardium, don't panic. As long as the pericardium surrounds enough of the heart to form a bowl around it, you can still do washes over the heart.
Choose a point on the pericardium approximately halfway between the sternum and the heart, preferably more rostral than cordal, and push the Teflon tip of the quick cath through both layers of the pericardium. Once the tip is in place, gently fill the pericardial space with two to three milliliters of the HBSS. When the sack has been filled, switch the tip to the empty 10 cc syringe and place the tip back into the pericardial space.
Using the same entry hole, aspirate as much of the buffer as possible, making sure the tip does not come too close to the walls of the pericardial sac while providing suction, especially on the underside of the heart. Then transfer the buffer into the 15 milliliter tube upon fluid extraction. Keeping the tube at four degrees Celsius fill and then empty the pericardial sac with two to three more milliliters of HBSS.
Approximately three to four more times transferring the recovered HBSS to the 15 milliliter tube. After each wash, then remove the heart, lungs, and or any other tissue needed for other studies. Keeping the tubes on ice.
When not in use centrifuge the collected immune cell samples for 10 minutes at 200 times gravity. At four degrees SIUs, remove the supinate and then reconstitute the pellet. In one milliliter of HBSS shown.
Here are examples of histological studies to determine cell morphology and maturity after cardiac immune cell isolation. Cardiac mast cells stained with toine blue and AUM blue and seranin O are presented on the left and right respectively. Here the results from a functional study where cardiac mast cells isolated from the epicardial region of the heart were stimulated with known activators are shown.
The isolated cardiac mast cells maintain the ability to release histamine are treatment with 10 micrograms per milliliter of compound 48 80 or 10 micromolar of substance P.In the next two figures, representative Scatterplots from a flow cytometric analysis of isolated cardiac immune cells are shown in the first scatterplot CD four positive T cells have been identified in the pericardial isolate. In the second scatterplot CD eight positive T cells are detected. When attempting this procedure, it is important to remember to protect the integrity of the pericardium when injecting the buffer solution and when aspirating the buffer solution off containing the immune cells, this will increase the yield of cells obtained.
