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Jeffrey Dunmire, Benjamin Shekhtman, Eric Mintz, Rachida Bouhenni; Light-Independent Expression of Circadian Rhythm Genes in the Iris-Ciliary Body Complex. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5825.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported rhythmic expression of circadian clock genes in the iris-ciliary body (I-CB) complex of mice entrained to 12-hour light-dark cycles (ARVO 2013). Although the components for an intrinsic circadian clock were detected in the I-CB, it remained unknown whether the oscillations of gene expression were endogenous or simply responding to the daily light-dark cycle. Here, we investigated the potential for a peripheral circadian clock in the I-CB that can operate independent of photic input.
C57BL/6J mice (n=18) were housed in constant darkness for two weeks and then sacrificed at circadian times (CT) 2, 6, 10, 14, 18, and 22 (n=3 per group). Eyes were enucleated, flash-frozen in isopentane, and stored at -80°C. Following dissection of I-CB tissue, RNA was extracted, reverse transcribed, and expression of Bmal1, Per1 and Per2 was assessed by quantitative real-time PCR using TaqMan primer-probe sets.
The circadian clock genes Bmal1, Per1 and Per2 were found to be rhythmically expressed in I-CB during constant darkness. One-way ANOVA showed that differences in expression among circadian times were significant for Bmal1 (p<0.001), Per1 (p<0.01), and Per2 (p<0.001). The amplitude of oscillation, as the maximum fold change in expression, was 5.19 +/- 0.93, 4.47 +/- 1.23, and 6.18 +/- 1.71 for Bmal1, Per1 and Per2, respectively.
A local circadian clock has been identified in the mouse I-CB that functions independent of any light cues. The oscillation of clock gene expression seen in constant darkness approximates the pattern previously observed in light-dark entrained animals. This finding suggests the possibility for an intrinsic circadian control mechanism that could affect aqueous humor dynamics and daily rhythms of IOP variation.
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