Circadian Intraocular Pressure Rhythm Is Generated by Clock Genes

Abstract

purpose. The present study in a mouse model was undertaken to reveal the role of the circadian clock genes Cry1 and Cry2 in generation of 24-hour intraocular pressure (IOP) rhythm.

methods. IOP was measured at eight time points daily (circadian time [CT] 0, 3, 6, 9, 12, 15, 18, and 21 hours), using a microneedle method in four groups of C57BL/6J mice (groups 1 and 3, wild-type; groups 2 and 4, Cry-deficient [Cry1 ^−/− Cry2 ^−/−]). During the IOP measurements, mice in groups 1 and 2 were maintained in a 12-hour light–dark cycle (LD), whereas mice in groups 3 and 4 were kept in a constant darkness (DD) that started 24 to 48 hours before the measurements. Circadian IOP variations in each group were evaluated by one-way analysis of variance (ANOVA) and Scheffé tests.

results. In wild-type mice living in LD conditions, pressures measured in the light phase were significantly lower than those in the dark phase. This daily rhythm was maintained under DD conditions with low pressure in the subjective day and high pressure in the subjective night. In contrast, Cry-deficient mice did not show significant circadian changes in IOP, regardless of environmental light conditions.

conclusions. These findings demonstrate that clock oscillatory mechanisms requiring the activity of core clock genes are essential for the generation of a circadian rhythm of intraocular pressure.

Previous studies have demonstrated that intraocular pressures (IOPs) of rabbits, rats, chicks, marmosets, and mice have a biphasic pattern when the animals are maintained in a 12-hour light–dark (LD) cycle. ^{1 2 3 4 5} Because this IOP rhythm in rabbits and rats is maintained in an environment of constant darkness, it has been suggested that day–night IOP variations are related to circadian rhythms in these species. ^{1 2}  

Several factors have been reported to be associated with 24-hour IOP rhythm. Sympathetic nerves are thought to play an important role because neuronal impairment abolishes day–night variations in IOP. ^{6 7 8 9} Other studies have shown that aqueous humor concentrations of norepinephrine and melatonin have 24-hour profiles that are synchronized with changes in IOP. ^{7 9 10} Furthermore, several adrenergic receptors were also involved in the nocturnal IOP elevation in rabbits based on studies in which topical selective adrenergic agents were used. ⁹ However, the molecular mechanism that generates 24-hour IOP rhythm remains unknown. 

In mammals, circadian oscillation is driven by a transcription–translation-based core feedback loop of a set of clock genes that is dynamically regulated by clock proteins. ^{11 12} Previous studies have shown that the absence of Cry1 and Cry2 genes, members of the family of plant blue-light receptors (cryptochromes), in mice results in complete loss of behavioral rhythm. ^{13 14 15} A previous study investigating the effects of lesions of the suprachiasmatic nuclei indicated that the central circadian clock is involved in 24-hour IOP rhythm in rabbits. ¹⁶ Therefore, we hypothesized that if the IOP rhythm is controlled by the circadian clock, the rhythm would be abolished in animals genetically deficient in these two clock genes. To examine this hypothesis, we measured circadian IOP changes in Cry-deficient (Cry1 ^{− / −} Cry2 ^{− / −}) mice and compared results with those for wild-type mice. 

Materials and Methods

Animals

Four groups of C57BL/6J mice (groups 1 and 3, wild-type; groups 2 and 4, Cry-deficient [Cry1 ^{− / −} Cry2 ^{− / −}]) were used in this study. Cry-deficient mice were generated as described previously. ¹⁷ Wild-type mice were obtained from a local supplier (Charles River Japan, Inc., Yokohama, Japan). Mice were housed at 23°C with food and water ad libitum. All experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Initial animal age in each of the four groups ranged from 8 to 12 weeks. Body weight ranged from 25 to 37 g in wild-type mice and 19 to 27 g in Cry-deficient mice at the time IOP measurements were begun. 

All mice were maintained in a 12-hour LD condition at least 2 weeks before IOP measurements were begun. During the period of IOP measurements, mice in groups 1 and 2 remained in LD conditions, whereas mice in groups 3 and 4 were kept in constant darkness (DD) conditions that started 24 hours before the beginning of measurements. 

IOP Measurements

Mice (wild-type, n = 80; Cry-deficient, n = 60) were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (9 mg/kg). IOP was measured by the microneedle method as described previously. ¹⁸ Briefly, a glass microneedle was made with a pipette puller and connected to a pressure transducer that was inserted into the anterior chamber through the cornea using a micromanipulator. An IOP measurement for each mouse was obtained for one eye selected at random 4 to 6 minutes after administration of anesthesia when the IOP under anesthesia remained stable. ¹⁸  

When IOP measurements were to be performed repeatedly in the same mouse, the interval of measurements was at least 1 week, to avoid any effect of general anesthesia, and the eye to be used for measurement was selected alternately to reduce the effect of prior measurement in the same eye. The mean number of IOP measurements in the same eye was 2.41 ± 1.59 (range, 1–6). Our preliminary data indicated that IOPs were not significantly different between eyes in the same mouse and did not vary significantly in repeated measurements (data not shown). 

IOP measurements at circadian time (CT) 0, 3, 6, and 9 hours (light phase) in mice maintained under LD conditions were performed with a fiber-optic illuminator, whereas those obtained at CT 12, 15, 18, and 21 hours (dark phase) under LD conditions and at all time points for mice maintained under DD conditions were performed under dim red light to minimize any interference to the dark condition. 

