Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Exp Eye Res. 2019 Jan 7;181:5–14. doi: 10.1016/j.exer.2019.01.008

Effects of time-of-day on inhibition of lens-induced myopia by quinpirole, pirenzepine and atropine in chicks

Debora L Nickla 1, Kelsey Jordan 1, Jane Yang 1, Puneet Singh 1
PMCID: PMC6443441  NIHMSID: NIHMS1518676  PMID: 30629959

Abstract

Injections of the D2 dopamine receptor agonist quinpirole or the acetylcholine muscarinic receptor antagonists pirenzepine and atropine prevent the development of negative-lens-induced myopia in chicks by inhibiting ocular growth. Because ocular growth is diurnally rhythmic, we hypothesized that the efficacy for inhibition may depend on time of day. Chicks wore monocular −10D lenses for 5 days, starting at 12d of age. The light cycle was 12L/12D. The lens-wearing eye received daily intravitreal injections for 4 days, of 20 μl quinpirole (10 nmol), at the following times: 7:30 EST (lights-on; morning; n=12), 12:00 (mid-day; n=13), or 19:30 (evening; n=17). The same protocol was used for pirenzepine (0.2 μmol) and atropine (18 nmol), at the following times: 8:30 EDT (lights-on; n=10, n=18), 14:00 (n=10, n=12), or 20:30 (n=18, n=16). Saline injections were done in separate groups of birds for all groups as controls, and the data combined (n=28). Ocular dimensions were measured using A-scan ultrasonography on treatment day 1 at 12:00, and again on day 5 at 12:00; growth rate is defined as the change in axial length over 96 hr. For quinpirole and pirenzepine, subsets (n’s in Methods) of mid-day and evening groups were measured at 6 hr intervals on day 5 (from 12:00 to 12:00) to obtain rhythm parameters for axial length and choroidal thickness; for atropine, only the mid-day group was measured. Refractions were measured on day 5 with a Hartinger’s refractometer. For quinpirole and pirenzepine, mid-day injections were more effective at inhibiting ocular growth than evening (Exp-fellow: quinpirole: −68 vs 118 μm/96hr; post-hoc Bonferroni p=0.016; pirenzepine: 79 vs 215 μm/96hr; p=0.046). There were no between-group statistically significant differences for atropine. For quinpirole, the mid-day amplitude of the axial rhythm was smaller than for evening (95 vs 142 μm; p<0.05), but there were no time-dependent effects on the rhythms for pirenzepine. For atropine, the amplitude of the axial-length rhythm was significantly larger than that for pirenzepine at mid-day. We conclude that there is a phase-dependent efficacy for quinpirole and pirenzepine, with mid-day injections being most effective. There are no consistent time-dependent alterations in rhythm parameters for any of the drugs.

Keywords: dopamine, acetylcholine, axial length, myopia, choroid, diurnal rhythms

INTRODUCTION

Myopia, a condition in which images of distant objects are focused in front of the photoreceptors as a result of abnormal axial elongation, is becoming epidemic, especially in parts of Asia (Goh and Lam 1994; Lin and al. 1996; Fan et al. 2004; He et al. 2004; Lin et al. 2004; Qian et al. 2009; Pan et al. 2012). However, despite decades of study, prophylactic pharmaceutical treatments against childhood myopia are limited to atropine, an anti-muscarinic drug whose mechanism was initially believed to reside in its inhibition of accommodation by blockade of ciliary muscle receptors (review: (Bedrossian 1971; Bedrossian 1979)). Animal models have since disproven this hypothesis, as atropine is effective at inhibiting form-deprivation myopia in chickens (Stone et al. 1991), which have nicotinic as opposed to muscarinic cholinergic receptors in iris and ciliary muscle, and in the squirrel, a non-accommodating mammal (McBrien et al. 1993a; McBrien et al. 1993b). However, the site and mode of action remain unknown (McBrien et al. 2013).

There is evidence for roles for muscarinic cholinergic (Stone et al. 1991; McBrien et al. 1993b; Cottriall and McBrien 1996; Luft et al. 2003) and dopaminergic signaling (review: (Feldkaemper and Schaeffel 2013) in the etiology of myopia development from animal models, and, for muscarinic signaling, from clinical data (Chia et al. 2012). In the chick model, both atropine (non-specific muscarinic antagonist) and pirenzepine (cm2/cm4 receptor antagonist) are effective inhibitors of deprivation-induced (Stone et al. 1991; Luft et al. 2003; Diether et al. 2007) and negative lens-induced myopia (Schmid and Wildsoet 2004; Nickla et al. 2013). In children, clinically-available 1% atropine is effective at inhibiting myopia, but its wide acceptance is limited by the side effects of cycloplegia, mydriasis and photophobia (Shih et al. 1999). In the United States, Myopia Control Centers are using 0.01%, but the published results on effectiveness are variable (Chia et al. 2012). Pirenzepine had also been reported to inhibit myopia in children (Siatkowski et al. 2004; Tan et al. 2005), but clinical trials were stopped.

Dopamine is a retinal neuromodulator found in amacrine and interplexiform cells. Its synthesis and release are diurnally rhythmic, being high during the day and low at night, and as such mediates the light-adaptive changes in retinal circuitry that ensure optimal visual function during the light part of the cycle. In the chick model, intravitreal injections of dopamine agonists inhibit deprivation-induced (McCarthy et al. 2007) and lens-induced (Schmid and Wildsoet 2004; Nickla et al. 2010) myopia, and antagonists prevent the vision-induced inhibition of excessive eye growth in form-deprived eyes (McCarthy et al. 2007), suggesting that dopamine mediates the effects of unobstructed vision in preventing myopia.

