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Chi Hoang Viet Vu, Motoko Kawashima, Kokoro Sano, Wataru Nakamura, Takahiro J Nakamura, Kazuo Tsubota; Circadian clock regulates tear secretion in the lacrimal gland. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4930.
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Tear film parameters were observed to have circadian variations, however the role of clock genes in controlling tear secretion has not been elucidated. Previously, we showed circadian variations of clock genes expression in the lacrimal gland (LG) of mice. In this study, we investigated the effects of knocking out core clock genes, Cryptochromes, on tear secretion and mRNAs expression of clock genes in the LG of mice.
Male Cry1-/- Cry2-/- mice were maintained under a 12 hours of light and 12 hours of darkness (LD) with the light on at 08:00 and light off at 20:00, ad libitum access to food, prior at least 2 weeks before the beginning of the experiment. Experiments were performed every 4 hours for 24 hours starting from zeitgeber time (ZT) 0 which was defined as the time of light onset. For constant darkness (DD) studies, after being kept at the LD cycle for at least 2 weeks, animals were transferred to DD and kept under DD condition for 48 hours. Samples were collected under dim red light and at the same time as those collected in LD cycle. Thirteen Cry 1-/- Cry 2 -/- mice (12-18 weeks old, n = 7 for LD group and n = 6 for DD group) were used for measuring tear volume by cotton thread test for 30s. Thirty three Cry 1-/- Cry 2 -/- mice (9-12 weeks old, n = 2-3 per time point) were used for collecting lacrimal gland samples. Total RNA was extracted from the LGs of mice, then real time PCR was performed for major clock gene mRNAs including Bmal1, Clock, Per1, Per2 and Per3.
In Cry 1-/- Cry 2 -/- mice, disruption in diurnal and circadian variations of tear secretion were observed in both LD and DD conditions (one way ANOVA, F [5, 78] = 20.253, P = 0.211 for LD condition; F [5, 66] = 1.162, P = 0.337 for DD condition. Expression of clock genes in the lacrimal gland also showed complete loss of oscillation regardless of environmental light conditions (LD condition; one-way ANOVA, Bmal1: F [5, 9] = 0.662, P = 0.661; Clock: F [5, 9] = 0.679, P = 0.651; Per1: F [5, 9] = 0.317, P = 0.891; Per2: F [5, 9] = 0.302, P = 0.900; Per3: F [5, 9] = 0.409, P = 0.831; DD condition; one-way ANOVA, Bmal1: F [5, 12] = 1.661, P =0.692; Clock: F [5, 12] = 0.812, P = 0.563; Per1: F [5, 12] = 0.812, P = 0.563; Per2: F [5, 12] = 0.927, P = 0.497; Per3: F [5, 12] = 1.128, P = 0.397).
These results suggest that the circadian clock regulates tear secretion in the LG.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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