Cholesterol

Dlin-MC3-DMA (MC3) was purchased through BioFine International Inc. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was acquired from Avanti Polar Lipids. Cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) was purchased from MP Biomedicals and NOF America Corporation, respectively. Pierce D-Luciferin was purchased from Thermo Fisher Scientific. Firefly luciferase (L-7202), Cre (L-7211) and mCherry (L-7203) mRNA were all purchased from Trilink Biotechnologies.

Silastic capsules were utilized for sex steroid replacement. Capsules consisted of a 10 mm long section of Silastic tubing (Dow Corning, Midland, MI) filled with either 17β‐estradiol (Sigma‐Aldrich, St. Louis, MO), cholesterol (MP Biomedicals, Solon, OH), or a mixture of the two chemicals. Control animals received capsules filled with 100% cholesterol; experimental groups received capsules filled with a 10:90 mixture of 17β‐estradiol and cholesterol, a 50:50 mixture of 17β‐estradiol and cholesterol, or 100% 17β‐estradiol. Prepared capsules were sealed at both ends with weatherproof silicone caulk, washed in 70% ethanol, and rinsed with sterile water. Capsules containing cholesterol were stored at −20°C until use; capsules containing 100% 17β‐estradiol were stored at room temperature until use.
Capsules were surgically implanted between the skin and fascia on the dorsum of the animals. Briefly, a small incision was made on the dorsolateral aspect of the neck. Hemostats were used to create a pocket between the skin and muscle tissue and the capsule was placed inside the formed cavity. The skin incision was closed with a wound clip. All surgeries were performed under light isoflurane anesthesia and with sterile techniques as described above. All animals were allowed a 2‐day postprocedure recovery period to facilitate efficient release of the steroid from the capsule.