There is mounting evidence that circadian/diurnal ocular rhythms play a role in eye growth regulation (emmetropization), and by extension, that altered rhythms underlie the development of ametropias (Stone et al. 2013; Chakraborty et al. 2018). Many ocular processes are diurnally rhythmic, including processes believed to be involved in the regulation of eye growth, which is itself rhythmic, such as choroidal thickness (Nickla et al. 1998) and scleral proteoglycan synthesis (Nickla et al. 1999). Some visual conditions that experimentally alter ocular growth rates are associated with alterations in certain parameters (phase and amplitude) (Papastergiou et al. 1998; Nickla 2005; Nickla et al. 2017a; Nickla et al. 2017b). Notably, retinal dopamine levels are decreased in chick eyes developing myopia, but only during the day, when levels are normally high (Stone et al. 1989). It stands to reason that increasing daytime, but not nighttime, dopamine levels would ameliorate the myopia, but this has not been explicitly tested.

The purpose of this study was to determine whether the growth-inhibiting effects of two different classes of myopia-inhibiting drugs, dopaminergic and cholinergic, depend on the time-of-day of administration. We chose quinpirole, a D2 dopamine receptor agonist, and two muscarinic antagonists: atropine (non-selective) and pirenzepine (cm2/cm4 selective) (Tietje and Nathanson 1991; Jakubik and Tucek 1994). We hypothesized that their efficacy would be dependent on the time of delivery, and perhaps, if altered retinal rhythms are involved in myopia development, that there might be consistent time-dependent effects on the rhythms in axial length or choroidal thickness. Parts of this manuscript have been published in Abstract form (Jordan et al. 2017).

Methods

Subjects:

White Leghorn chicks (Gallus gallus domesticus; Cornell-K strain) were hatched in an incubator and raised from day one in temperature-controlled brooders. The light cycle was 12L/12D; the light level in the brooders at chick height was ~500 lux (Light meter; International Light Technologies). Lighting was fluorescent (Sylvania Octron XP; 4100K). Food and water were supplied ad libitum. Care and use of the animals conformed to the ARVO Resolution for the Care and Use of Animals in Research.

General paradigm:

For all experiments, −10 D lenses mounted onto Velcro rings were attached to matching rings around the right eyes of 12-day-old chicks for the duration of the experiment. Lenses were briefly removed once daily in the morning, mid-day or evening for 4 days, and eyes received an intravitreal injection of the drug dissolved in saline. Saline injections (20 μl, 0.75% NaCl w/v) were given in separate groups of birds in all experiments (n=30), and these data were combined as there were no statistically significant between-group differences as a function of time of day. Injections used a Hamilton syringe with a 30G needle under isofluorane (in O2) inhalation anesthesia; injections went through the skin of the lids into the superior temporal sclera, after removal of feathers and cleaning the skin with alcohol. The needle remained in place for 30 seconds before withdrawal, while the skin around the site was held tightly together with forceps to minimize leakage. The lenses were replaced upon removal of the needle, and prior to awakening from the anesthesia. Axial dimensions were measured using high-frequency (30 MHz) A-scan ultrasonography under inhalation anesthesia (for details see (Nickla et al. 1998): on day 1 starting at 12:00, and again on day 5 starting at 12:00. Subsets for each drug (numbers and groups noted below) and saline controls (mid-day; n=7) were measured at 6-hour intervals starting at 12:00, for 24 hours (12:00, 18:00, 0:00, 6:00 and 12:00) to obtain the parameters of the diurnal rhythms in axial length and choroidal thickness. Measurements at night were done under a photographic safe light; they typically took ~ 5 minutes per eye, after which the birds were returned to the dark cage. Refractive errors were measured at the end of the experiment with a Hartinger’s refractometer, without cycloplegia.

Drugs:

Quinpirole:

Quinpirole (10 nmol; (McCarthy et al. 2007; Nickla et al. 2010) was injected at the following times: 7:30 EST (morning; n=12), 12:00 (mid-day; n=13) or 19:30 (evening; n=17). Sub-sets in the mid-day and evening groups (n=6 in each) were measured at 6-hr intervals on the last day. The light cycle was 7:30-19:30.

Pirenzepine:

Two concentrations of pirenzepine were tested at mid-day to determine dosage: 100 mM in the syringe (dose=2 μmol; n=11; (Luft et al. 2003; Nickla et al. 2013) and 10 mM (dose 0.2 μmol; n=10). The higher dose inhibited growth of the lens-wearing eyes to below that of fellow controls (Exp-fellow: −83 μm; paired t-test p=0.027), so we chose the lower dose for the experiment. Injections were done at 8:30 EDT (morning; n=10), 14:00 (mid-day; n=10), or 20:30 (evening; n=18). Subsets in the mid-day (n=6) and evening (n=8) groups were measured at 6-hr intervals on the last day. The light cycle was 8:30-20:30.

Atropine:

Atropine (dose=18 nmol) was injected at the following times: 8:30 EDT (morning; n=18), 14:00 (mid-day; n=12), or 20:30 (evening; n=16). Subsets in the midday group only (n=7) were measured at 6-hr intervals on the last day, because there was no significant effect of “time of day”. The light cycle was 8:30-20:30.

