Mice, Induction of Atherosclerosis, and Tissue Preparation

All procedures performed on mice were in conformance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and approved by the Institutional Animal Research and Use Committee of Shantou University.

C57BL/6 WT mice were bought from Vital-River (Beijing, China). ApoE^–/– mice were purchased from Slac (Shanghai, China), while TP^–/– mice were produced as described previously (Li et al., 2017). ApoE^–/– and TP^–/– mice (both were of C57BL/6 genetic background) were first cross-bred to yield apoE^±/TP^± and then to apoE^–/–/TP^–/– mice. Genotyping was performed by PCR of tail biopsy described previously (Li et al., 2016, 2017).

Mice were housed in the Animal Center of Shantou University Medical College (SPF grade; Temp: 20–26; Humility: 40–70%) with standard day/night (12/12) cycles and free accesses to food and drink. Atherosclerosis was induced in male apoE^–/– or apoE^–/–/TP^–/– mice by 12 week of atherogenic diet containing 1.25% cholesterol (Research Diets Inc., New Brunswick, NJ, Canada), starting from the beginning of 9th week of age. Male WT or TP^–/– mice of comparable age fed on normal chow were used as non-atherosclerotic negative controls to indicate the abnormality developed in atherosclerotic conditions. Mice were killed by CO₂ inhalation. With the help of a binocular microscope, aortas were isolated and dissected free of adherent tissues. For functional studies, the abdominal aorta was examined for spots of atherosclerotic lesions (the outer layer shows loss of gloss with grayish appearance under the light microscope) and vessels were cut into 1-mm long rings. Rings with atherosclerotic lesions were used to study vasomotor reactions of atherosclerotic conditions.