June 2010
Volume 51, Issue 6
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Physiology and Pharmacology  |   June 2010
Aqueous Humor Dynamics during the Day and Night in Juvenile and Adult Rabbits
Author Affiliations & Notes
  • Min Zhao
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Joseph J. Hejkal
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Carl B. Camras
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Carol B. Toris
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Corresponding author: Carol B. Toris, Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE 68198-5840; ctoris@unmc.edu
Investigative Ophthalmology & Visual Science June 2010, Vol.51, 3145-3151. doi:10.1167/iovs.09-4415
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      Min Zhao, Joseph J. Hejkal, Carl B. Camras, Carol B. Toris; Aqueous Humor Dynamics during the Day and Night in Juvenile and Adult Rabbits. Invest. Ophthalmol. Vis. Sci. 2010;51(6):3145-3151. doi: 10.1167/iovs.09-4415.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To determine the day and night differences in intraocular pressure (IOP), aqueous flow, outflow facility, uveoscleral outflow, and central corneal thickness (CCT) in juvenile and adult rabbits.

Methods.: Studies were performed on twelve 3-month-old and ten 12-month-old male New Zealand White rabbits. Daytime measurements were made between 9 AM (3 hours after lights on) and 3 PM, and nighttime measurements were made between 11 PM and 5 AM. IOP was measured by pneumotonometry and aqueous flow by fluorophotometry. Outflow facility was determined by both fluorophotometry and tonography. Uveoscleral outflow was calculated by the Goldmann equation. CCT was measured by ultrasound pachymetry. Repeated-measures ANOVAs and Student's two-tailed t-tests were used for statistical comparisons.

Results.: When nighttime versus daytime readings were compared, IOP, aqueous flow and uveoscleral outflow were higher, fluorophotometric outflow facility was lower, and CCT was thinner in both age groups. When the juvenile rabbits were compared to adult rabbits, IOP was lower, aqueous flow and uveoscleral outflow were higher, and fluorophotometric outflow facility and CCT were not different during the day or night. Tonographic outflow facility did not change in a 24-hour period in the juvenile rabbits.

Conclusions.: The increased IOP at night in rabbits can be explained mainly by a decrease in outflow facility. An increase in aqueous flow at night is counterbalanced by an increase in uveoscleral outflow. Although the rates of aqueous flow and uveoscleral outflow slow with maturity, their relative day/night differences remain the same.

