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.
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