Validation experiment designed to validate causal mechanisms targeting P2RY12 in ApoE-deficient mice. Primary outcome: lipid accumulation and VSMC-derived foam cell formation
This experiment investigated the role of P2RY12 receptor in atherosclerosis using ApoE-deficient mice fed a high-fat diet. The study examined how P2RY12 receptor inhibition affects lipid accumulation and VSMC-derived foam cell formation. Mice were treated with P2RY12 receptor inhibitors and assessed for atherosclerotic plaque formation, lipid levels, and cellular changes in vascular smooth muscle cells. The researchers used various staining techniques including Oil Red O, elastic van Gieson, and Masson trichrome to evaluate plaque composition and foam cell formation.
Animals: ApoE-/- mice (C57BL/6 background), 8-10 weeks old, n=15 per group (vehicle, clopidogrel 10mg/kg/day, ticagrelor 100mg/kg/day). Diet: High-fat diet (60% kcal from fat, Research Diets D12492) for 12 weeks. Treatment: Daily oral gavage starting week 0. Euthanasia at week 12. Controls: ApoE-/- mice on normal chow diet (n=10), WT C57BL/6 on HFD (n=10). Histology: (1) Aortic root serial sections (5μm), stain every 5th section. (2) Oil Red O for lipid area (% of total plaque). (3) α-SMA immunostaining for VSMC content. (4) BODIPY-cholesterol for foam cells. (5) Elastic van Gieson for elastin. (6) Masson trichrome for collagen. Plasma: Measure total cholesterol, LDL-c, HDL-c, triglycerides at weeks 0, 6, 12. Autophagy: Western blot aortic tissue for LC3-II/I ratio, p62 levels.
...Quantitative predictions: (1) Vehicle HFD group: aortic root plaque area 400,000-500,000 μm², 60-70% lipid content. (2) Clopidogrel: 25-35% reduction in plaque area, 15-20% reduction in lipid content. (3) Ticagrelor: 30-40% reduction in plaque area, 20-25% reduction in lipid content. (4) VSMC-derived foam cells (α-SMA+/BODIPY+ double positive) reduced by 40-50% in treatment groups. (5) LC3-II/I ratio increased 2-3 fold, p62 decreased 50-60% in treatment groups. (6) No significant change in plasma LDL-c levels across groups (validates mechanism is independent of systemic lipid lowering).
Primary endpoint: Plaque area reduction >25% in P2RY12 inhibitor groups vs vehicle (p<0.05, one-way ANOVA with Dunnett post-hoc). Secondary endpoints: (1) Foam cell reduction >30% (p<0.05). (2) Autophagy marker changes: LC3-II/I >1.5-fold increase AND p62 <50% decrease (p<0.05). (3) Effect size maintained when LDL-c is used as covariate (ANCOVA, p<0.05). Exclusion criteria: Mice with <300 mg/dL LDL-c at week 6 excluded from analysis.
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