Intraocular pressure (IOP) is not constant but rather varies during a 24-hour period and is predictable on a population basis. All animals, including rabbits and humans, in which IOP has been studied, exhibit distinct circadian rhythms. 116 During the night, IOP peaks in rabbits, 19 when they are generally most active, 17 and it is at its lowest in humans when they are generally least active (provided all measurements are taken with the person seated). 1016  
What causes these circadian rhythms in IOP is not entirely clear, but it relates to circadian changes in aqueous humor dynamics. The various parameters of aqueous humor dynamics include aqueous flow, outflow facility, uveoscleral outflow, and episcleral venous pressure. Regarding circadian rhythms, much is known about inflow but very little is known about outflow. It is well established that throughout a 24-hour period, aqueous flow is greatest when IOP is greatest. The peak occurs at night in rabbits 18,19 and during the day in humans. 20,21 Outflow has not been studied at night in rabbits, but considering that outflow facility in healthy humans may decrease during the sleep period (nighttime) compared with the active period (daytime) (Fan S, et al. IOVS 2008;49:ARVO E-Abstract 1568), in the present study, we investigated whether outflow facility also decreases during the sleep period (daytime) in rabbits. Episcleral venous pressure has been shown to vary considerably throughout the day in humans, especially with postural changes. 22 Episcleral venous pressure has not been studied extensively in conscious rabbits due to the difficulty in making accurate measurements, but since a rabbit's posture changes relatively little throughout a 24-hour period, published values were used in the present study and were assumed to remain relatively stable. Uveoscleral outflow has not been studied at night in rabbits, but in one study in humans (Fan S, et al. IOVS 2008;49:ARVO E-Abstract 1568), no significant night/day variation was noted, suggesting that uveoscleral outflow does not contribute to circadian rhythms of IOP, at least in humans. In the present study, we investigated whether uveoscleral outflow shows the same lack of circadian change in rabbits. 
One additional factor with significant circadian changes that should be considered in the study of aqueous humor dynamics is CCT. CCT has been found to be thicker during the sleep period in humans and cats, 2326 and it reportedly affects the tonometric measurement of IOP, with relatively higher IOPs in eyes with thicker corneas. 2735 In addition, both in children 36 and adults, 30,31,33,37 CCT is greater in patients with ocular hypertension than in healthy subjects. For these reasons, day and night measurements of CCT were collected in rabbits in the present study. 
Characterization of the day- and nighttime differences in aqueous humor dynamics in animal models will help in understanding the mechanisms responsible for circadian IOP changes. Ultimately, this information should aid in the identification of abnormal rhythms of IOP and the development of more effective medications for lowering nocturnal IOP in glaucoma patients. As an initial step in this direction, we investigated circadian rhythms of aqueous humor dynamics in rabbits of two different ages. 
Materials and Methods
Animals
Studies were performed on 22 male New Zealand White rabbits. Twelve were 3 months of age (juveniles) and 10 were 12 months of age (adults) at the start of the study. The animals were conditioned on a 12-hour light-dark cycle, with lights on at 6 AM. The rabbits were conditioned to the light/dark cycle for a minimum of 2 weeks before the experiments started. All animal procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Nebraska Institutional Animal Care and Use Committee. 
Daytime Measurements
IOP and Aqueous Flow Measurements.
All the measurements and scans were performed in conscious rabbits gently restrained by hand. Daytime measurements were obtained in standard room lighting. IOP1 was measured at 9 AM with a pneumatonometer (model 30 Classic; Reichert Ophthalmic Instruments, Depew, NY) after topical application of 0.5% proparacaine HCl. For the daytime measurements of aqueous flow, 17 hours before the measurements commenced, 7 drops of 15-μL 10% fluorescein were topically applied to each eye of the juvenile rabbits at 5-minute intervals, and 5 drops were similarly applied to the eyes of the adult rabbits. As in older humans, older rabbits required fewer drops than younger rabbits to achieve a similar amount of fluorescein in the eye. 38 Daytime measurements were taken between 9 AM and noon. The fluorescence of the cornea and anterior chamber was measured in duplicate with a scanning ocular fluorophotometer (Fluorotron Master; OcuMetrics, Mountain View, CA). Scans were repeated at 45-minute intervals for four sets of scans. These data were used to determine aqueous flow (F a). 39  
Outflow Facility Measurements.
After the fourth set of fluorophotometric scans, IOP2 was measured at noon, followed by an intramuscular injection of acetazolamide (16 mg/kg) to reduce IOP by reducing aqueous flow. One, 1.75, and 2.5 hours later, fluorophotometric scans and IOP measurements were repeated. Fluorophotometric outflow facility (C fl) was calculated as the ratio of the change in aqueous flow to the change in IOP. 40  
On a separate day, outflow facility was evaluated again in the juvenile rabbits by the method of 2-minute constant-pressure tonography. 41 Conscious rabbits were placed in their normal, squatting posture. One tonometer probe (sensing probe) was applied horizontally to the central cornea to measure spontaneous IOP. A second probe (pressure applicator) was applied to the peripheral cornea to raise IOP by 7 to 25 mm Hg and maintain it at that level for 2 minutes. During this time, the sensing probe recorded IOP. At the end of the 2-minute test, the pressure applicator was removed, and spontaneous IOP was measured again. The Langham and Edwards 41 pressure–volume table for rabbits was used to determine the volume change corresponding to the pressure change. Tonographic outflow facility was calculated as the ratio of the change in aqueous volume to the change in IOP during the 2-minute measurement. Measurements were made at 5 AM, noon, 3 PM, and midnight. 
Uveoscleral Outflow Calculation.
Uveoscleral outflow (F u) was calculated by the modified Goldmann equation   where C was either fluorophotometric (C fl) or tonographic (C ton) outflow facility and P ev was episcleral venous pressure. When C fl was used, the IOP in the equation was IOP2; when C ton was used, IOP was the spontaneous pressure at the start of tonography. F u was calculated with either of two values of P ev (10 mm Hg 42 and 12 mm Hg 43 ). 
CCT Measurement.
CCT was measured in both groups of rabbits at 2:30 and 11:30 PM by ultrasound pachymetry (Pacscan Series 300; Sonomed, Inc., Lake Success, NY). The reported CCT is the average of five consecutive measurements taken in quick succession. Measurements were made on a day separate from the day of fluorophotometry. 
Nighttime Measurements
Nighttime measurements were performed within 2 weeks, either before or after the daytime measurements. All the measurements were made under dim red light, with the same instruments, procedures, and animals as in the daytime study. For the nighttime aqueous flow measurements, 5 drops of 15-μL 10% fluorescein were instilled in each eye of juvenile rabbits and 4 drops in adult rabbits at 5-minute intervals starting at 14 hours before the measurements began. The measurements commenced at 11 PM, and the study concluded at 5 AM. 
Statistical Analysis
Data are presented as the mean ± SEM. Repeated-measures analysis of variance (ANOVA) was used to analyze the effects of age and time on IOP, time effects on tonographic outflow facility, and uveoscleral outflow calculated from tonographic outflow facility. Student's two-tailed paired t-tests were used to compare, within groups, the times of measurement of aqueous flow, central corneal thickness, fluorophotometric outflow facility, and fluorophotometric uveoscleral outflow. Student's two-tailed unpaired t-tests were used to compare these parameters across age groups. Differences were considered statistically significant at P < 0.05. 
Results
Effects of Maturity
Both day- and nighttime IOPs in the juvenile rabbits were significantly (P < 0.01) lower than the corresponding IOPs in the adult rabbits (Table 1, Fig. 1). Day- and nighttime aqueous flows were lower (P < 0.001) in the adult rabbits than in the juvenile rabbits (Table 2, Fig. 2). Fluorophotometric outflow facility was not significantly different between the two age groups during the day and at night (P > 0.05, Table 2, Fig. 3). Uveoscleral outflow (calculated with the fluorophotometric outflow facility) significantly decreased with maturity during the day (P < 0.001) and night (P < 0.0001, Table 2, Fig. 4). Differences in CCT between adult and juvenile rabbits during the day and at night did not reach statistical significance (Table 3, Fig. 5; P = 0.08 and P = 0.06, respectively) when evaluated by unpaired t-test. However, when the same day- and nighttime CCT data were combined and analyzed by repeated-measures ANOVA, the effect of maturity on CCT was significant (P = 0.02). 
Table 1.
 
