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1 From the Department of Biological Science and Disease, New England College of Optometry, Boston, Massachusetts; and the 2 School of Optometry, University of California at Berkeley, Berkeley, California.
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Abstract |
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METHODS. IOP was measured by applanation tonometry in 14 untreated marmosets ranging in age from 24 to 259 days. High-frequency A-scan ultrasonography was used to measure ocular dimensions (axial length and choroidal thickness) in 12 marmosets ranging in age from 24 to 572 days. Four monkeys were measured when they were juveniles (<110 days of age) and again later, when they were adolescents. Measurements were typically made at 12-hour intervals, although three animals were measured at 6-hour intervals. Nine monkeys had both IOP and axial dimensions measured in the same experiment.
RESULTS. There was a diurnal rhythm in IOP in the marmosets: IOP was higher during the dark period and lower during the light period (mean change, 3.6 mm Hg; P < 0.005). There were also rhythms in axial length and choroidal thickness. The rhythm in axial length was dependent on age, with faster-growing eyes of juveniles increasing in length during the day and decreasing at night (+25 µm vs. -22 µm; P < 0.001) and slower-growing eyes of adolescents showing the opposite pattern (-27 µm vs. +46 µm; P < 0.0001). The choroid thickened during the night and thinned during the day, at all ages measured (+19 µm vs. -16 µm; P < 0.0001).
CONCLUSIONS. Diurnal rhythms in IOP, axial length, and choroidal thickness exist in primates. Age-related differences in the phase relationships of these rhythms may be associated with differences in the rates of ocular growth.
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Introduction |
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Recent evidence in chickens7 8 suggests that some ocular diurnal rhythms may play a role in emmetropization (the visual regulation of ocular growth rate to attain emmetropia). It has been shown, for instance, that axial length fluctuates in a diurnal rhythm, increasing during the day and decreasing during the night.7 8 9 This rhythm has also been found in rabbits; however, the phase of the rhythm is opposite that of chicks, so that rabbit eyes increase in length during the night and decrease during the day.10 In both species, the phase of the rhythm in axial length generally coincides with that of the rhythm in IOP,10 11 which in chicks is high during the day11 and in rabbits is high during the night.12 13 14 15 This coincidence in phase is consistent with IOP playing a role in the diurnal fluctuations in axial length. However, various lines of evidence indicate that, although IOP may be one factor involved, it is not the sole one.10 11 Other possible underlying factors are the diurnal rhythm in scleral extracellular matrix synthesis16 17 or the rhythm in choroidal thickness.7
In chicks, the thickness of the choroid, the vascular tissue behind the retina, undergoes diurnal oscillations—normally, thickening during the night when the eye is shortest and thinning during the day when the eye is longest. These rhythms in axial length and choroidal thickness are thus in antiphase, thereby increasing the amplitude of the fluctuations in vitreous chamber depth and, consequently perhaps, refractive error. Although the functional significance of these two rhythms is as yet unknown, it has been speculated that they form part of a negative-feedback loop through which the associated fluctuations in refractive error are sensed and translated into changes in ocular growth.16 Further evidence that they may be involved somehow in emmetropization is that their phase relationships appear to vary in a consistent manner, depending on experimentally induced changes in ocular growth rate. Specifically, in eyes that are growing slower than normal, the rhythms shift into phase with one another,7 18 whereas in faster-growing eyes, they shift completely out of phase.7 Whether these phase shifts are a cause or a result of the changes in ocular growth rate is unknown. However, the rhythm in choroidal thickness could be a component of a rhythm in choroidal "stiffness" that may modulate the mechanical influence of IOP on the sclera (see, for instance, Ref. 19 ). This is supported by the finding that thicker choroids synthesize more glycosaminoglycans (GAGs, presumably proteoglycans) than thinner ones,20 and that increased proteoglycan synthesis is associated with increased stiffness in some connective tissues.21
The purpose of the present study was to determine whether rhythms in axial length, choroidal thickness, and IOP are present in the marmoset model of eye growth, and if so, to determine whether the phase relationships of these rhythms showed similar growth-rate-dependent differences as in the chick. We measured juvenile animals with fast-growing eyes and adolescent animals with slow-growing eyes, and we found diurnal fluctuations in axial length, choroidal thickness, and IOP. Furthermore, the phase at which axial elongation is at its maximum differs with age (and ocular growth rate). Parts of these results have been reported in abstract form.22 23 24
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Methods |
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To minimize the possible effect of brief exposures to light on circadian rhythms, all nighttime measurements were made under a dark yellow photographic safe light that provided approximately 0.5 lux at the monkey’s eye. The animals were immediately returned to the dark after measurement. The same protocol was used for the ultrasound measurements, described later.