Data Analysis:

Ocular dimensions:

Ocular growth rate was defined as the change in axial length (front of the cornea to the front of the sclera) over the 96 hour period, from noon to noon, and the data are expressed as interocular differences (Exp-Fellow) in the growth rates. The data for choroidal thickness and anterior chamber (front of cornea to front of lens) are similarly expressed. Data for the interocular differences in rate of change for vitreous chamber depth (back of lens to front of retina) are included in Table 1, which shows values with standard errors. Refractive error was measured on day 5 only; data are as expressed as interocular differences in the end refraction. For ocular dimensions and refractive error, statistical analyses of the time-of-day effect used one-way ANOVAs with post-hoc Bonferroni tests on the three experimental (i.e. different times of day) conditions. Conditions for parametric ANOVAs were met (Lavene’s test for homogeneity of variance and Shapiro-Wilk for normality). Two-sample t-tests were used to test differences between drug groups and saline injection controls (two-sample planned comparisons).

Interocular differences in rates of change (X-C Δ) for ocular dimensions, and interocular difference in end refractive errors (X-C RE), with standard errors and ANOVA levels of significance across the three injection times for all three drug groups.

QUINPIROLE PIRENZEPINE ATROPINE ANOVA
AM MID PM AM MID PM AM MID PM Q P A
X-C Δ Axial 0 (49) −68 (38) 118 (37) 115 (51) 79 (26) 215 (37) 59 (32) −28 (40) 33 (49) ** * n.s.
X-C Δ Vitre 102 (31) 45 (52) 103 (31) 210 (51) 210 (24) 296 (41) 115 (39) 31 (38) 58 (54) n.s. n.s. n.s.
X-C RE −2.8 (0.5) −1.6 (0.9) −3.4 (0.6) −3.8 (0.9) −3.5 (0.7) −5.6 (0.8) −1.9 (1.8) −1.6 (0.8) −3.4 (1.0) n.s. n.s. n.s.
X-C Δ Chor −71 (18) 0 (5) −48 (14) −66 (17) −48 (13) −10 (19) −43 (13) −20 (9) −90 (21) ** n.s. **
X-C Δ AC −89 (28) −77 (22) −51 (25) −55 (16) −83 (11) −58 (22) 34 (14) 14 (29) 42 (19) n.s. n.s. n.s.
Open in a new tab
*

p<0.05

**

p≤0.01; n.s.: non-significant

Axial: axial length; Vitre: vitreous chamber depth; Chor: choroid thickness; AC: anterior chamber depth; X: experimental eye; C: fellow eye

Diurnal rhythm parameters:

The diurnal rhythm in eye length, as determined by the 6-hour interval measurements on the last day of the experiment, was assessed in 2 ways: first, we calculated the change over each of the four 6-hour intervals (“Interval” data), which includes the “steady state” eye growth occurring over the 24 hour period (i.e the slope of the longitudinal data). Second, the “steady state” growth rate (regression) was subtracted from the data for each individual eye, as described in Nickla et al. (Nickla et al. 1998). To control for the large between-animal variability, these data were normalized to their mean, and the means were plotted as line graphs (“linear regression data”), with standard errors. These data were fitted to a sine wave with a fixed 24-hour period (Nickla et al. 1998). For choroidal thickness, the longitudinal data did not require a subtraction of the regression, because unlike eye length, choroidal thickness did not change in any discernable way (i.e. the slope is about zero). The data were normalized to the mean for consistency with the presentation for axial length. For both the “interval” data and the “longitudinal” data (Figures 2, 3 and 5), two-way ANOVAs for repeated measures were used to assess whether “interval” or “time of day” (respectively) accounted for the variance between the experimental groups only (mid-day and evening for drugs only, not saline). For the “interval” data, to determine between-group differences for each interval, when only two experimental groups were tested (quinpirole mid-day vs. evening) two-sample t-tests were used. When three experimental groups were tested (pirenzepine mid-day, pirenzepine evening, and atropine mid-day), an ANOVA was used with post-hoc Bonferroni corrections. Rhythm amplitude was assessed as the difference between the daytime and night time maxima and minima for both axial length and choroid thickness.

Figure 2.

Figure 2.

Open in a new tab

Axial length rhythm parameters for quinpirole. A. Changes in axial length over 6-hour intervals for mid-day (white bars), evening (black bars) and saline controls (grey bars). A 2-way ANOVA showed that “interval” accounts for the difference between the two experimental groups. B. Mean normalized longitudinal data with the regression subtracted (see methods) for mid-day (blue lines), evening (red lines) and saline controls (green lines), and the fitted sine waves having a fixed 24-hour period. C. Amplitude as derived from the difference between the daytime maximum and nighttime minimum. Error bars are sems. *p<0.05

Figure 3.

Figure 3.

Open in a new tab

Choroidal thickness rhythm parameters for quinpirole. A. Changes in choroidal thickness over 6-hour intervals for mid-day (white bars) and evening (black bars). A 2-way ANOVA showed no significant between-group difference. B. Mean normalized longitudinal data for the mid-day (blue lines), evening (red lines) and saline controls (green lines), and the fitted sine waves having a fixed 24-hour period. A 2-way ANOVA showed that the “time” accounts for the difference between the mid-day and evening groups. Error bars are sems.

Figure 5.

Figure 5.

