Male apoe mice

The studies were approved by the Cedars-Sinai Institutional Animal Care and Use Committee. Male apoE−/−mice were purchased from Jackson Laboratory at 6 weeks of age and housed in a specific pathogen-free facility, kept on a 12-h day/night cycle, and had unrestricted access to water and food.

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.

The research was approved by the Local Animal Ethics Committee of Southeast University. The housing and care of animals and all the procedures done in the study were performed in accordance with the guidelines and regulations of the local Animal Ethics Committee of Southeast University. PAR-2^-/- female mice (Jackson Laboratory, Bar Harbor, ME, United States), at least 5 generations backcrossed to C57BL/6, were outcrossed to ApoE^-/- male mice (Jackson Laboratory, Bar Harbor, ME, United States), which had been backcrossed 11 generations to C57BL/6. PAR-2 wild-type and PAR-2^-/- mice among ApoE^-/- mice were designated as Control and PAR-2^-/-, respectively. Male Control and PAR-2^-/- mice were fed a lard-containing diet comprising 21% lard and 0.15% added cholesterol for 12 weeks starting at 6 weeks of age. All mice were housed in the same room and exposed to the same light dark cycle.

WT and Apoe^−/− male mice, both of a C57BL/6J genetic background, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained on a 12 h light/dark cycle at room temperature. Animals were fed ad libitum with a STD (5.3% calories from fat; D.Rod18.H07, LASvendi GmbH, Soest, Germany) or HFD (60% calories from fat; TD 06414, Envigo Teklad, Barcelona, Spain) from weaning until euthanasia at 8 or 18 weeks. For each period of study, three groups (5-14 mice/group) were established: STD-fed WT, STD-fed Apoe^−/− and HFD-fed Apoe^−/−. Sacrifice of 16 h-fasted mice was performed after 8 or 18 weeks on the respective diets. All animal experimentation was conducted in accordance with the accepted standards of animal use approved by the Complutense University of Madrid Committee.