Abstract
Abstract: :
Purpose: There are distinct 24–hour rhythms of total protein level in aqueous humor in diurnally active humans and in nocturnally active laboratory rabbits. The rabbits’ pattern showing a diurnal–to–nocturnal decrease of total aqueous humor protein is different from the pattern in humans (aqueous flare). The diurnal/nocturnal change pattern of aqueous humor protein in nocturnally active laboratory mice is unknown. The present study compares total protein concentrations in aqueous humor at various times of the day in the C57BL/6 mouse strain. Methods: Thirty–two young adult C57BL/6 mice (about 25 g) of either sex were entrained to a daily 12–hour diurnal/light (6 AM to 6 PM) and 12–hour nocturnal/dark cycle for at least two weeks. Eight mice were sacrificed at each of 4 time points: 8 AM, noon, 8 PM, and midnight. In each mouse, micro–liter specimens of aqueous humor were collected from both eyes and mixed. Concentrations of total protein in the mixed aqueous humor samples were determined using the Bradford method in microplates. One–way ANOVA was used to determine a statistical difference in the protein concentrations among the 4 time points. Post–hoc multiple comparisons were performed using the Bonferroni t–test. Results: Levels of aqueous humor protein were 0.516±0.049 mg/ml (mean±SEM, N=8), 0.490±0.042 mg/ml, 0.306±0.033 mg/ml, and 0.330±0.041 mg/ml at 8 AM, noon, 8 PM, and midnight, respectively. The protein level at 8 AM was significantly higher than the levels at 8 PM and midnight. The protein level at noon was significantly higher than the level at 8 PM. Conclusions: Total protein level in aqueous humor is significantly higher during the early diurnal period than during the early nocturnal period in C57BL/6 mice. This diurnal/nocturnal pattern is similar to the pattern observed in laboratory rabbits. There is likely a significant secretion of proteins into aqueous humor during the early diurnal period. The observed reduction of aqueous humor protein level during the early nocturnal period also suggests a corresponding increase in the rate of aqueous humor formation.
Keywords: aqueous • protein purification and characterization • circadian rhythms