2.5. Examinations, measurements, and microscopic studies

The body weight of each animal was measured every 3 d whilst they were on the lithogenic diet. At the end of each week until the tenth week of the diet, animals were killed after fasting for 12 h, cholecystectomies were performed (six mice from each group at each time point) and gallbladder bile was collected through a small hole made at the fundus of the gallbladder with an 18-gauge needle. The bile was observed immediately using polarizing light microscopy for the presence of mucin strands, liquid and solid crystals, and sandy and true gallstones. Mucin was observed as non-birefringent amorphous strands. Arc-like, helical, tubular crystals, plate-like cholesterol monohydrate (ChM) crystals as well as small, aggregated, and fused liquid crystals were defined according to a previous study by Wang and Carey (1996). Sandy stones, which were irregularly shaped, were easily disintegrable agglomerates of ChM crystals embedded in mucin gel. As visualized under the microscope, individual cholesterol monohydrate crystals projected clearly from the edges of sandy stones, macroscopically displaying a yellow color. True gallstones were hard, ball-like objects with smooth curved surfaces. Because of scattered and absorbed light, they were opaque and black in color when observed using polarizing light microscopy.

Blood was collected from mouse orbital sinus just prior to euthanization and serum was obtained from the whole blood sample after centrifugation at 3300g for 20 min at 4 °C. Bile was stored at −20 °C, and serum samples, livers, and gallbladders were stored at −80 °C until further analyses.

Serum cholesterol levels were determined using a mouse cholesterol kit. Serum T3, thyroxine (T4), and thyroid-stimulating hormone (TSH) levels were determined using mouse enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s protocols.

RNA was extracted from frozen livers using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcriptions were carried out with the iScript cDNA synthesis kit (Bio-Rad, Berkeley, CA, USA), according to the manufacturer’s instruction. Real-time polymerase chain reaction (RT-PCR) was performed with iQ™ SYBR Green Supermix (Bio-Rad, Berkeley, CA, USA) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Eight crucial hepatic lithogenic genes (Trβ, Rxr, Lxrα, Rxr, Cyp7α1, Fxr, Abcg5, Abcg8) that have been reported to be associated with cholesterol gallstone susceptibility were studied to explore the genetic effects on gallstone formation in mice with different thyroid hormone levels.