Alzet model 2004

Male ApoE^−/− mice on a C57BL/6J background strain were housed under barrier conditions with food and water. Thirty-six mice (24-week-old male) were randomly divided into 3 groups (n = 12, each): AngII alone group (infusion of AngII, 1000 ng/kg/min, Sigma, St. Louis, MO, USA), AngII + Cur group (100 mg/kg/d, Sigma-Aldrich, St. Louis, MO, USA), and control group (infusion of normal saline). The mice were anaesthetized by intraperitoneal injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Miniosmotic pumps (Alzet Model 2004, Durect Corp, Cupertino, CA, USA) were implanted subcutaneously in mice to deliver AngII (1000 ng/kg/min) or saline for 28 days according to the literature as described previously [5 (link), 8 (link), 12 (link)]. Curcumin was dissolved in 1% carboxymethyl cellulose and was applied by daily oral gavage in AngII + Cur group. The curcumin treatment was initiated 1 week before AngII infusion and lasted for 28 days. At the end of experiment, the mice were anesthetized with xylazine 20 mg/kg and ketamine 100 mg/kg and the aortas were isolated for pathology examination.

Male apoE (-/-) mice (Jackson Laboratories, Bar Harbor, ME) were housed in a pathogen-free animal facility, fed normal chow, and kept on a 12-hour day/night cycle with ad libitum access to food and water. At 10 weeks of age, an osmotic pump (Alzet model 2004, DURECT Corporation, Cupertino, CA) was subcutaneously implanted under anesthesia in the back to continuously infuse AngII (Sigma-Aldrich, St. Louis, MO) at a dose of 1000ng/Kg/min for 4 weeks [9 (link), 10 (link)]. Analgesic was injected subcutaneously prior to the pump implantation to alleviate post-procedure pain. Mice were observed post implantation for recovery from anesthesia and pump implantation under a heat lamp. Mice were checked daily for the duration of the experiment. For the immunization study, a separate group of mice were subcutaneously immunized with p210/cBSA/Alum conjugate in the dorsal area at 7 weeks of age, followed by a booster at 10 and 12 weeks of age [7 (link), 8 (link)]. Mice receiving cBSA/alum or PBS served as control groups. AngII pump implantation was performed on the mice at 10 weeks of age, as described above. Mice were sacrificed at 14 weeks of age. The method of euthanasia was overdose of inhalational anesthesia followed by pneumothorax to assure death.

Male ApoE-KO mice received saline or angiotensin II (Ang II, 1000 ng/kg/min) at 9–10 weeks old via mini-osmotic pumps (Alzet Model 2004, DURECT Corp.) for 28 days to induce AAA. During the 28 days, ApoE-KO mice were fed with HFD (0.2% (w/w) cholesterol and 20% (w/w) fat, Test Diet AIN-76A, Hubbard, OR). WT mice were fed with a normal chow diet (5% fat, Labdiet 5001).

Male ApoE^−/− mice (8-12 weeks old) were from Vital River Laboratories (Distributor of Jackson Laboratory, Beijing, China) and housed under specific pathogen-free conditions on a 12-hr light/12-hr dark cycle with food and water freely available. All ApoE^−/− mice were fed with a high fat diet (0.25% cholesterol and 15% cocoa butter) and divided randomly into three groups (n = 30 in each group). The control group received continuous subcutaneous infusion of saline for 28 days via an osmotic pump (Alzet, model 2004, Durect Corp., Cupertino, CA, USA), the AngII group received infusion of AngII (1.44 mg/kg/day, Lintai Biological Technology Company, Xi’an, China) plus intraperitoneal injection with 4% ethanol and the melatonin group received infusion of AngII (1.44 mg/kg/day) plus intraperitoneal injection of melatonin in 4% ethanol (30 mg kg⁻¹ day⁻¹, Sigma, St Louis, MO, USA) [31 ]. After 28 days of infusion, all ApoE^−/− mice were euthanized and their aortic tissues removed for further analysis. The animal experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shandong University.

Following 2 days of baseline pressure recording, the 11 remaining mice were randomized to receive either AngII (1,000 ng·kg^-1·min^-1; n = 6) or saline (n = 5) via continuous infusion with subcutaneous osmotic mini-pumps (Alzet model: 2004; DURECT Corporation, Cupertino, CA) for 28 days. Pumps were implanted under isoflurane anesthesia, as described previously[12 (link)]. All efforts were made to minimize suffering. Telemetry data were collected continuously for the duration of the 28-day infusion.