Open in a new tab

Axial length rhythm parameters for pirenzepine (pirenz) and atropine. A. Changes in axial length over 6-hour intervals for pirenzepine at mid-day (white bars) and evening (black bars), for mid-day atropine (dark grey bars), and for saline controls (light grey bars). In the 6:00-12:00 interval, ANOVA showed a significant difference for the three experimental groups: p=0.002. B. Mean normalized longitudinal data with the regression subtracted (see methods) for the mid-day (blue lines) and evening (red lines) pirenzepine groups, the mid-day atropine group (black line), and saline controls (green line) with the fitted sine waves having a fixed 24-hour period fit. C. Amplitude as derived from the difference between the daytime minima and nighttime maxima. The amplitude for atropine was larger than that for mid-day pirenzepine. Error bars are sems. * p<0.05

Results

I. Quinpirole

Ocular dimensions and refractive error

Quinpirole inhibited ocular growth rate in lens-wearing eyes of the mid-day injection group only, relative to that of saline controls (Figure 1A: Exp-fellow/96hr: −68 vs 112 μm). An ANOVA shows that the effect for mid-day differed significantly from that for evening (p=0.007; −68 μm vs 118 pm/96hr; post-hoc Bonferroni p=0.004) but did not differ from morning. Neither the morning nor evening groups differed significantly from saline controls (0 and 118 μm vs 112 pm; p>0.05 for both), nor did they differ from one another. For vitreous chamber depth as well, the interocular difference data was similar for morning and evening groups (Exp-fellow: 102 and 103 μm respectively, Table 1), supporting a greater efficacy for themid-day group relative to both morning and evening groups. The interocular differences in growth rate for both the evening and saline groups were significant (paired t-test: p=0.007 and 0.038 respectively).

Figure 1.

Figure 1.

Open in a new tab

Ocular dimensions for quinpirole. A. Interocular differences (Exp-fellow) in growth rates (change in axial length/96 hrs) for the three experimental groups (morning, mid-day and evening) and saline controls. ANOVA for experimental groups: p=0.007. B. Growth rates (change in axial length/96 hrs) for experimental groups and saline controls. ANOVA for experimental groups: p=0.005. C. Interocular differences (Exp-fellow) in refractive error (D) on day 5 for experimental groups and saline controls. D. Interocular differences (Exp-fellow) in choroidal thickness changes for the three experimental groups and saline controls. ANOVA for experimental groups: p=0.002. E. Interocular differences (Exp-fellow) in anterior chamber depth for quinpirole and pirenzepine. Error bars are sems. *p<0.05; **p<0.005.

Similar to the interocular difference data, the growth rate data (change in axial length, Figure 1B), also show a greater growth-inhibition for mid-day versus evening (ANOVA p=0.005; 287 vs 483 μm; post-hoc Bonferroni p=0.004), and those for mid-day differ significantly from saline controls (287 vs 485 μm; p<0.05) while those for evening does not. On day 5, the mean refractive error for the mid-day group was less myopic than that of the other groups (Figure 1C; −1.6 D vs −2.8 D and −3.4 D), but the differences were not significant (ANOVA p=0.1). Choroids in both morning and evening groups thinned in response to the lenses (Figure 1D: −71 μm and −48 μm; paired t-test p<0.05 for both) but those in the mid-day group did not; the effects differed significantly for midday versus morning (ANOVA p=0.002; 0 μm vs −71; p=0.002) and between mid-day and saline controls (t-test, p<0.01). Finally, there was a significant injection effect on anterior chamber growth (Figure 1E; black bars), which was significantly less in injected than in fellow eyes in all groups (all paired t-tests: p<0.05); there were no significant between-group differences (ANOVA p>0.1).

Rhythms in axial length and choroidal thickness

Figure 2A shows the changes in axial length over the successive 6-hour intervals for both mid-day and evening groups and the mid-day saline controls. There was a main effect of “time interval” between the two experimental groups (black vs white bars: 2-way ANOVA F=3.61(3,48); p=0.022). In the interval 18:00-0:00, the evening group (black bars) showed a significantly larger decrease in axial length than did the mid-day group (white bars) (t-test; p=0.005). There were no other significant between-group differences at any other interval. This larger excursion for the evening group can also be seen in the mean normalized longitudinal data in figure 2B, which show a larger fluctuation for evening (red line) compared to mid-day (blue line) (mean sine wave amplitude: 121 μm vs 36 μm). A 2-way ANOVA for the longitudinal data shows that time of day accounts for the difference between mid-day and evening groups (Figure 2B: F(4,64)=5.1, p=0.001). Note that the mean sine wave amplitude for saline controls was similar to that of evening (green lines; 117 μm); this similarity between evening and saline groups was also seen in the interval data (compare black and grey bars, figure 2A). Assessing amplitude as the difference between the daytime and nighttime maxima and minima showed a significantly smaller amplitude for mid-day compared to evening (Figure 2C: 95 μm vs 142 μm; p<0.05). The acrophases (peaks) for mid-day versus evening (means of individual eyes: 18:30 vs 15:25; p>0.1), were not statistically significant.

Figure 3A shows the changes in choroidal thickness over the 6 hour intervals for both groups; a 2-way ANOVA (repeated measures) shows no significant between-group differences as a function of time interval (p>0.1). The mean normalized longitudinal data (Figure 3B; red and blue lines) show that both mid-day and evening groups are well-fitted to a 24-hour sine function, and are similar to that of saline controls (green lines). A 2-way ANOVA shows no significant interaction between the two experimental groups. There is also no significant difference in amplitude (mid-day vs evening: 72 μm vs 45 μm; p>0.1; data not shown).

