Atorvastatin

Sixty-week-old male apoE^−/− mice with a C57Bl/6 J background were purchased from Nanjing Biomedical Research Institute (Nanjing University, Nanjing, China) and fed with regular rodent chow (containing 20.5% protein, 4.68% fat, 1.23% calcium, and 0.91% phosphorus). Twenty-four mice were randomly allocated to two groups. Twelve mice (atorvastatin group) were orally gavaged with 10 mg kg⁻¹ day⁻¹ atorvastatin (Pfizer, New York, USA) dissolved in DMSO (dimethyl sulfoxide) for 12 weeks. The other mice served as the control group and received an equivalent amount of DMSO as placebo. The mice were maintained in groups under a strict 12 h light/dark cycle in a temperature-controlled environment (25 °C) with free access to food and water. The procedures involving animals and their care were conducted in conformity with the guidelines of the department of laboratory animal science of Fudan University. All animal experiments were conducted with the approval of the Animal Resources Committee of Huadong Hospital, Fudan University (2012-03-FYS-GJJ-01).

Atorvastatin was a gift of Pfizer Inc. Atorvastatin was prepared in PBS and 5% (vol/vol) ethanol (pH 7.6). Triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical Co. (St. Louis, MO) and 3-diaminobenzidine tetrahydrochloride from DAKO, Inc. (Carpinteria, CA). Rat anti-mouse neutrophil antibody was purchased from Serotec, Inc. (Raleigh, NC) and rabbit polyclonal antibody against a peptide corresponding to the 25 COOH-terminal amino acids of P-selectin was a gift from Dr. S. A. Green (Univ. of Virginia, Charlottesville, VA).

At week 8, The normal control group rabbits (n = 8) were fed on normal chow and received saline orally every day for 4 weeks. The experimental atherosclerotic rabbits have distributed among 3 groups with 8 rabbits in each group: the atherosclerosis model group received saline orally every day for 4 weeks; the desipramine group received desipramine (Sigma, MO, USA) at a dose of 4 mg/kg/day for 4 weeks; atorvastatin group received atorvastatin (Pfizer, NY, USA) at a dose of 2.5 mg/kg/day for 4 weeks. The calculation of the drug dose was based on the body surface area formula for therapeutic human dosing (desipramine of 200 mg/day and atorvastatin of 40 mg/day). Drugs were dissolved in saline and administrated by oral gavage. An equal volume of saline was administered in the normal and model groups. The study protocol was summarized in Fig 1A. The investigators who performed followed examinations such as lipid profile, ASM activities, and imaging, are blinded to the animal group.

As described in the previous study, we established an SDH rat model by subdural injection of autologous blood 10 (link). Rats were anesthetized with isoflurane (5% induction, 2% maintenance) and prepared by shaving the hair and disinfecting the scalp. A 1-cm incision along the midline exposed the dorsal skull, and a 1.5 mm diameter, 1 mm deep bone window was drilled 3 mm to the right of the bregma. The dura mater was carefully torn using microscopic tweezers, and 400 µL of autologous blood from the femoral vein was injected into the subdural space at a rate of 50 µL/min using a trace injection pump (RWD Bioscience Co., Ltd., Shenzhen, China) equipped with 14G indwelling needles. The indwelling needle was left in place for 10 min to prevent blood leakage before removal. The bone window was sealed with medical glue, and the scalp incision was disinfected, sutured, and postoperative pain was alleviated with intraperitoneal injection of ibuprofen (10 mg/kg). A sham group underwent the same surgical procedure without blood injection, and all surgeries were performed using sterile medical instruments.
For atorvastatin treatment, rats received daily intragastric administration of atorvastatin (Pfizer, US; 3 mg/kg, dissolved in saline). This dosage was consistent with our previous studies 23 (link), 34 (link). The control group received an equal volume of saline.

Atorvastatin was purchased from Sigma (USA) for cell experiments. The powder was dissolved into DMSO (Sigma-Aldrich, USA) at 50 mM. Atorvastatin was purchased from Pfizer for animal experiment. ERK^T185/Y187, CDK^T14 were obtained from Thermo Fisher Scientific. BRCA1^S1189 was purchased from Abcam. ERK, CDK1, and BRCA1 were obtained from Cell Signaling Technology.