Hamsters were sacrificed by CO2 asphyxiation at 22:00 on the last day, after all hamsters had been in darkness for at least 2 h. Blood samples were collected immediately from the heart after sacrifice,stored at 4?C for 30 min, and then centrifuged at 3000 rpm for 15 min at 4?C. Serum MT levels were estimated using an enzyme-linked immunosorbent assay (Shanghai Hengyuan Biological Technology Co., Ltd., H-40277, Shanghai, China). The HGs were removed, with lengths and weights then recorded. The HGs from five hamsters were fixed by immersion and processed for light and electron microscopy. The right HGs were immersed in 10% buffered formalin for paraffin section embedding, and the left HGs were immersed in glutaraldehyde-paraformaldehyde for transmission electron microscopy (TEM). The remaining HGs from 15 hamsters in each group were frozen in liquid nitrogen and stored in a refrigerator at −80℃ for subsequent Western blotting, real-time polymerase chain reaction (RTPCR), and immunofluorescence histochemical analyses. In each experiment, unilateral HGs from 10 hamsters were used for quantification. All procedures were carried out in accordance with approved guidelines.
