Longitudinal stability of the diurnal rhythm of intraocular pressure in subjects with healthy eyes, ocular hypertension and pigment dispersion syndrome

The Erlangen Glaucoma Registry

The primary study, the Erlangen Glaucoma Registry (EGR; ClinicalTrials.gov number NCT00494923), is concerned with subjects at risk for glaucoma and patients with manifest glaucoma, its special focus being the predictive value of diagnostic tests. It is a local registry, located at the University Eye Hospital Erlangen. The study was approved by the ethics committee of the Friedrich-Alexander-University Erlangen-Nuremberg and conducted in accordance with the tenets of the declaration of Helsinki. The first examinations took place in 1991. The EGR has been supported by the Deutsche Forschungsgemeinschaft (DFG) until July 2009, first by the DFG Clinical Research Group and then, since 1997, by the SFB 539 grant. Subjects who agreed to undergo extensive diagnostic testing at regular follow-up visits were recruited from the glaucoma clinic of the University Eye Hospital Erlangen. All subjects gave written informed consent.

Complete clinical examinations were performed annually; they included slit-lamp examination, standardized perimetry, dilated fundus examination, standardized photography of the optic nerve head, and a 24-hour IOP profile (see below for details). Intermittent examinations were scheduled as needed. When indicated, patients were treated conservatively or surgically according to the current clinical standards. Careful slit-lamp examination of the anterior segment was performed to detect PDS and to look for signs of other ocular conditions, especially ones that may cause secondary glaucoma, like pseudoexfoliation syndrome. Standardized automatic white-on-white perimetry was performed with an Octopus 500 perimeter (Haag-Streit, Switzerland) using the G1 program. When patients were not experienced in sensory tests, the first three visual fields were not included in the analysis. Standardized 15°-fundus photographs centered on the optic disc were performed annually. They were graded in a masked fashion by two glaucoma specialists for signs of glaucomatous atrophy. Each year, the complete series of photographs was analyzed for loss of neuroretinal rim tissue or progressive optic disc cupping by the specialists.

These data were entered into a database and checked for internal consistency on a regular basis.

Selection of eligible patients and study groups

For this retrospective analysis, data from the EGR was used with permission of the principal investigators and the medical director of the University Eye Hospital Erlangen. Only subjects without signs of manifest glaucoma (i.e. normal visual field and normal appearing optic nerve) were included and only if regular follow-up of more than four years had been documented and at least four complete IOP profiles had been performed without local or systemic antiglaucomatous therapy. Patients with manifest glaucoma or with other eye disease (except cataract) were excluded. Likewise, shift workers were not included in the study. Eligible subjects from the Erlangen Glaucoma Registry with appropriate diagnoses and length of follow-up were identified by querying the database. Inclusion and exclusion criteria were then verified by the authors using data from the database or from the clinical records of these participants. None of the subjects included in this retrospective analysis had signs of glaucomatous optic disc changes or visual field defects. Subjects with pseudoexfoliation, rubeosis iridis, angle closure, aphakia, uveitic or congenital glaucoma were excluded. Furthermore, no subject had undergone intraocular surgery, except cataract extraction.

The selected subjects were divided into the following three groups: 1) normal subjects with normal intraocular pressure and without signs of pigment dispersion (normals); 2) subjects with ocular hypertension who showed no signs of pigment dispersion, but three intraocular pressure readings ≥ 23 mmHg (OHT); and 3) subjects with at least two signs of pigment dispersion syndrome, regardless of IOP (PDS). Clinical signs of PDS include melanin pigment in the corneal endothelium (Krukenberg spindle), melanin pigment on the iris and the lens, or typical radial transillumination defects of the iris.

The subjects that were included in the “normal” group had been referred to us because they were classified as glaucoma suspects elsewhere. Commonly, the reason was an elevated IOP measured by the referring ophthalmologist before inclusion in the EGR. Many of these subjects had a positive family history for glaucoma. These subjects were concerned and chose to continue participating in the study despite being informed that there were no signs of either OHT or glaucoma after the first few visits.

Intraocular pressure profiles

IOP measurements, including those at night, were performed in a sitting position using Goldmann applanation tonometry. Diurnal intraocular pressure profiles were performed annually and consisted of a series of at least six measurements. Physical activity, exact timing of the sleep-wake-cycle, and consumption of tobacco, caffeine or alcoholic beverages were not recorded. In general, subjects were instructed not to stay up and wait for the midnight recording, but rather go to bed at their usual time. The subjects underwent further morphological and sensory tests during the first half of the day between IOP measurements, but all tests requiring dilatation of the pupil were performed on the second day after the last IOP measurement.

