Analysis of Circadian Rhythm Gene Expression With Reference to Diurnal Pattern of Intraocular Pressure in Mice

Abstract

Citation: Dalvin LA, Fautsch MP. Analysis of circadian rhythm gene expression with reference to diurnal pattern of intraocular pressure in mice. Invest Ophthalmol Vis Sci. 2015;56:265756: -266356: . DOI:10.1167 PURPOSE. To determine the expression of circadian rhythm clock genes in the iris-ciliary body complex of mice and their association with the diurnal pattern of intraocular pressure (IOP). METHODS. Thirty wild-type C57BL/6 mice were acclimated to a 12-hour light-dark cycle. Intraocular pressure was measured with a rebound tonometer at six time points daily (circadian time [CT] 2, 6, 10, 14, 18, and 22 hours) for five consecutive days. On day 6, mice were euthanized at CT 2, 6, 10, 14, 18, and 22. Eyes were flash-frozen or fixed in 4% phosphate-buffered paraformaldehyde. Total RNA was extracted from the iris-ciliary body complex, and RNA expression of circadian rhythm genes Bmal1, Clock, Cry1, Cry2, Per1, and Per2 was assessed by quantitative real-time PCR. Fixed eyes were paraffin embedded, and immunohistochemistry was performed to localize corresponding proteins (BMAL1, CLOCK, CRY1, CRY2, PER1, and PER2). Linear regression analysis was performed to correlate gene expression with IOP. RESULTS. Intraocular pressure demonstrated a consistent circadian pattern. The clock genes Bmal1, Clock, Cry1, Cry2, Per1, and Per2 showed a circadian pattern of expression in the iris-ciliary body complex of mice. Bmal1, Clock, Cry1, Per1, and Per2 gene expression demonstrated statistically significant correlations with diurnal variations of IOP. BMAL1, CLOCK, CRY1, CRY2, PER1, and PER2 proteins were found to be expressed locally in the nonpigmented epithelium of the ciliary body. CONCLUSIONS. Expression patterns of candidate circadian rhythm genes correlates with the diurnal pattern variation of IOP in mouse eyes, indicating a possible mechanism of IOP regulation through these genes. Keywords: aqueous outflow, iris-ciliary body complex, clock genes, intraocular pressure, circadian rhythm E levated intraocular pressure (IOP) is a strong risk factor for primary open-angle glaucoma (POAG), and current standard of care focuses on the reduction in IOP as the primary modality to reduce optic nerve damage and prevent vision loss. 1 Characterization of molecular regulators of IOP would help identify novel molecules that can be used as therapeutic targets for the treatment of POAG. Intraocular pressure is the result of a balance between secretion and outflow of aqueous humor, a colorless fluid in the anterior segment of the eye. 2 Three variables are of interest in this balance: the rate of aqueous humor formation, the resistance to outflow, and the episcleral venous pressure. The rate of formation and resistance to outflow are the primary targets for current drug therapy aimed at lowering IOP. The ciliary body is responsible for aqueous humor production, primarily via active secretion from its nonpigmented epithelium. Aqueous humor secretion is known to vary in a circadian manner, decreasing by up to 50% during sleep. 8 Epinephrine may play a role in this change, but the mechanism behind decreased secretion remains poorly understood. 9 Outflow facility also decreases slightly at night. 10,11 However, the decrease in outflow does not appear to be significant enough to compensate for the decrease in aqueous secretion, and the cause of this decrease in outflow remains unknown. 10 While the decrease in aqueous humor secretion and the slight decrease in aqueous humor outflow suggest a nocturnal normality or slight decrease in IOP, multiple studies have repeatedly demonstrated a sinusoidal pattern of IOP that peaks at night when animals are maintained on a 12-hour light-dark cycle. 12-16 Interestingly, IOP is higher at night irrespective of diurnal or nocturnal habits of the animals, which suggests that IOP is regulated by light intensity and not by the level of activity. 17 This circadian pattern of IOP is abolished in the absence of a distinct light-dark cycle