II. Pirenzepine & Atropine

Ocular dimensions and refractive error

At the dose used, pirenzepine was not fully effective at inhibiting ocular growth rate in the experimental eyes at any of the three injection times: all interocular differences were statistically significant (Exp-fellow, p<0.05), and none differed from those of saline controls (p>0.05 for all) (Figure 4A; black bars). However, an ANOVA on the experimental groups showed that the growth rate of the mid-day group was significantly less than that of the evening group (Figure 4A, black bars: ANOVA p=0.032; 79 μm vs 215 μm; post-hoc Bonferroni p=0.046; Table 1), evincing a partial effect of the mid-day pirenzepine injection. This partial effect is consistent with results from a previous report using the same dose at noon (Nickla et al. 2013). For vitreous chamber depth, the interocular difference data did not differ as a function of injection time (Table 1), and there were no between-group differences in growth rates (Figure 4B, black bars; ANOVA p=0.1). Refractive errors at 5 days also showed no significant differences (Figure 4C, black bars; ANOVA p>0.1), and there no between-group differences in the changes in choroidal thickness (figure 4D, black bars; ANOVA p>0.1). Similar to quinpirole, there was a significant injection effect on anterior chamber growth (Figure 1E; white bars); anterior chambers of injected eyes in all groups grew significantly less than did fellow eyes (all paired t-tests: p<0.05), and there were no significant between-group differences (ANOVA p>0.1).

Figure 4.

Figure 4.

Open in a new tab

Ocular dimensions for pirenzepine (black bars) and atropine (white bars). A. Interocular differences (Exp-fellow) in growth rates (change in axial length/96 hrs) for the three experimental groups (morning, mid-day and evening) and saline controls that were combined for both drugs. For pirenzepine, mid-day growth differed from that of evening (ANOVA p=0.032; post-hoc Bonferroni p<0.05). For atropine, an ANOVA for the three experimental groups did not show significant differences, but mid-day growth was less than that of saline controls (p<0.05). B. Growth rates (change in axial length/96 hrs) for all experimental groups and saline controls combined for both drugs. There were no significant between-group differences for either drug by ANOVA. C. Interocular differences (Exp-fellow) in refractive error (D) on day 5 for both drugs. There were no significant between-group differences for either drug by ANOVA. D. Interocular differences (X-C) in the rate of choroidal thickness changes for all three experimental groups and saline controls for both drugs. ANOVA for experimental groups: p=0.01. Error bars are sems. *p<0.05

Atropine, in contrast to pirenzepine, prevented the excessive lens-induced ocular elongation in all groups, as the interocular differences were not significant for any injection time (Figure 4A, white bars). Furthermore, there were no significant between-group differences (ANOVA p>0.1). However, only the effects in the mid-day group differed significantly from those of saline controls (Exp-fellow: −28 vs 152 μm/96 hr; ANOVA p=0.011; post-hoc Bonferroni p=0.018). There were also no significant between-group differences in growth rates (Figure 4B; white bars, ANOVA p>0.1) or in refractive errors on day 5 (Figure 4C, white bars). However, choroids thinned significantly more in the evening group compared to the mid-day group (Figure 4D, white bars; ANOVA p=0.01: −90 vs −20 μm; post-hoc Bonferroni p=0.01). There was no significant “injection effect” on growth of the anterior chamber (data not shown).

Rhythms in axial length and choroidal thickness

Figure 5A shows the changes in axial length over the 6-hour intervals for mid-day and evening pirenzepine groups, mid-day atropine, and mid-day saline controls. For pirenzepine (black and white bars), one-way ANOVAs showed no significant effect as a function of time in either group, and there was no significant between-group effect (2-way ANOVA, p=0.7). For atropine, however (dark grey bars), there was a significant effect of interval (one-way ANOVA, p<0.0001). In the interval 6:00-12:00, atropine-injected eyes grew faster than those of both pirenzepine groups (ANOVA p=0.002; post-hoc Bonferroni p<0.05 for both comparisons). The mean normalized longitudinal data (Figure 5B; red and blue lines) show that axial length functions for both pirenzepine groups were sinusoidal, and reasonably fitted to a 24-hr sine function, similar to that of saline controls (green line); a 2-way ANOVA for the two experimental groups showed no significant interaction between group and time of day (p>0.1). The mean sine wave acrophases for both groups were similar to those of saline controls (Figure 5B: evening, mid-day and saline: 15:35, 16:10, 13:45, respectively). The same was also true for atropine (14:45). Note, however, that the mean sine-wave amplitudes for both pirenzepine groups were smaller than those of saline controls and atropine (45 μm and 41 μm vs. 118 μm and 132 μm, respectively). Statistical analysis of the maxima vs minima data showed no differences between the effects of pirenzepine and saline (Figure 5C: mid-day, evening, saline: 74 μm, 67 μm, 110 μm respectively; ANOVA p=0.4). However, a comparison of the mid-day pirenzepine versus the mid-day atropine showed a larger amplitude for atropine (145 μm vs 74 μm; two sample t-test, p=0.024).

Figure 6A shows the changes in choroid thickness over the 6-hour intervals for the three experimental groups, and figure 6B shows the mean longitudinal data. There were no significant between-group differences at any interval (Figure 6A), nor did any experimental group differ from that of saline controls (data not shown). Statistical analysis of the maxima vs minima data showed no significant differences in amplitude (mid-day, evening, saline, atropine: 48 μm, 42 μm, 25 μm, 36 μm respectively; ANOVA p>0.5; data not shown).

Figure 6.

Figure 6.