Most subjects were hospitalized for one day and one night for regular measurements at 08:00 h, 12:00 h, 17:00 h, 21:00 h, and 24:00 h. Subjects were accommodated in a hospital room, usually with one or two other patients. For the measurement at midnight, patients were woken by a nurse not more than 15 minutes before IOP was measured in the office a few meters from the patient’s room. The next day, an additional measurement was performed at 07:00 h immediately after the subject was woken.

Cosinor analysis

Cosine curves were characterized by three parameters: MESOR M, amplitude A, and acrophase φ. The MESOR is the Midline Estimating Statistic Of Rhythm, i.e. the value about which fluctuation occurs. The acrophase is the time of the highest point of the fitted curve. In Equation 1, φ is a negative value (unit: radians) that represents the phase angle of the lag behind the reference time (=midnight); however, in this paper, acrophase data are expressed as the time of day when the peak of the cosine curve occured (i.e. hours after midnight).Circular data, such as the calculated acrophases, had to be evaluated using specialized statistical methods: mean acrophases were calculated in two steps. First, X- and Y-coordinates of the points where the lines through the origin whose direction represented the acrophases in the clock plot intersected the unit circle were calculated. In a second step, all X- and Y-coordinates were averaged separately and the coordinates of the resulting point were converted back into polar coordinates. The angle between the resultant vector and the Y-axis now represented the mean acrophase, and its length r the dispersion of the angles from which it was calculated (r close to zero means high dispersion of the angles that were averaged, while r close to one signifies low dispersion). These steps are also illustrated in Figures 1 and 2. Angular standard deviation s’ was calculated from angular dispersion r according to the following equation.

$s\text{'}=\sqrt{\left(-2\cdot lnr\right)}$

(2)

To test for intra-individual stability of phase timing from year to year, the Rayleigh test was used (implemented in R in the Circular Statistics package by Ulric Lund and Claudio Agostinelli, 2007; circular: R package version 0.3-8). A significant test indicated the presence of a significant predominant long-term acrophase as opposed to a uniform, random distribution of acrophases around the circle.

To test for differences in mean acrophase between groups, a second-order parametric analysis of two samples of angles was performed using a method proposed by Batschelet and Hotelling (Figure 2). This test was implemented manually by the authors after the description in Zar [15].

Categorical data were analyzed using Fisher’s exact test, and comparisons of clinical parameters among groups were made using analysis of variance (ANOVA), followed by the Tukey HSD post-hoc test if the ANOVA was significant. Generally, a level of p ≤ 0.05 was considered significant.

Cosinor analysis

Cosinor-analysis of the 364 suitable IOP profiles available for these 52 subjects was generally feasible, and only three IOP profiles (1%) had to be excluded because the algorithm of the function nls for the least-squares method in the R statistics software was not able to calculate an adequate cosine model. On qualitative examination of the remaining IOP profiles, the cosine models seemed adequate to describe fluctuation of IOP and the fit seemed well constrained by the data in the majority of cases.

Reproducibility of cosinor analysis

The variability of the individual acrophases from visit to visit was higher than expected; circular standard deviation was between 48 minutes and as much as 7 hours 50 minutes with a median of 4 hours 2 minutes. Due to this variability, the Rayleigh test showed a statistically significant mean direction only in 19 of the 52 subjects (36%).

Age did not seem to influence either stability or timing of IOP rhythm. When subjects younger than the median age of 44.4 years were compared to older subjects, no statistically significant differences were found. This did not change when only subjects with significant Rayleigh tests were included, or when the lowest age quartile was compared to highest (data not shown).

Group statistics from averaged longitudinal data

The overall directions of individual long-term mean acrophase vectors showed a higher inter-individual variability than expected but were not random: Rayleigh test demonstrated, that its distribution was significantly different from a uniform distribution (p = 0.01). Individual mean acrophases ranged between 0:13 h and 18:44 h, most subjects (62%) having a mean acrophase between 06:00 h and 12:00 h. Sixteen subjects (31%) had an acrophase later than noon, and four subjects (8%) had an acrophase earlier than 06:00 h. However, in the PDS group, the majority of subjects had a mean acrophase later than 12:00 h (54% compared to 20% in other subjects, p = 0.05).

A detailed description of the estimated parameters of the fitted cosine curves of all subjects is presented in Table 2, and individual mean acrophases are displayed in a clock plot for each patient in Figure 3. Briefly, the average MESOR of the fitted cosine curves was 16.0 ± 2.3 mmHg and the mean amplitude was 1.9 ± 0.6 mmHg. Mean acrophase was 10:37 h with a circular standard deviation of 4 hours and 52 minutes. For differences among groups, see Table 2.

Subjects were examined annually; therefore each subject was examined at the same time every year. But annual examinations of different subjects were distributed uniformly around the year (Rayleigh test: p = 0.09), and differences in phase timing between subjects cannot be explained by seasonal variation.