IOP during the Day and Night in Juvenile and Adult Rabbits
Table 1.
 
IOP during the Day and Night in Juvenile and Adult Rabbits
9 AM Noon 11 PM 2 AM
Juvenile (n = 12) 14.5 ± 1.0 15.1 ± 0.6 19.3 ± 0.6 16.3 ± 0.5
Adult (n = 10) 16.8 ± 0.6 15.9 ± 0.5 20.9 ± 0.5 17.7 ± 0.4
Figure 1.
 
IOP during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars represent ± SEM; repeated-measures ANOVA.
Figure 1.
 
IOP during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars represent ± SEM; repeated-measures ANOVA.
Table 2.
 
Daytime and Nighttime Aqueous Measurements
Table 2.
 
Daytime and Nighttime Aqueous Measurements
Juvenile (n = 12) Adult (n = 10) P P
9 AM-3 PM 11 PM-5 AM P * 9 AM-3 PM 11 PM-5 AM P *
F a 2.24 ± 0.08 2.53 ± 0.1 0.017 1.46 ± 0.13 1.71 ± 0.15 0.042 0.000 0.000
C fl 0.20 ± 0.03 0.10 ± 0.02 0.047 0.14 ± 0.02 0.10 ± 0.02 0.023 0.23 0.81
F u1 1.29 ± 0.14 1.87 ± 0.17 0.013 0.63 ± 0.14 0.99 ± 0.07 0.023 0.00 0.000
F u2 1.62 ± 0.11 2.07 ± 0.14 0.006 0.92 ± 0.13 1.18 ± 0.09 0.054 0.00 0.000
Figure 2.
 
Aqueous flow during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05 and ***P < 0.001, using paired or unpaired t-tests.
Figure 2.
 
Aqueous flow during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05 and ***P < 0.001, using paired or unpaired t-tests.
Figure 3.
 
Fluorophotometric outflow facility during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, paired t-test
Figure 3.
 
Fluorophotometric outflow facility during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, paired t-test
Figure 4.
 