Open in a new tab

Choroidal thickness rhythm parameters for pirenzepine (pirenz) and atropine. A. Changes in choroidal thickness over 6-hour intervals for pirenzepine at mid-day (white bars) and evening (black bars), and mid-day atropine (grey bars). There were no significant between-group differences. B. Mean normalized longitudinal data for the mid-day (blue line) and evening (red line) pirenzepine groups, mid-day atropine (black line) and saline controls (green line). Error bars are sems.

In summary, the growth-inhibiting effects of both quinpirole and pirenzepine in negative-lens-wearing eyes were most effective when given at mid-day, however, there were no injection-time-dependent differences for atropine. For quinpirole, but not for pirenzepine, the more-effective mid-day injections were associated with a decrease in amplitude of the rhythm in axial length.

Discussion

We asked whether the efficacy of three different drugs, all of which inhibit the development of experimental myopia in animal models, depended on the time of day at which they were injected. We found that both quinpirole and pirenzepine were more effective when given in the middle of the day as opposed to evening; in fact, evening injections were ineffective. Although atropine appeared to be effective at all three injection times, only the mid-day injection effect differed from that of the saline controls. This is consistent with the inhibition of the minus lens-induced choroidal thinning for the mid-day group alone. For quinpirole only, the amplitude of the rhythm in axial length for the mid-day injection group was significantly smaller than that of the evening group.

Potential mechanisms

This study was prompted by prior studies that found time-of-day-dependent differences in the ocular growth responses to myopic versus hyperopic defocus, and to exposures to brief periods of vision in form-deprived eyes (Ohngemach et al. 2001; Nickla et al. 2017a; Nickla et al. 2017b). Specifically, brief myopic defocus is more effective at inhibiting ocular growth later in the day (Ohngemach et al. 2001; Nickla et al. 2017a), while brief hyperopic defocus is more effective at stimulating growth early in the day (Nickla et al. 2017b), suggesting diurnal variations in one or more of the growth processes involved in the signal cascade from retina through the choroid, to the sclera. This time-dependent process could occur at any of these tissue levels, as all three have diurnal oscillations in one or more processes: the retina has numerous diurnal rhythms, including dopamine and melatonin synthesis and release, horizontal cell coupling, and retinomotor movements (review: (Besharse and McMahon 2016). The choroid exhibits diurnal oscillations in thickness (Nickla et al. 1998; Papastergiou et al. 1998; Brown et al. 2009; Chakraborty et al. 2011), and retinoic acid synthesis (Mertz et al. 1999; Nickla and Mertz 2002). The chick sclera shows oscillations in extracellular matrix synthesis (Nickla et al. 1999). There are diurnal fluctuations in receptor binding for some ligands, which may explain the diurnally-dependent efficacies. For example, D2/D4 dopamine receptor-binding in photoreceptors is rhythmic in mice and rats (Bai et al. 2008; Klitten et al. 2008), being lower during the day and higher at night; hence it is possible that dopamine receptors on other retinal neurons (and in other species) such as those involved in the signal cascade regulating ocular growth, are also rhythmic.

For both quinpirole and pirenzepine, the efficacy at growth-inhibition in the lens-wearing eyes was greater for mid-day compared to evening, with evening being no different than the saline controls. One obvious visual difference between the two injection times is the presence of light in the hours following drug application for the mid-day group, as opposed to an absence of light in the evening group, suggesting that the processes initiated by the binding of the ligand may require light, or form (unobstructed) vision, to exert their effect. However, the report that quinpirole was equally effective at inhibiting eye growth in form-deprived chicks when given during a 3-hour (daytime) dark period as it was in the light (McCarthy et al. 2007) argues against this hypothesis. Nevertheless, it is possible that in the earlier study, the 3 hours in darkness (followed by light) was not long enough to oppose the effect of the drug, but the 12-hours of darkness in our experiment was sufficient. If the mechanism was unrelated to the presence or absence of light in the time following drug administration, would suggest that the efficacy was related to phase. Perhaps maximal efficacy requires the concurrence of ligand with a specific phase of another rhythmic process. For example, the choroid is thinnest at mid-day (Nickla et al. 1998), perhaps reflecting a lessening of a diffusion barrier between retina and sclera. Elucidation of the precise mechanism requires further study. The efficacy of atropine was independent of injection-time, at the sub-maximal dose used here (Schmid and Wildsoet 2004).

Rhythms in axial length and choroidal thickness:

We speculated that time-dependent differences in efficacy might be associated with alterations in the rhythm parameters for axial length or choroidal thickness; this was not the case. The only significant effect on rhythm parameter was a decrease in the amplitude of the rhythm in axial length in the mid-day (effective) quinpirole group, compared to that for the (ineffective) evening group. However, the (smaller) mid-day amplitude did not differ from those of both the effective and ineffective pirenzepine groups, refuting a mechanistic association between amplitude and efficacy of growth inhibition. Similarly, for atropine, the amplitude of the mid-day (most effective) injection was larger than that for the effective mid-day pirenzepine group, and similar to that of the ineffective evening quinpirole group. The lack of associations between growth rate and axial rhythm amplitude is consistent with previous reports that found no consistent correlations between the growth effects of brief periods of defocus and axial rhythm amplitude (Nickla et al. 2017a; Nickla et al. 2017b). There were no statistically significant effects on phase for either rhythm, for any of the three drugs.

In conclusion, the effectiveness of quinpirole and pirenzepine at inhibiting eye growth is optimal when drugs are given mid-day. By contrast, atropine is effective at all times, with a tendency toward greater efficacy at mid-day. These findings might support giving latitude for treatment times when prescribing low dose atropine for myopic children.