Comparison after exclusion of subjects without reproducible IOP rhythm in the long-term

For further analysis, subjects without a significant individual Rayleigh test were excluded. Estimates of MESOR and amplitude, as well as mean acrophase, did not change significantly after exclusion: MESOR was 16.2 ± 1.8 mmHg (not significantly different from that of excluded subjects) and amplitude was 2.1 ± 0.7 mmHg (again not significantly different compared to excluded subjects). After exclusion, the mean acrophase direction was 10:55 h with a circular standard deviation of 3 hours 2 minutes.

The individual circular standard deviation of acrophases between visits was lower in the remaining 19 subjects compared to the excluded subjects, ranging between 48 minutes and 3 hours 36 minutes, with a median of 2 hours 28 minutes. These subjects also had more similar mean acrophases (ranging between 08:13 h and 12:26 h), i.e. less inter-individual variation. Again, the distribution of mean acrophases was significantly different from a uniform distribution (p = 0.01). Fifteen subjects (79%) had an acrophase between 06:00 h and 12:00 h, and four subjects (21%) had an acrophase later than noon. Of the four subjects with an acrophase later than noon, three (75%) had PDS.Cosinor analysis of the remaining 19 subjects with significant Rayleigh test demonstrated significant differences among groups. Subjects with OHT had significantly higher MESORs and amplitudes compared to normal subjects (p = 0.001). In contrast, phase timing was not different (p = 0.17). In contrast, subjects with PDS had a significant phase lag of 2 h 7 min compared to subjects without PDS (12:28 h in the former compared to 10:21 h in the latter group, p = 0.05, Figure 4). When compared to normal subjects, the mean mesor of the PDS group was slightly lower and the amplitude was similar (p = 0.08 for MESOR, p = 0.9 for amplitude). Of the subjects with a significant Rayleigh test and PDS, 60% had an acrophase after noon compared to 7% in the subjects without PDS (p = 0.05).

Limitations

The presented study is a retrospective analysis of data that was not primarily intended for studying chronobiology of IOP. Measurements were not equally spaced throughout the 24 hours and no measurements were made between midnight and 08:00 h. Significant changes in IOP that may have occurred during this time period may have been systematically missed. Another limitation of the present study is the relatively small sample size. Thus, confirmation of the presented findings in a study with more subjects is desirable.

Furthermore, the time of sleep onset and offset was not recorded in our study, and no independent markers showing a circadian rhythm (such as body temperature or blood cortisol level) were monitored. As a result, we cannot be certain to what degree fluctuations were caused by circadian rhythms or by rhythmic environmental influences [26]. Short exposure of subjects to light may have taken place during the measurements at 21:00 h and 24:00 h. The influence of this exposure cannot be determined; however, in a study by Liu et al., short exposure to low levels of light did not seem to cause relevant disruption of the pattern of IOP fluctuation [37].

All IOP measurements, including those at midnight, were taken in a sitting position using Goldmann tonometry. It is known that posture does influence IOP, and there is a significant increase of IOP in the supine position [38]. Consequently, habitual variation of IOP (i.e. when all measurements are taken in the habitual position for that time of day) exhibits a different pattern due to the postural increase in IOP in the supine position at night [9, 10]. The lack of measurements in the supine position in our study may limit the validity of the absolute IOP measurements at night and may have lead to a different pattern of IOP variation, but it should not have influenced our primary end-point: the long-term stability of diurnal IOP curves.

In the final analysis, a bias may have been introduced by the exclusion of subjects with a non-significant Rayleigh test. On the other hand, the interpretation of long-term data from subjects with high variability in phase timing from year to year would have been difficult. Selection of any single IOP curve or comparison of accumulated individual IOP curves (that is individual mean acrophase) seemed pointless in subjects with an erratic pattern of IOP variation.

Further research

The interesting findings of our retrospective analysis should ideally be confirmed and further explored in prospective studies. These studies might include control of the wake-sleep-cycle as well as the light–dark-cycle and control of the activity of the subjects. The latter can be achieved with a wrist device. If possible, an independent marker of the circadian system, for example, body temperature or plasma cortisol levels, should also be determined several times per day.

Specific factors associated with PDS should be examined more closely. The role of physical activity on the diurnal fluctuation of IOP in subjects with PDS may be clarified by comparing IOP profiles under regular activity to IOP profiles under bedrest conditions, similar to the study by Buguet et al. [11]. In addition, the concentration of melanin granules in the anterior chamber using a laser flare-cell meter should be determined several times during the 24-h cycle [39, 40].

In the long term, continuous measurement of IOP would be desirable. Initial experiments have been conducted using a telemetry system with an intraocular manometer in non-human primates and a contact lens in humans [22, 25]. However, methods to analyze these complex IOP curves have not yet been established.