Uveoscleral outflow calculated from fluorophotometric outflow facility and P ev = 10 mm Hg during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; paired or unpaired t-tests.
Figure 4.
 
Uveoscleral outflow calculated from fluorophotometric outflow facility and P ev = 10 mm Hg during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; paired or unpaired t-tests.
Table 3.
 
Day- and Nighttime CCTs
Table 3.
 
Day- and Nighttime CCTs
Juvenile (n = 12) Adult (n = 10) P P P §
2:30 PM 11:30 PM P * 2:30 PM 11:30 PM P *
CCT 369 ± 5 333 ± 5 0.000 384 ± 7 349 ± 6 0.000 0.08 0.06 0.02
Figure 5.
 
CCT during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. ***P < 0.001, paired t-test.
Figure 5.
 
CCT during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. ***P < 0.001, paired t-test.
Day- and Nighttime Measurements
Both groups of rabbits exhibited significant differences in day- and nighttime IOP, aqueous flow, fluorophotometric outflow facility, and uveoscleral outflow (calculated from fluorophotometric outflow facility). Compared with daytime (9 AM) IOP, nighttime (11 PM) IOP significantly (P < 0.0001) increased (24% in the adult rabbits and 33% in the juvenile rabbits; Table 1, Fig. 1). Nighttime aqueous flow increased by 17% (P < 0.05) in the adult rabbits and 13% (P < 0.05) in the juvenile rabbits compared with daytime values (Table 2, Fig. 2). Compared with daytime levels, fluorophotometric outflow facility decreased at night by 29% (P < 0.05) in the adult rabbits and 50% (P < 0.05) in the juvenile rabbits (Table 2, Fig. 3). Uveoscleral outflow, calculated with two published values of episcleral venous pressure (10 and 12 mm Hg) 42,43 and fluorophotometric outflow facility in the Goldmann equation, increased at night by 57% (P ev = 10 mm Hg; P = 0.02) and 28% (P ev = 12 mm Hg, P = 0.05) in the adult rabbits and by 45% (P ev = 10 mm Hg, P = 0.01) and 28% (P ev = 12 mm Hg, P = 0.006) in the juvenile rabbits (Table 2, Fig. 4). 
The four measurements (collected at 5 AM, noon, 3 PM, and midnight) of tonographic outflow facility in the juvenile rabbits were not different when evaluated by repeated-measures ANOVA (P > 0.05, Table 4, Fig. 6). Uveoscleral outflow calculated from tonographic outflow facility and each of two episcleral venous pressure values, exhibited no significant difference between daytime and nighttime (P > 0.05, Table 4). 
Table 4.
 
Tonographic Outflow Facility and Corresponding Uveoscleral Outflow in Juvenile Rabbits
Table 4.
 
Tonographic Outflow Facility and Corresponding Uveoscleral Outflow in Juvenile Rabbits
Time 5 AM Noon 3 PM Midnight P
C ton 0.18 ± 0.02 0.19 ± 0.03 0.15 ± 0.03 0.24 ± 0.02 0.279
F ut1 0.79 ± 0.35 0.98 ± 0.27 1.03 ± 0.31 −0.10 ± 0.28 0.088
F ut2 1.15 ± 0.31 1.36 ± 0.24 1.32 ± 0.27 0.39 ± 0.24 0.082
Figure 6.
 
Tonographic outflow facility during the day and night in juvenile (3-month-old) rabbits. Vertical bars, ±SEM.
Figure 6.
 
