This method can help answer key questions in the pathophysiology of early atherosclerosis in mice. The main advantage of this technique is the efficiency, rapidity with which aldosterone induces the development of atherosclerosis plaques in adult growth. Visual demonstration of this method is critical as some technical steps are hard to be learned through text instruction alone.
Demonstrating the procedure will be Caterina Mammi, a staff scientist from my lab. Wearing surgical gloves, attach the mini-pump filling tube to a one milliliter syringe and load the tube with 125 microliters of the aldosterone or vehicle solution. Insert the filling tube through the hole at the top of the mini-pump until it can advance no further and slowly fill the pump with the aldosterone solution.
Stop filling the pump when a bead of fluid rises out of the pump body and carefully remove the filling tube. Insert the flow regulator into the body of the mini-pump, taking care that the regulator is seeded tightly against the pump body and prime the filled pump in a beaker containing sterile saline solution at 37 degrees Celsius, for up to 24 hours before implantation. To implant the pumps, use a razor to remove the hair from the surgical site and disinfect the exposed skin with 70%isopropyl alcohol soaked, non-woven pad material.
Next, make a 0.8 to one centimeter incision through the skin, but not the underlying tissues and use forceps to open the incision. Using a trocar in the other hand, spread the skin to create a pocket for the pump from the incision toward the scapula and insert the pump into the incision, with the head of the regulator oriented toward the front of the mouse, gently pushing the pump completely into the pocket so that there is enough skin to close the wound without tension or stretching. Once the pump has been inserted, suture the incision with surgery wire and use a clean swab to apply topical, 10%povidone iodine ointment.
Then allow the mouse to recover on a warming stage with monitoring until full recumbency. At the appropriate experimental end point, perfuse mouse via the left ventricle using gravity perfusion with 10 to 15 milliliters of ice cold DPBS, followed by 40 to 50 milliliters of 10%of neutral buffered formulate. After 10 to 20 minutes, place the mouse under stereo-microscope and use forceps and scissors to remove the excess periaortic and pericardial adipose tissue.
Hold the fixed heart with forceps and cut perpendicularly to the access of the aortic arch, as closely as possible to the heart, without causing damage. Once the heart has been separated from the aorta, use a scalpel to cut transversely along a straight line, joining the lower points of the right and left atrium and fill the upper portion of the open heart with optimal cutting temperature compound. Place the resected tissue with a cryo-section plastic mold with the axis of the aorta placed perpendicularly to the base of the rectangular mold and cover the tissue with fresh OCT.
Backlight the mold to ensure that the tissue is properly oriented, then carefully place the mold on a metal plate on dry ice, wrapping the entire mold with silver foil for minus 80 degrees Celsius storage when the OCT turns white. When the cryo-stat reaches minus 20 degrees Celsius, mount the frozen heart block onto the specimen holder with the ventricular facing outward and insert the specimen holder into the specimen orienting head. Adjust the orientation of the specimen holder to section the block with the aortic root perpendicular to the blade and cut the block until the aortic root is reached.
Collect serial sections onto glass slides, monitoring the anatomical aortic root profile, until all three aortic valves appear. Then collect six to eight, 10 micrometer thick sections or until one of the three valves disappears or is no longer intact. For oil red O neutral fat staining, fix the sections in 70 milliliters of 37%formaldehyde for five minutes, followed by a thorough wash in tap water.
After two to three minutes, blot off access water and stain the sections in 70 milliliters of 0.3%oil red O for 10 minutes. At the end of the incubation, wash the slides in tap water for 10 minutes and stain the cells for one minute in 70 milliliters of Meyer's hematoxylin, followed by a one minute water wash and a one minute emersion in 70 milliliters of 0.5%lithium carbonate. Then wash the cells in water for one final minute and mount the sections with an aqueous mounting medium for imaging by light microscopy.
After four weeks of treatment, apolipoprotein-E knockout mice displayed a significant increase in lipid content, compared to control-wild type animals. Moreover, aldosterone treatment increases the macrophage content in knockout animals as evidenced by mack-3 staining and indicating a more inflamed atherosclerotic plaque in these animals compared to vehicle treated mice. However, aldosterone treatment does not alter collagen content of atherosclerotic plaques.
While attempting this procedure, it's important to remember that this method is validated only for apolipoprotein mice which are naturally prone to develop atherosclerosis when fed an atherogenic dye. Therefore, it cannot be applied to different animal models. Following this procedure, other methods like infra-staining can be performed to characterize atherosclerosis in other aortic region and to quantify the total of atherosclerotic burden.
After its development, this technique paved the way for researchers in the field of atherosclerosis to explore early mechanism of atherogenesis, such as inflammation and endothelial disfunction. Don't forget that working with volatile compounds such as isophane and formalin can be extremely hazardous and that precautions, such as wearing safety lab face mask should always be taken while performing this procedure.