Highlights.

Quinpirole is most effective at inhibiting eye growth when given at noon.

Pirenzepine is most effective at inhibiting eye growth when given at noon.

Noon injections of quinpirole “normalizes” the amplitude in the axial rhythm.

Atropine is effective at inhibiting eye growth at all three times of day.

Acknowledgements:

This work was funded by NIH-NEI-013636 and NIH-NEI-025307. The authors thank Dr. Shanta Sarfare, Kristen Totonelly and Jonathan Elin-Calcador for collecting some of the data, and Dr. Chris Taylor for advice on statistics.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Bai L, Zimmer S, Rickes O, Rohleder N, Holthues H, Engel L, Leube R and Spessert R (2008). Daily oscillation of gene expression in the retina is phase-advanced with respect to the pineal gland. Brain Res. 1203: 89–96. [DOI] [PubMed] [Google Scholar]
  2. Bedrossian RH (1971). The effect of atropine on myopia. Ann. Ophthalmol 3: 891–897. [PubMed] [Google Scholar]
  3. Bedrossian RH (1979). The effect of atropine on myopia. Ophthamol. 86: 713–717. [DOI] [PubMed] [Google Scholar]
  4. Besharse J and McMahon D (2016). The retina and other light sensitive ocular clocks. J. Biol. Rhythms 31: 223–243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown JS, Flitcroft DI, Ying G, Francis EL, Schmid GF, Quinn GE and Stone RD (2009). In vivo human choroidal thickness measurements: Evidence for diurnal fluctuations. Invest Ophthalmol Vis Sci 50: 5–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chakraborty R, Ostrin L, Nickla D, Iuvone P, Pardue M and Stone RA (2018). Circadian rhythms, refractive development, and myopia. Ophthal Physiol Opt 38: 217–245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chakraborty R, Read SA and Collins MJ (2011). Diurnal variations in axial length, choroidal thickness, intraocular pressure, and ocular biometrics. Invest. Ophthalmol. Vis. Sci 52: 5121–5129. [DOI] [PubMed] [Google Scholar]
  8. Chia A, Chua W and Cheung Y (2012). Atropine for the treatment of childhood myopia: safety and efficacy of 0.5%, 0.1% and 0.001% doses. Ophthalmology 119: 347–354. [DOI] [PubMed] [Google Scholar]
  9. Cottriall CL and McBrien N (1996). The m1 muscarinic antagonist pirenzepine reduces myopia and eye enlargment in the tree shrew. Invest Ophthalmol Vis Sci 37: 1368–1379. [PubMed] [Google Scholar]
  10. Diether S, Schaeffel F, Lambrou GN, Fritsch C and Trendelenburg A (2007). Effects of intravitreally and intraperitoneally injected atropine on two types of experimental myopia in chicken. Exp. Eye Res. 84: 266–274. [DOI] [PubMed] [Google Scholar]
  11. Fan DS, Lam DSC, Lam R and al., e. (2004). Prevalence, incidence and progression of myopia of school children in Hong Kong. Invest. Ophthalmol. Vis. Sci 45: 1071–1075. [DOI] [PubMed] [Google Scholar]
  12. Feldkaemper M and Schaeffel F (2013). An updated view on the role of dopamine in myopia. Exp. Eye Res. 114: 106–119. [DOI] [PubMed] [Google Scholar]
  13. Goh WS and Lam CS (1994). Changes in refractive trends and optical components of Hong Kong Chinese aged 19-39 years. Ophthal Physiol Opt 14: 378–382. [PubMed] [Google Scholar]
  14. He M, Zeng J, Liu Y, Xu J, Pokharel G and Ellwein L (2004). Refractive error and visual impairment in urban children in southern China. Invest. Ophthalmol. Vis. Sci 45: 793–799. [DOI] [PubMed] [Google Scholar]
  15. Jakubik J and Tucek S (1994). Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation. Brit. J Pharmacol. 113: 1529–1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jordan K, Totonelly K and Nickla D (2017). The efficacy of the dopamine D2 agonist quinpirole at inhibiting ocular growth in chicks is dependent on time of day. ARVO E-Abstract#5461.
  17. Klitten LL, Rath MF, Coon SL, Kim J-S, Klein DC and Moeller M (2008). Localization and regulation of dopamine receptor D4 expression in the adult and developing rat retina. Exp. Eye Res. 87: 471–477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin LL and al. e. (1996). Epidemiological study of ocular refractions among school children (aged 6 through 18) in Taiwan. Invest. Ophthalmol. Vis. Sci 37: S1002. [Google Scholar]
  19. Lin LL, Shih YF, Hsiao CK and J CC (2004). Prevalence of myopia in Taiwanese schoolchildren: 1983–2000. Ann. Acad. Med. Singapore 33: 27–33. [PubMed] [Google Scholar]
  20. Luft W, Ming Y and Stell W (2003). Variable effects of previously untested muscarinic receptor antagonists on experimental myopia. Invest Ophthalmol Vis Sci 44: 1330–1338. [DOI] [PubMed] [Google Scholar]
  21. McBrien NA, Moghaddam HO, New R and Williams LR (1993a). Experimental myopia in a diurnal mammal (Sciurus carolinensis) with no accommodative ability. J. Physiol 469: 427–441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McBrien NA, Moghaddam HO and Reeder AP (1993b). Atropine reduces experimental myopia and eye enlargement via a nonaccommodative mechanism. Investigative Ophthalmology & Visual Science 34(1): 205–215. [PubMed] [Google Scholar]