We did not calibrate the tonometer for the marmoset eye; therefore, the data may not be an accurate representation of the actual IOP for marmosets. Because the purpose of the study was to ascertain the existence (or absence) of a diurnal rhythm in IOP, relative measures were sufficient, and we therefore considered the killing of a monkey for calibration purposes unwarranted.
Data Analysis.
The tonometer (Tono-pen; O&O Mentor) provides confidence interval information based on successive readings. The data shown for each eye per time point represent the averages of at least two readings, with confidence intervals of less than 0.05%. Most of the data were obtained at 12-hour intervals over 48 hours (i.e., two cycles and four measurements per eye). The data for corresponding time points over two cycles (for example, 6 AM) were averaged for presentation and analyses. The paired data from right and left eyes (regardless of time of day, n = 10 animals) correlated significantly (r = 0.83; P < 0.01). Thus, in statistical analyses, the averages for the two eyes of individual animals were pooled with the individual eye data for the four cases in which only one eye was measured. A paired t-test was used to compare changes during the day versus those during the night.
Axial Dimensions
Measurements.
Axial dimensions were measured in 12 untreated monkeys at ages ranging from 24 to 572 days (mean, 177) using high-frequency A-scan ultrasonography in animals under halothane anesthesia (for details, see Ref. 7
). Four of these 12 monkeys were measured as juveniles (ages, 24–52 days) and again as adolescents or adults (ages, 327–572 days; monkeys O3, P3, T3, and H3; Table 1
). Measurements were generally performed at 6 AM and 6 PM over 48 hours; however, 2 monkeys (M1, J1) were measured at 8 AM, 2 PM, and 8 PM over 48 hours, and one (R1) at 6-hour intervals starting at 8 AM for 24 hours (Table 1)
. In some of these animals, IOP and ultrasonography were performed at the same time (n = 7; Table 1
). In these cases, tonometry was always performed first to avoid introducing measurement artifacts associated with the ultrasonography procedure.
For ease of presentation of the data, when referring to trends over 12-hour intervals, "day" is used to refer to the interval spanning the largest part of the light cycle and "night" the interval spanning the dark cycle. For instance, in the 6 AM-to-6 PM measurement schedule, the day is defined as 6 AM to 6 PM and includes 1.5 hours of darkness; in the 8 AM-to-8 PM schedule, the day includes only 0.5 hours of darkness.
Data Analysis.
We defined axial length as the distance from the front of the cornea to the front of the sclera, with one exception (monkey Z1). Because the corneal peak could not be unambiguously identified in this animal, the distance from the back of the lens to the front of the sclera was used instead. Because the identification of the retina-choroid boundary is sometimes difficult, choroidal thickness was defined as the sum of the retinal and choroidal thicknesses. This indirect measure reflects true choroidal thickness, because retinal thickness is fairly constant over the ages used in this study (Troilo D, Nickla DL, unpublished data, 2000). Data for choroidal thickness in two animals (M1 and J1) was excluded, based on the poor quality of the ultrasound traces.
Because a wide range of ages, and hence eye sizes, were used in this study, to best illustrate the fluctuations in length as a function of time, the data for each eye per time point were normalized to the mean of all measurements for that eye. The data shown in the line graphs (Figures 3A 4A 4B and 7) represent the mean of normalized data for all eyes. In this way, the large age-related variability in eye size was eliminated. The bars represent the standard errors for the normalized data.
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Data collected more frequently than the Nyquist limit (12-hour intervals for a 24-hour period) can be subjected to an analysis that yields phase and amplitude (for example, Fig. 2B ). To assess these parameters, a sine wave having a period of 24 hours (the diurnal period) can be fit to the data. Because most of our data were collected at 12-hour intervals, this analysis was possible on only a small subset of data, and no statistics were possible. Despite the absence of accurate "phase" determinations for our 12-hour interval data, we found differences in the temporal pattern of these various rhythms that were related to age. We take the liberty of referring to these pattern differences as "phase" differences.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Rhythms in Axial Length and Choroidal Thickness
Marmoset eyes exhibited diurnal rhythms in axial length and choroidal thickness, extending earlier findings in chicks to a primate model of eye growth. We found that the diurnal pattern for the rhythm in axial length differed between juvenile and adolescent monkeys (n = 8 animals for both groups, Figures 3
4 and 5
), whereas that for the rhythm in choroidal thickness did not (Fig. 6)
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Adolescent Monkeys.
