4.4. Induction of Atherosclerosis in a Mouse Model

Eight-to-twelve-week-old ApoE^−/− mice (C57BL/6 background, Jackson Laboratory) were randomly assigned and intranasally administered vehicle (endotoxin-free water) or NaVO₃ (4 mg/kg) once weekly with or without intraperitoneal injection of NAC (150 mg/kg or 250 mg/kg) three times a week for 12 weeks. This method followed the guidelines of experimental atherosclerosis studies described in the AHA (American Heart Association) Statement [45]. Following this, the mice were killed, and blood samples were collected for vanadium, ROS, and cytokine analysis. Mouse urine was collected on week 4 and 6 for urine vanadium level measurements. The aortas were harvested for analysis of atherosclerotic lesion. In addition, the lung, kidney, liver, and heart were collected for histological determination. To be the more specific in terms of the measurement of the atherosclerotic area in mouse lesion sites from the ascending aorta to the end of the arch, we made three 4-µm sections on each slide, with a total of 100 slides in a sequential manner. We picked one slide from every 10 slides and 10 slides for each group to perform H&E (hematoxylin and eosin) or immunohistochemistry (IHC) staining. The mouse experiments were approved by the Institutional Animal Care and Use Committee, National Health Research Institutes.