  23. McCarthy CS, Megaw P, Devadas M and Morgan I (2007). Dopaminergic agents affect the ability of brief periods of normal vision to prevent form-deprivation myopia. Exp. Eye Res. 84: 100–107. [DOI] [PubMed] [Google Scholar]
  24. Mertz J, Howlett M, McFadden S and Wallman J (1999). Retinoic acid from both the retina and choroid influences eye growth. Invest Ophthalmol Vis Sci 40: S849. [Google Scholar]
  25. Nickla D (2005). The phase relationships between the diurnal rhythm in axial length and choroidal thickness and the association with ocular growth rate in chicks. J. Comp. Physiol. A 192: 399–407. [DOI] [PubMed] [Google Scholar]
  26. Nickla D, Jordan K, Yang J and Totonelly K (2017b). Brief hyperopic defocus or form deprivation have varying effects on eye growth and ocular rhythms depending on the time-of-day of exposure. Exp. Eye Res. 161: 132–142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nickla D and Mertz J (2002). All-trans-retinoic acid in chick suprachoroidal fluid shows a diurnal rhythm. Invest Ophthalmol Vis Sci ARVO supplement: E-Abstract 208.
  28. Nickla D, Thai P, Zanerkia-Trahan R and Totonelly K (2017a). Myopic defocus in the evening is more effective at inhibiting eye growth than defocus in the morning: Effects on rhythms in axial length and choroidal thickness in chicks. Exp. Eye Res. 154: 104–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nickla DL, Rada JA and Wallman J (1999). Isolated chick sclera shows a circadian rhythm in proteoglycan synthesis perhaps associated with the rhythm in ocular elongation. J Comp Physiol [A] 185(1): 81–90. [DOI] [PubMed] [Google Scholar]
  30. Nickla DL, Totonelly K and Dhillon B (2010). Dopaminergic agonists that result in ocular growth inhibition also elicit transient increases in choroidal thickness in chicks. Exp. Eye Res. 91: 715–720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nickla DL, Wildsoet C and Wallman J (1998). Visual influences on diurnal rhythms in ocular length and choroidal thickness in chick eyes. Exp. Eye Res. 66: 163–181. [DOI] [PubMed] [Google Scholar]
  32. Nickla DL, Zhu X and Wallman J (2013). Effects of muscarinic agents on chick choroids in intact eyes and eyecups: evidence for a muscarinic mechanism in choroidal thinning. Ophthalmic Physiol. Optics 33: 245–256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohngemach S, Feldkaemper MP and Schaeffel F (2001). Pineal control of the dopamine D2-receptor gene and dopamine release in the retina of the chicken and their possible relation to growth rhythms of the eye. J. Pineal Res. 31: 145–154. [DOI] [PubMed] [Google Scholar]
  34. Pan CW, Ramamurthy D and Saw S-M (2012). World-wide prevalence and risk factors for myopia. Ophthal Physiol Opt 32: 3–16. [DOI] [PubMed] [Google Scholar]
  35. Papastergiou GI, Schmid GF, Riva CE, Mendel MJ, Stone RA and Laties AM (1998). Ocular axial length and choroidal thickness in newly hatched chicks and one-year-old chickens fluctuate in a diurnal pattern that is influenced by visual experience and intraocular pressure changes. Exp. Eye Res. 66: 195–205. [DOI] [PubMed] [Google Scholar]
  36. Qian YS, Chu RY, He JC and al., e (2009). Incidence of myopia in high school students with and without red-green vision deficiency. Invest. Ophthalmol. Vis. Sci 50: 1598–1605. [DOI] [PubMed] [Google Scholar]
  37. Schmid KL and Wildsoet C (2004). Inhibitory effects of apomorphine and atropine and their combination on myopia in chicks. Optometry Vis. Sci. 81: 137–147. [DOI] [PubMed] [Google Scholar]
  38. Shih YF, Chen CH, Chou AC, Ho TC, Lin LL and Hung PT (1999). Effects of different concentrations of atropine on controlling myopia in myopic children. J. Ocular Pharm. Therapeut. 15: 85–90. [DOI] [PubMed] [Google Scholar]
  39. Siatkowski RM, Cotter S, Miller JM, Scher CA, Crockett RS and Novack GD (2004). Safety and efficacy of 2% pirenzepine ophthalmic gel in children with myopia. Arch Ophthamol. 122: 1667–1674. [DOI] [PubMed] [Google Scholar]
  40. Stone RA, Lin T and Laties AM (1991). Muscarinic antagonist effects on experimental chick myopia. Experimental Eye Research 52: 755–7588. [DOI] [PubMed] [Google Scholar]
  41. Stone RA, Lin T, Laties AM and Iuvone PM (1989). Retinal dopamine and form-deprivation myopia. Proc. Nat. Acad. Sci 86(2): 704–706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stone RA, Pardue M, Iuvone P and Khurana T (2013). Pharmacology of myopia and potential role for intrinsic retinal circadian rhythms. Exp. Eye Res. 114: 35–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tan DTH, Lam DS, Chua WH, Shu-Ping DF and Crockett RS (2005). One-year multicenter, double-masked, placebo-controlled, parallel saftey and efficacy study of 2% pirenzepine ophthalmic gel in children with myopia. Ophthalmology 112: 84–91. [DOI] [PubMed] [Google Scholar]
  44. Tietje KM and Nathanson NM (1991). Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. J Biol Chem. 266: 17382–17387. [PubMed] [Google Scholar]

RESOURCES