In marked contrast to eyes of juvenile monkeys, eyes of adolescent monkeys decreased in length during the day (6 AM–6 PM) and increased during the night (6 PM–6 AM; compare Figs. 4 and 3
; see Fig. 5
). Figure 4
shows separately the axial length data from eyes measured at 12-hour intervals—at 6 AM and 6 PM (n = 9 eyes, Fig. 4A
)—and more frequently—at 8 AM, 2 PM, 8 PM, and 2 AM (n = 6 eyes, Fig. 4B
). The mean raw diurnal changes in axial length, shown in the insets of the figures, exhibited similar diurnal patterns of changes in length (-20 µm vs. +38 µm; Fig. 4A
, inset; -49 µm vs. +68 µm, Fig. 4B
, inset; both differences are significant, t-test; P < 0.0001 and P < 0.01, respectively). For an estimation of phase, sine waves were fit to the mean data of the eyes that were sampled more frequently than at 12-hour intervals (R1, solid lines, J1 and M1, dashed lines; Fig. 4B
). The period of the best-fit sine wave was approximately 24 hours for both sets of data, and the mean phases were similar (peaks at 5:30 AM, 6:00 AM).
As an illustration of the consistency of the age-related change in the phase of the rhythm in axial length, Figure 5 shows the data for two of the four monkeys measured as juveniles and again as adolescents. The data for the two eyes of each animal are averaged in this representation. The juvenile and adolescent patterns were approximately antiphase in both cases.
Choroidal Thickness
The choroid showed a diurnal rhythm, thickening during the night (6 PM–6 AM or 8 PM–8 AM) and thinning during the day (6 AM–6 PM or 8 AM–8 PM; Fig. 6
). This pattern was the same in juvenile and adolescent monkeys (Fig. 6A)
. The averaged data from the two eyes of one monkey (R1, an adolescent) measured on a different schedule is shown by the solid symbols and dashed lines. The phase and amplitude were similar to that of both other groups. In juvenile monkeys (n = 8), the mean day-versus-night change in choroidal thickness was -12 µm versus +18 µm (P < 0.0001), whereas in adolescent monkeys (n = 6), the mean change was -22 µm versus +21 µm (P < 0.0001; Fig. 6B
). The mean diurnal change in choroidal thickness for all monkeys was -16 µm versus +19 µm (P < 0.0001).
The net result of the age-related differences in the rhythms in axial length was that in juvenile monkeys, the rhythms in axial length and choroidal thickness were (approximately) out of phase (Fig. 7A , top), whereas in adolescent monkeys they were (approximately) in phase (Fig. 7A , bottom). Similarly, the phase relationship of the rhythms in axial length and IOP showed age dependence (Fig. 7B) . In adolescents, the two rhythms were approximately in phase, whereas in juveniles they were approximately out of phase.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Diurnal Rhythm in IOP
Diurnal rhythms in IOP have been documented in many species, including rats,27
rabbits,13
15
28
chicks,8
11
and humans.29
30
31
The phase and amplitude of the rhythm in IOP differs between species.
We found that in our marmosets, IOP was higher during the dark phase and lower during the light phase of the diurnal cycle. This was true, regardless of whether IOP was measured in the dark at 6 AM (most of our data), at midnight (three monkeys), or 8 PM (one monkey). Humans29 30 31 and rhesus macaques32 reportedly show an increase in IOP in the early morning. We did not sample IOP at sufficient intervals to determine a precise phase or amplitude. However, the one monkey measured at 6-hour intervals showed an approximately sinusoidal rhythm with an amplitude of approximately 6 mm Hg. This is similar to the amplitude of 5 mm Hg reported in humans30 and rhesus monkeys,32 but is lower than the 10-mm Hg amplitude in rats,27 33 rabbits,12 14 15 34 35 and chickens (8 mm Hg11 ). The sinusoidal nature of the rhythm in this one animal is not necessarily true in all cases. Indeed, if the rhythm were sinusoidal with a peak at 11 PM (as in Fig. 2B ), then the 6 AM and 6 PM measurement times would be very close to the mesor (5 AM and 5 PM), and hence unlikely to yield significant differences. That we found significant differences at these time points suggests that the phase and/or shape of the rhythm in this particular animal did not represent the common trend. Finally, we do not know whether this rhythm was circadian, driven by light and dark, or a combination of the two (as in the chickens11 ).
