The quantification of atherosclerotic lesions in apoE and other types of hyperlipidemic mice is key to assessment of various genetic factors and new therapies. Our protocol provides step-by-step instructions for analysis of atherosclerotic burden in qualitative and quantitative manner in a single mouse. 16 weeks after inducing atherosclerosis, before perfusing the heart, load a 10 milliliter syringe with 10 milliliters of DPBS and attach a 25 gauge needle.
Lift the abdominal skin of the experimental mouse with tweezers and use fine scissors to cut the skin from the base of the abdomen to the top of the neck. Open the abdominal wall below the ribcage and use the tweezers to lift the sternum to access the diaphragm. Cut the diaphragm and cut away the ribcage to expose the thoracic cavity.
Next, make a small incision in the right atrium of the heart and slowly inject the entire volume of DPBS into the apical left ventricular puncture. The liver and kidney will turn light brown in color. When all of the saline has been perfused, use a nonwoven sponge to clean the chest cavity of any extraneous blood and fluid.
After the chest cavity has been wiped, remove the non-experimental organs and cut the clavicle with tweezers and fine scissors. Place the mouse under a stereo microscope and use the tweezers and spring scissors to dissect the aorta. When the aorta has been isolated, cover the tissue with a saline-soaked nonwoven sponge and dissect the aortic branches including the brachiocephalic, carotid, subclavian, renal, common iliac, and femoral arteries.
When all of the arteries branching from the aorta have been isolated, use tweezers and spring scissors to carefully dissect and remove the adventitial adipose and connective tissue around the aorta and aorta branches. A successful isolation of aorta and aortic branches requires practice and patience. To fix the vascular tree, first load a 10 milliliter syringe with 4%formaldehyde in 1X DPBS and attach a 25 gauge needle.
Remember to always prepare 4%formaldehyde inside a fume hood. Insert the needle into the apical left ventricular puncture and slowly inject the entire volume of fixative. Clean the chest cavity of any extraneous fluids with a nonwoven sponge as demonstrated and use tweezers and micro dissecting spring scissors to separate the heart from the aorta.
When the tissues have been separated, isolate and excise the aorta and its major vessel from one millimeter above the carotid artery to the end of the femoral artery and place the vessel in a container of DPBS. For Oil Red O staining of the unopened whole aorta, use Minutien pins to secure the vessel onto a wax Petri dish and rinse the vessel with fresh DPBS. Pour 25 milliliters of fresh 0.45 micrometer pore-filtered Oil Red O solution into the dish for a 60-minute stain at room temperature.
Submerge the tissue with 60%isopropanol for a 20-minute wash at room temperature. At the end of the incubation, rinse the vessel three times with fresh distilled water for five minutes per wash and place the dish under the dissecting microscope. Use tweezers and spring scissors to gently clean all of the perivascular adipose tissue around the aorta to avoid any false background Oil Red O staining and transfer the vessel to a clean glass microscope slide.
Oil Red O will label the lipid-rich plaque red, leaving the non-plaque containing areas pale in color. Then acquire digital high-resolution micrographs with a light microscope equipped with a camera and save the images preferably in tagged image file format. For en face mounting of the aortic tissue sample, transfer the vessel to a wax Petri dish and cover the tissue with fresh DPBS.
Sever the carotid and subclavian arteries of the aortic arch and the iliac arteries in the abdominal aorta one to two millimeters after the bifurcations. Sever the renal arteries. Next, use micro dissecting spring scissors to longitudinally open the aorta preparation along the inner curvature and iliac arteries and cut the three branches of the aortic arch along the greater curvature until the base level of the inner curvature.
Then use Minutien pins to secure the aorta flat against the dish without stretching with the lumen side facing up and cover the tissue with fresh DPBS. Compared to apolipoprotein E knockout mice, the aortic walls of smooth muscle cell TGF-beta-R2 knockout apolipoprotein E mice demonstrate severe atherosclerosis, making it difficult to dissect the lesions. In addition, the aneurysms are particularly extensive below the suprarenal aorta, highly reminiscent of advanced human aortic aneurysms.
Here, a representative image of an unopened aorta from a high-cholesterol high-fat diet-fed TGF-beta-R2 knockout apolipoprotein E mouse stained with Oil Red O is shown. The mouse developed both an ascending and an abdominal aortic aneurysm with accelerated atherosclerotic lesion formation observed in the brachiocephalic, carotid, subclavian, iliac, femoral, and renal arteries. This en face Oil Red O staining of TGF-beta-R2 knockout apolipoprotein E mouse tissue reveals severe aneurysmal enlargement and marked elongation of the entire aorta compared to the apolipoprotein E knockout mouse group.
It is important to be able to distinguish Oil Red O stained plaques from false background Oil Red O stained perivascular adipose tissues. Atherosclerosis plaques are three-dimensional phenomena. After en face aortic 2D lesion quantification, we recommend plaque set analysis such as aortic roots and the brachiocephalic artery.
This protocol can also be used to decide whether a particular genetic factor, intervention, or treatment affects atherosclerosis progression or regression in these models.
