Study investigates atherosclerotic lesion development in the aortic arch and root of LDLR knockout mice, offering a protocol for isolation, staining, and analysis to assess atherosclerosis severity plaque composition. Besides histological examination used in this study, practical techniques for assessing atherosclerosis also include biochemical markers, molecular biology tests, and vascular ultrasound. The precision of the dissection procedure poses the greatest technical hurdle in performing in vivo aortic stripping within the mouse model of atherosclerosis.
To begin, place the euthanized mice in the supine position on a foam board covered with one to two layers of absorbent paper. Fix the limbs with pins to stabilize the body. Spray 75%ethanol over the abdomen of the mice to clean and moisten the fur.
Use forceps to grasp the ventral skin of the mouse. Then using fine scissors, cut the skin from the base of the abdomen to the top of the neck. Open the abdominal wall and use forceps to lift the sternum.
Cut through both the diaphragm and ribs to expose the thoracic cavity. Remove the esophagus and trachea to facilitate cleaning of the carotid arteries. Carefully remove organs including the liver, lungs, spleen, gastrointestinal tract, and pancreas while preserving the kidneys and the abdominal aorta.
When dissecting the mesenteric arteries, lift the intestines and make incisions away from the aorta to avoid damage. Now, position the mouse under a stereo microscope to provide a clear observation field and align a cold light source with the dissection area. Using a 10 milliliter syringe filled with PBS, slowly inject into the left ventricular apex with a needle and observe the dilation of the aorta.
Gently wipe away blood and fluid in the dissection area with tissue paper to fully expose the field of view. Next, grasp the diaphragmatic segment of the thoracic aorta with forceps, and use spring scissors to cut through the connective tissue between the aorta and the thoracic muscle wall. Gently pull the heart with forceps and lift the thoracic aorta.
Observe the branches of the aortic arch and dissect the adventitial fat and connective tissue around the aortic arch and its branches upwards along the thoracic aorta. Dissect the abdominal aorta and common iliac arteries downward along the thoracic aorta. Trim the heart tissue and cut the lower half of the heart along a plane parallel to the atria, and place the trimmed heart in a suitable storage medium.
Before dissecting the aortic arch, place a small dark-colored gasket underneath it to create contrast. Capture a photograph of the aortic arch area. Cut the common iliac arteries one to two millimeters below their bifurcation, and trim the small branches of the aorta along the spine to free it.
After detaching the renal arteries near the kidneys, cut off the three branch vessels in the neck from their distal ends. Finally, cut the ascending aorta near the heart. Prepare one milliliter of 4%paraformaldehyde solution in a 1.5 milliliter centrifuge tube in advance.
Place the aorta into the prepared tube, and fix it at room temperature for at least 24 hours. Next, transfer the aorta to a Petri dish containing PBS to prevent it from drying out. Under a stereo microscope, carefully remove any remaining adventitial fat using forceps and spring scissors to reduce interference in plaque quantification.
Using spring scissors, carefully cut open the aorta along its inner longitudinal axis. Sequentially, cut open the three branches of the aortic arch along the lateral side to the level of the curvature of the aortic arch, allowing it to spread out fully. At this stage, either proceed with Oil Red O staining, or store the aorta in 4%paraformaldehyde for future analysis.
Place the cut open aorta into a six well plate. Add one milliliter of sterile double distilled water to each well and wash for five minutes on a shaker. Place the six well plate in a fume hood and air dry for 20 minutes until no visible watermarks remain.
Add one milliliter of freshly prepared Oil Red O working solution to each well. After shaking the plate for 20 minutes, remove the Oil Red O solution from the wells. Add two milliliters of sterile double distilled water to each well and wash for five minutes.
After three washes, keep the aorta in double distilled water. Transfer the aorta onto a slide, and put a black rubber pad underneath the slide to enhance contrast. Spread the aorta out while viewing under the stereo microscope.
Now, place the slide on a white paper to further enhance contrast. Position a ruler beside the slide and acquire a photograph using a camera. Store the treated aorta in a 1.5 milliliter centrifuge tube containing one milliliter of PBS to keep it moist.
The body weights of LDLR knockout mice fed with a Western style diet increased significantly compared to those fed with the chow diet. Plasma triglyceride and total cholesterol levels were significantly higher in the Western style diet group than in the control group. Aortic Oil Red O staining showed severe lipid accumulation in the arteries of LDLR knockout mice fed with the Western style diet compared to those on a chow diet.
Increased lipid deposits correlated with more severe atherosclerotic lesions. The aortic root lesional area and necrotic core were significantly larger in Western diet fed mice compared to chow diet fed mice. Quantification of the aortic root lesional area and necrotic area confirmed that the Western diet exacerbated atherosclerotic plaque formation in LDLR knockout mice.