Tonographic outflow facility during the day and night in juvenile (3-month-old) rabbits. Vertical bars, ±SEM.
The 2:30 PM measurement of CCT was significantly higher than the 11:30 PM measurement in both the juvenile and adult rabbits (P < 0.001, Table 3, Fig. 5). 
Discussion
The existence of circadian variations in IOP in humans has been known for decades, 44,45 and it is generally accepted that there is a nocturnal increase in habitual IOP (the nocturnal supine IOP is higher than diurnal seated IOP). However, if the subject's posture is the same for both daytime and nighttime measurements, the literature is inconsistent as to whether IOP actually decreases 1016 or increases 46,47 at night. In either case, it is thought that much of the optic nerve damage in glaucoma occurs at night, due to IOP spikes and elevations or lower systemic blood pressure. 4850 The importance of understanding the mechanisms underlying the circadian rhythm of IOP is evident. 
Circadian rhythms of aqueous humor dynamics are responsible for the circadian rhythms of IOP. Reductions in outflow facility and uveoscleral outflow and increases in aqueous flow and episcleral venous pressure can increase IOP, and opposite changes in these parameters can reduce IOP. Aqueous humor production is reduced at night in healthy subjects, 20,44 in patients with open-angle glaucoma, 51 and in patients with normal-tension glaucoma. 52 In a recent study, it was reported that, in young, healthy adults in both seated and supine positions, nocturnal IOP was lower than diurnal IOP. Corresponding to the IOP decrease in the nocturnal period, aqueous flow is reduced, and outflow facility may be reduced as well. 16 Similar results were found in middle-aged, healthy volunteers (Fan S, et al. IOVS 2008;49:ARVO E-Abstract 1568). 
In the present study, both age groups of rabbits exhibited a nocturnal increase in IOP and a nocturnal increase in aqueous flow. These results are similar to those in previous studies 19,19,53 and indicate that rabbits, as nocturnal animals, exhibit circadian rhythms of IOP and aqueous humor dynamics that are 12 hours out of phase with those of humans. 20,44,51,52  
Of interest, the outflow facility measured by fluorophotometry was lower at night than during the day in both groups of rabbits, just as in humans (Fan S, et al. IOVS 2008;49:ARVO E-Abstract 1568). The rabbits were expected to have a rhythm of outflow facility that was 12 hours out of phase with that of humans, as found with IOP and aqueous flow. This expectation was not supported by the data. Rabbits have significant morphologic differences in their outflow pathways when compared with primates and humans. In humans, the three layers of the trabecular meshwork and inner wall of Schlemm's canal provide the major resistance to aqueous drainage. The outer layer of trabecular meshwork is connected to the tendons of the ciliary muscle. As a consequence, any mechanism that relaxes or contracts the ciliary muscle may modulate the space between the plates of the meshwork and regulate resistance to outflow. The rabbit does not have a true Schlemm's canal or highly developed trabecular meshwork and scleral spur, 54,55 and thus the functional relationship between the ciliary muscle and trabecular outflow mechanism is not strong. If ciliary muscle contraction regulated by an unknown mechanism is involved in the circadian change in trabecular outflow in humans, this apparently is not the case in rabbits, which suggests that the mechanisms controlling circadian changes in outflow facility are different in rabbits and humans. 
Outflow facility was evaluated further by a 2-minute tonography method in the juvenile rabbits. This method showed no significant difference among measurements taken at four time points throughout a 24-hour period. This finding is different from those with the fluorophotometric method. Of interest, a study of healthy volunteers found similar inconsistencies. 56 In the human study, the nocturnal fluorophotometric outflow facility was less than one half that measured during the daytime, but day versus night differences in tonographic outflow facility were not found. 56 Certain inherent differences in the two methods may account for these inconsistencies. First, the tonography technique assumes that the displacement of fluid from the eye across the trabecular meshwork by the weight of the tonometer probe is the only factor that accounts for the IOP decrease, but pseudofacility cannot be ruled out. In addition, scleral rigidity can confound the measurement of tonography. The fluorophotometric method avoids the problems of pseudofacility and scleral rigidity because IOP does not have to be elevated for the assessment. Second, the fluorophotometric method measures rather than assumes any change in aqueous flow, whereas, the tonographic method relies on reference tables 41 to estimate aqueous volume changes from the IOP changes. Third, tonography is a 2-minute measurement, whereas the fluorophotometric method provides an average value over the 5-hour assessment period. The fluorophotometric method assumes, rightly or wrongly, that all parameters in the Goldmann equation are relatively stable during the long assessment period and that IOP and aqueous flow are the only parameters affected by acetazolamide. These assumptions are not relevant for the tonographic method. Finally, the tonographic method was technically more difficult and the animal was aroused more during this measurement than during the fluorophotometric procedure, which may have affected the results. The discrepancy between the results of the two methods in the present study of rabbits warrants further investigation, especially since similar discrepancies have been found in a previous study of humans. 
