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Jeffrey Dunmire, Lauren Dalvin, Rachida Bouhenni, Deepak Edward; Expression of Circadian Rhythm Genes in the Mouse Iris-Ciliary Body Complex. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1994.
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It is well known that intraocular pressure (IOP) exhibits a circadian rhythm. However, the molecular basis for this circadian pattern of IOP remains to be understood. We hypothesized that a local circadian clock exists in the iris-ciliary body complex, where clock gene expression may contribute to daily IOP variation. We investigated the temporal and spatial expression of circadian clock genes in iris-ciliary body of mice.
C57BL/6J mice were entrained to a 12-hour light-dark cycle for two weeks and then sacrificed at Zeitgeber Times (ZT) 2, 6, 10, 14, 18, and 22 (n=4 per group), where ZT 12 is the beginning of the dark cycle. Eyes were enucleated and either flash-frozen or fixed in 10% neutral-buffered formalin. Following dissection of iris-ciliary body tissues, RNA was extracted and assessed by real-time PCR in an array format profiling 84 circadian clock or clock-controlled genes. Immunohistochemistry (IHC) was performed on formalin fixed eyes to confirm protein expression and identify localization.
Among the 84 genes tested in the PCR array, 35 showed expression with varying degrees of circadian oscillation. The circadian clock genes Bmal1, Clock, Cry1, Cry2, Per1, Per2, and Per3 were all found to be rhythmically expressed. Expression levels were highest for Bmal1 and Clock, with peak expression between ZT 2-6, and their oscillation was antiphase to Cry1, Cry2, Per1, Per2, and Per3. Other well known clock-controlled genes that showed a clear pattern of rhythmic expression included Dbp, Rev-erbA-α, Rev-erb-β, Tef, and Rorc. The amplitude of oscillation was greatest for Dbp, with a 17-fold difference in expression between ZT 10 and ZT 22. IHC showed strong staining of CLOCK and BMAL1 at ZT 14 in the non-pigmented ciliary epithelium. Expression was also detected in the anterior surface of the iris, but to a much lesser degree. Protein localization was both nuclear and cytoplasmic, with increased distribution to the cytoplasm at ZT 14 compared with ZT 2.
The components of an intrinsic circadian clock were identified in the mouse iris-ciliary body complex with temporal changes in clock gene expression and immunolocalization of their protein products. This finding is a first step toward elucidation of clock-controlled pathways that may be involved in aqueous humor dynamics and an understanding of diurnal IOP variation.
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