Diurnal Rhythms in Axial Length and IOP
We found that in our marmosets, the eyes showed diurnal fluctuations in axial length, extending this phenomenon to a primate model of eye growth. In chicks, eyes elongate more during the day than during the night,7
8
9
whereas in rabbits, the reverse is true.10
In normal (untreated) eyes of both these species, the time of maximal elongation approximately coincides with the time of highest IOP, implicating a possible influence of the rhythm in IOP on the rhythm in axial length. Indeed, IOP has long been postulated to play a role in ocular development and ocular growth as a source of inflationary pressure (see, for example, Refs. 36
37
38
). In the marmosets, however, this phase correspondence held true only in the adolescent animals, with their slower-growing eyes increasing in length during the night, when IOP was highest. The faster-growing eyes of juveniles, on the other hand, increased in length during the day, when IOP was lower. This age-related difference in the phases of the rhythm in axial length is not associated with a concurrent age-related phase difference in the rhythm in IOP that might account for this (Fig. 7B)
. These findings support the notion that IOP is not the only (or the main) influence on axial length, in agreement with several other studies showing a noncorrespondence in the phases of these two rhythms.10
11
In rabbits, for instance, the rhythms in IOP and axial length are only approximately in phase: IOP peaks early in the dark phase,14
15
whereas axial length peaks late in the dark phase.10
Furthermore, in form-deprived myopic chick eyes, the rhythm in IOP is no longer synchronized to the light-dark cycle, yet the phase of the rhythm in axial elongation does not change accordingly.11
Evidence for the probable involvement of various other ocular growth-related rhythms in emmetropization make the findings of phase differences in IOP and axial length less paradoxical than they at first might appear. Specifically, the synthesis of extracellular matrix glycosaminoglycans in chick sclera exhibits a circadian rhythm that presumably influences the rhythm in axial elongation.16 17 It is plausible that the rate of biosynthesis in the sclera is dependent on the phase of the rhythm in IOP, because cyclic forces have been shown to effect changes in growth activities in several connective tissues (for a review, see Ref. 39 ). Furthermore, it is known that the proteoglycan content of connective tissue influences its mechanical stiffness (e.g., cartilage21 ). Thus, it is reasonable to assume that there is also a rhythm in scleral compliance associated with the rhythm in GAG synthesis. The net effect of IOP on eye size is thus determined, at least in part, by the phase relationships between the rhythms in IOP and in scleral compliance. Finally, if choroidal thickness changes are associated with changes in stiffness (in chick, thicker choroids are associated with increases in proteoglycan synthesis20 ), then the rhythm in choroidal thickness could also play a significant role in modulating the influence of IOP on the sclera (and hence eye size.19 ) In summary, in any of these scenarios, the net effect of IOP on eye size is dependent on the phase relationships of these various other rhythms. Some of these rhythms—for example, the rhythm in GAG synthesis—are presumably more active in young, fast-growing eyes. We speculate that in older animals in which eye growth has almost ceased, the coincidence between the peaks in IOP and axial length may reflect an increasing contribution of IOP to the changes in axial length.
Diurnal Rhythms in Choroidal Thickness
We find that the thickness of the choroid in the marmosets also showed a diurnal rhythm, increasing during the night and decreasing during the day, similar to the rhythm in normal chicken eyes.7
8
Unlike the rhythm in axial length, the choroidal thickness rhythm did not differ as a function of age. The net consequence of the age-dependent phase shift in the rhythm in axial length and the age independence of the phase of the rhythm in choroidal thickness is that the phase relationship between these two rhythms changed with age. Specifically, in younger, faster-growing eyes the two rhythms were approximately out of phase, whereas in older, slower-growing eyes they were approximately in phase. This finding has an interesting parallel in chickens: In chicks as well, in rapidly growing eyes, the two rhythms are typically out of phase, whereas in slower-growing eyes they are in phase.7
18
This similarity between species supports the notion that the phase relationships between these rhythms influence ocular growth rate.7
A true test of the relevance of these different phase relationships to growth rate changes in marmosets would be to study these rhythms in eyes in which the growth rates had been experimentally altered by spectacle lenses or diffusers.
We conclude that diurnal rhythms in IOP, axial length, and choroidal thickness exist in primates, supporting the hypothesis that these rhythms may subserve similar functions in the control of eye growth across species. The similarities between the different phase relationships for axial length and choroidal thickness as a function of ocular growth rate in both chicks and marmosets further support this idea.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 10, 2001; revised March 5, 2002; accepted March 22, 2002.
Commercial relationships policy: N.
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.
Corresponding author: Debora L. Nickla, New England College of Optometry, 424 Beacon St., Boston MA 02115; nicklad{at}ne-optometry.edu.
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) right eyes; (
) left eyes.
, left y-axis) and adolescents (
, right y-axis, n = 10) as a function of time of day. In juveniles, these rhythms were approximately out of phase, whereas in adolescents they were in phase.
) as a function of age (axial length data from a separate group of marmosets