Uveoscleral outflow (calculated with C fl and P ev = 10 mm Hg in the Goldmann equation) increased approximately 50% at night in the juvenile and adult rabbits, damping the nocturnal increase in IOP. The finding that uveoscleral outflow (Table 2), significantly increased at night in both groups of rabbits is the first report of daily fluctuations in uveoscleral outflow in any species. Episcleral venous pressure was not measured and was assumed to remain constant throughout the 24-hour period. However, if rabbits did have higher values of episcleral venous pressure during their active period (night) then the difference between day- and nighttime fluorophotometric uveoscleral outflow would be even greater than we report. For example, if P ev were 2 mm Hg lower in the sleep period than the active period in the mature rabbits (Table 2), F u would be 0.63 ± 0.14 μL/min in the sleep period and 1.18 ± 0.09 μL/min in the active period. A day-to-night difference in uveoscleral outflow was not found in healthy middle-aged humans (Fan S, et al. IOVS 2008;49:ARVO E-Abstract 1568). This contrast may reflect differences in age and/or species. It should be noted that when uveoscleral outflow was calculated with tonographic C instead of fluorophotometric C in the Goldmann equation, no significant change was seen between day and night although there appeared to be a dip at midnight (Table 4). Fluorophotometric C would not detect this dip, as it is a measurement over a period of hours. Whether this dip is real requires further investigation. How C was obtained can partly explain the significant day/night differences in uveoscleral outflow with one method of calculation but not with the other. The mechanism underlying the nighttime increase in uveoscleral outflow and the reasons for the different results with different methods of determination merit further investigation. 
In both groups of rabbits, the central cornea was thinner during the night compared with that during the day. The circadian variation of CCT in rabbits is approximately 12 hours out of phase with that in humans and cats, in which CCTs are thicker at night than during the day. 2326 This is the first study in which day- and nighttime CCTs were compared in rabbits. In an earlier study, it was found that CCT in rabbits decreased from morning to evening, which was explained by dehydration (tear evaporation) during the day and hydration at night (rabbits in that study were observed to sleep at night with eyes closed). 57 This contrasts with the CCT results in the present study. In rabbits, the circadian rhythms of locomotion, food and water intake, and excretion are easily affected by external stimuli other than light. 17 Since our rabbits were observed to be more active at night than during the day, differences in the rabbits' housing conditions may explain the disparity. In addition to corneal dehydration and tear evaporation, other factors are believed to affect CCT. Corneal swelling during sleep has been ascribed to the reduction in oxygen tension, which exists with prolonged eye closure. 24 Although not measured in this study, a reduction in oxygen tension may partially explain the higher daytime CCT in our albino rabbits. 
Central corneal thickness has emerged as an important predictive factor for the development of glaucomatous damage. 58 It also is thought to affect the accuracy of applanation tonometry. 29,31,33 Because applanation tonometry estimates IOP by quantifying the force needed to flatten an area of the cornea, the force with which the cornea resists flattening affects the measurement. Thus, applanation tonometers, such as the Goldmann tonometer, pneumatonometer, and handheld tonometer (Tono-Pen; Mentor, Melville, OH), also are influenced by CCT. 29,3133,35 If CCT did affect the IOP measurements, nighttime IOP in rabbits may be even higher than the values reported, because a thinner nighttime CCT should result in a lower IOP reading. Thus, the IOP differences between day and night would be greater than we report herein. 
The present study also showed an increase in CCT with maturity during the day and at night that was significant by repeated-measures ANOVA, but failed to reach significance by unpaired t-test. The process of maturation and eye growth may contribute in part to the noted age-related changes in aqueous humor dynamics and CCT. The 3-month-old rabbits were juveniles and were 27% smaller by weight than the mature adult animals. The younger animals also were growing rapidly, having gained 12% in body weight during the 2 weeks before the study. New Zealand White rabbits are reported to live for 7 to 13 years, 59 yet an increase in age of only 8 months showed significant changes in IOP, aqueous flow, and uveoscleral outflow. Once maturity is reached, aqueous humor dynamics may change at a slower rate. 
In conclusion, rabbits exhibit day- and nighttime differences in aqueous flow, outflow facility, and uveoscleral outflow that contribute to the IOP fluctuations throughout a 24-hour period. The changes in CCT are independent of changes in IOP. Maturity affects IOP, aqueous flow, and uveoscleral outflow in rabbits. The rabbit is an intriguing animal model for studies of factors regulating the daily changes in IOP. 
Footnotes
 Supported by Research to Prevent Blindness.
Footnotes
 Disclosure: M. Zhao, None; J.J. Hejkal, None; C.B. Camras, None; C.B. Toris, None
The authors thank Robin High, MBA, MA (Department of Biostatistics, University of Nebraska Medical Center), for lending statistical expertise, and Lisa Stapp and Tara Rudebush (Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center), for technical help with the animals. 
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Figure 1.
 