The number of IOP measurements for wild-type mice at each time point was 23 under LD and 13 under DD conditions compared with 10 under LD and 10 under DD conditions for Cry-deficient mice. 

Statistical Analysis

IOP data are expressed as the mean ± SEM. Circadian IOP variations in each group were evaluated by one-way ANOVA and Scheffé tests. Comparisons of IOPs at each time point between groups 1 and 2 or groups 3 and 4 were performed by the Mann-Whitney test. P < 0.05 was considered statistically significant. 

Results

In wild-type mice, IOPs measured under LD conditions showed a significant 24-hour rhythm. IOPs were low during the light phase and high during the dark phase (Fig. 1) . Furthermore, the biphasic IOP pattern in wild-type mice was maintained under DD conditions (Fig. 2) . In contrast, Cry-deficient mice did not show significant circadian IOP changes under either LD or DD conditions (Figs. 1 2) . 

When IOPs measured at each time point were compared between wild-type and Cry-deficient mice, the IOPs of Cry-deficient mice under LD conditions were significantly higher in the light phase (CT 3, 6, and 9 hours) and significantly lower in the dark phase (CT 15 hours) than IOPs obtained from wild-type mice (Fig. 1) . Under DD conditions, IOPs of Cry-deficient mice were lower at CT 12, 15, and 18 hours than those of wild-type mice. 

Discussion

The present study was undertaken to reveal the role of endogenous clock genes in the generation of daily IOP rhythm in mice. To achieve this, we used arrhythmic Cry-deficient (Cry1 ^{− / −} Cry2 ^{− / −}) mice, which show complete loss of oscillation of clock genes in the suprachiasmatic nucleus (SCN) and peripheral organs. ^{19 20} Before examining the IOP of these arrhythmic mice, we characterized the circadian nature of IOPs in wild-type mice, which are nocturnally active, because it had not been fully addressed in this species. 

Only one report deals with this subject, and those authors demonstrated the presence of a 24-hour IOP rhythm under LD conditions and loss of this rhythm under constant exposure to light (LL). ⁵ This result raises two possibilities: Either the IOP rhythm is raised by environmental LD conditions, or the rhythm is raised by an endogenous clock that is masked by LL conditions. In the present study using wild-type mice, we found that IOPs measured during the light phase were significantly lower than those obtained during the dark phase under LD conditions. This finding is quite similar to that reported in National Institutes of Health (NIH) Swiss white mice using the same procedure for IOP measurement. ⁵ Under constant dark conditions, we found that biphasic IOP variations were maintained in wild-type mice. This result demonstrates that the daytime-low and nighttime-high pattern of IOP rhythms in mice is generated by the circadian clock, as has been reported in rats and rabbits. ^{1 2 16} To the best of our knowledge, this is the first report that demonstrates that IOPs are generated by a circadian clock in the mouse, a species in which genetic and molecular analyses are useful. 

Although mice may allow researchers to investigate the mechanism underlying IOP control at the molecular level through molecular targeting techniques, accurate IOP measurement in mice has been problematic, because the eye is far smaller than that of humans. To date, several noninvasive and invasive methods of IOP measurement in mice have been reported. ^{5 21 22 23 24} Noninvasive methods allow repeated IOP measurements in the same eye and require no general anesthesia, but they demand highly skillful techniques and lack direct calibration of the pressure. Invasive methods, including the microneedle method, are supposed to be more accurate than noninvasive methods because the pressure can be directly calibrated during the IOP measurement. However, the interval between repeated IOP measurements by the microneedle method should be at least 1 week, to allow healing of the corneal perforation. ⁵ In this study, we waited at least 2 weeks to measure IOP repeatedly in the same eye, to avoid any effect of prior measurement, and IOPs obtained from the same eye were not significantly different in repeated measurements. 

To reveal the relationship between IOP rhythm and the core oscillating genes of the biological clock, we investigated 24-hour profiles of IOP in Cry-deficient mice because they completely lack a circadian pacemaker. ^{13 19} In this study, IOP measurements of Cry-deficient mice were constant throughout the day regardless of environmental light conditions. The results of the present study indicate that Cry genes are involved in generating the IOP circadian rhythm and that IOP is one of various physiological and behavioral systems that are controlled by an internal, self-sustaining molecular oscillating mechanism. 

 Submitted for publication February 21, 2006; revised April 26, 2006; accepted July 25, 2006.

 Disclosure: A. Maeda, None; S. Tsujiya, None; T. Higashide, None; K. Toida, None; T. Todo, None; T. Ueyama, None; H. Okamura, None; K. Sugiyama, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Twenty-four-hour IOP pattern of C57BL/6J mice maintained in the LD. In contrast to wild-type mice, which showed a biphasic IOP pattern, Cry-deficient mice had no significant IOP changes. *Time points when IOP of wild-type and Cry-deficient mice were significantly different. Data are expressed as the mean ± SEM.

Figure 2.

 

Circadian IOP pattern in C57BL/6J mice maintained in the DD cycle. IOP measurements were performed after mice were kept for 24 to 48 hours in darkness. Wild-type mice maintained the biphasic IOP pattern under DD conditions, suggesting the presence of an IOP circadian rhythm in wild-type mice. Cry-deficient mice had no significant circadian IOP changes. *Time points when IOP of wild-type and Cry-deficient mice were significantly different. Data are expressed as the mean ± SEM.

The authors thank Makoto Aihara and Takashi Ota (Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan) for technical advice in measurement of IOP.