IOP during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars represent ± SEM; repeated-measures ANOVA.
Figure 1.
 
IOP during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars represent ± SEM; repeated-measures ANOVA.
Figure 2.
 
Aqueous flow during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05 and ***P < 0.001, using paired or unpaired t-tests.
Figure 2.
 
Aqueous flow during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05 and ***P < 0.001, using paired or unpaired t-tests.
Figure 3.
 
Fluorophotometric outflow facility during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, paired t-test
Figure 3.
 
Fluorophotometric outflow facility during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, paired t-test
Figure 4.
 
Uveoscleral outflow calculated from fluorophotometric outflow facility and P ev = 10 mm Hg during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; paired or unpaired t-tests.
Figure 4.
 
Uveoscleral outflow calculated from fluorophotometric outflow facility and P ev = 10 mm Hg during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; paired or unpaired t-tests.
Figure 5.
 
CCT during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. ***P < 0.001, paired t-test.
Figure 5.
 
CCT during the day and night in juvenile (3-month-old) and adult (12-month-old) rabbits. Vertical bars, ±SEM. ***P < 0.001, paired t-test.
Figure 6.
 
Tonographic outflow facility during the day and night in juvenile (3-month-old) rabbits. Vertical bars, ±SEM.
Figure 6.
 
Tonographic outflow facility during the day and night in juvenile (3-month-old) rabbits. Vertical bars, ±SEM.
Table 1.
 
IOP during the Day and Night in Juvenile and Adult Rabbits
Table 1.
 
IOP during the Day and Night in Juvenile and Adult Rabbits
9 AM Noon 11 PM 2 AM
Juvenile (n = 12) 14.5 ± 1.0 15.1 ± 0.6 19.3 ± 0.6 16.3 ± 0.5
Adult (n = 10) 16.8 ± 0.6 15.9 ± 0.5 20.9 ± 0.5 17.7 ± 0.4
Table 2.
 
Daytime and Nighttime Aqueous Measurements
Table 2.
 
Daytime and Nighttime Aqueous Measurements
Juvenile (n = 12) Adult (n = 10) P P
9 AM-3 PM 11 PM-5 AM P * 9 AM-3 PM 11 PM-5 AM P *
F a 2.24 ± 0.08 2.53 ± 0.1 0.017 1.46 ± 0.13 1.71 ± 0.15 0.042 0.000 0.000
C fl 0.20 ± 0.03 0.10 ± 0.02 0.047 0.14 ± 0.02 0.10 ± 0.02 0.023 0.23 0.81
F u1 1.29 ± 0.14 1.87 ± 0.17 0.013 0.63 ± 0.14 0.99 ± 0.07 0.023 0.00 0.000
F u2 1.62 ± 0.11 2.07 ± 0.14 0.006 0.92 ± 0.13 1.18 ± 0.09 0.054 0.00 0.000
Table 3.
 
Day- and Nighttime CCTs
Table 3.
 
Day- and Nighttime CCTs
Juvenile (n = 12) Adult (n = 10) P P P §
2:30 PM 11:30 PM P * 2:30 PM 11:30 PM P *
CCT 369 ± 5 333 ± 5 0.000 384 ± 7 349 ± 6 0.000 0.08 0.06 0.02
Table 4.
 
Tonographic Outflow Facility and Corresponding Uveoscleral Outflow in Juvenile Rabbits
Table 4.
 
Tonographic Outflow Facility and Corresponding Uveoscleral Outflow in Juvenile Rabbits
Time 5 AM Noon 3 PM Midnight P
C ton 0.18 ± 0.02 0.19 ± 0.03 0.15 ± 0.03 0.24 ± 0.02 0.279
F ut1 0.79 ± 0.35 0.98 ± 0.27 1.03 ± 0.31 −0.10 ± 0.28 0.088
F ut2 1.15 ± 0.31 1.36 ± 0.24 1.32 ± 0.27 0.39 ± 0.24 0.082
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