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Recent evidence indicates that moderate levels of blue light are sufficient to suppress the nighttime rise in serum melatonin in humans, suggesting that luminous screens may be deleterious to sleep cycles and to other functions. Little is known however, about the effects of exposures to blue light on ocular physiology. We tested the effects of transient blue light exposures of various illuminances on ocular growth rates and ocular rhythms in chicks. 10-day old chicks were exposed to narrow band blue light (460 nm) of specific illuminance for 4 h in the evening (ZT8-ZT12) or the morning (ZT0-ZT4) for 9 days; for the remainder of the day they were in white light (588 lux). For the evening, four illuminances were tested: 0.15 lux (n = 15), 200 lux (radiometrically matched to white controls; n = 16), 600 lux (photometrically matched to white controls; n = 15) or 1000 lux (n = 8). The 600 lux condition was also tested using a 2-h duration (n = 8). The 200 and 600 lux conditions were extended to 14 and 21 days (n = 8 each). For morning exposures, 200 lux (n = 9), 600 lux (n = 9) and 1000 lux (n = 8) were tested. Controls remained in white light (n = 23). Ocular dimensions were measured by A-scan ultrasonography on days 1 and 9 to assess growth rates. On day 8 or 9, measurements were made at 6-h intervals over 24 h starting at noon to assess rhythm parameters. Evening exposure to blue light stimulated ocular growth rates relative to controls for all except the bright condition (0.15 lux, 200 lux, 600 lux vs bright and white respectively: 845 μm, 838 μm, 898 μm vs 733 μm and 766 μm; p 0.05 for all comparisons). 2 h exposures to 600 lux were similarly effective (915 μm vs 766 μm; p 0.05). Morning exposures only resulted in growth stimulation for the 200 lux condition (200 lux vs white: 884 μm vs 766 μm; p 0.05). Furthermore, for this group only, growth of the anterior chamber had a significant contribution to the overall effect (vs white: p 0.05), and choroids showed significant thickening. For evening exposures to 200 and 600 lux, the growth stimulatory effect lasted for 14 days (p = 0.01); by 21 days only the 600 lux group still differed (p 0.0001). Evening exposures caused circadian disruptions in the choroidal thickness rhythms, and morning exposures disrupted both axial and choroidal rhythms. Exposure to 4 h of blue light at lower illuminances (less than 1000 lux) at transition times of lights-on and lights-off stimulates ocular growth rates and affects ocular rhythms in chicks, suggesting that such exposures may be deleterious to emmetropization in children.

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 h. For quinpirole and pirenzepine, subsets (n's in Methods) of mid-day and evening groups were measured at 6 h 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/96h; post-hoc Bonferroni p = 0.016; pirenzepine: 79 vs 215 μm/96h; 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 were no consistent time-dependent alterations in rhythm parameters for any of the drugs.

Evidence suggests that the relevant variable in the anti-myopigenic effect of increased time spent outdoors is the increase in light intensity. Because light is the strongest Zeitgeber, it is plausible that the effects of bright light exposure depend on time of day, and may impact circadian rhythms. In these studies, we asked whether the effects on eye growth rates and ocular rhythms of brief daily exposures to bright light differed depending on time of day in eyes developing myopia in response to form deprivation (FD) or negative lens-induced hyperopic defocus (LENS). We also studied the effects of concurrent exposures to brief hyperopic defocus and bright light. Exp. 1: Starting at 12d, chicks wearing monocular diffusers or −10 D lenses were exposed to 3 daily hours (hr) of bright light (30K lux) in the morning (FD: n=12; LENS: n=7) or evening (FD: n=21; LENS: n=7) for 6 daily exposures. Controls wore diffusers or lenses but weren’t exposed to bright light (FD: n=14; LENS: n=9). Exp. 2: Untreated chicks were exposed to bright light in the morning (n=12) or evening (n=14) for 6 exposures. Controls were not exposed to bright light (n=11). Exp. 3: Chicks were exposed to 2 hr simultaneous monocular hyperopic defocus and bright light in the morning (n=11), mid-day (n=7) or evening (n=8) for 5 exposures. “Not bright” lens-wearing controls were data from published work (Nickla et al., 2017). High frequency A-scan ultrasonography was done at the start and end to measure growth rates. The FD group in Exp. 1 and the morning and evening groups in Exp. 3 were measured at 6-hr intervals over the final 24 hr to determine parameters for the rhythms in axial length and choroidal thickness. Brief bright light in the evening inhibited eye growth in eyes wearing diffusers or lenses relative to controls (X vs C: FD: 316 vs 468 μm; p=0.026; LENS: 233 vs 438 μm; p=0.03); morning bright light had no effect. There was no differential effect of time of day of exposures on the rhythm in axial length; for choroid thickness, “time” accounted for the variance between groups (2-way ANOVA interaction p=0.027). In untreated eyes, bright light in the morning had a small but significant inhibitory effect relative to evening exposures (803 μm vs 679 μm; post-hoc p=0.048). Eyes exposed to simultaneous hyperopic defocus and bright light were significantly more inhibited relative to “not bright” controls for the morning exposures (interocular differences: −207 vs 69 μm; p

Purpose Despite extensive research, mechanisms regulating postnatal eye growth and those responsible for ametropias are poorly understood. With the marked recent increases in myopia prevalence, robust and biologically-based clinical therapies to normalize refractive development in childhood are needed. Here, we review classic and contemporary literature about how circadian biology might provide clues to develop a framework to improve the understanding of myopia etiology, and possibly lead to rational approaches to ameliorate refractive errors developing in children. Recent findings Increasing evidence implicates diurnal and circadian rhythms in eye growth and refractive error development. In both humans and animals, ocular length and other anatomical and physiological features of the eye undergo diurnal oscillations. Systemically, such rhythms are primarily generated by the 'master clock' in the surpachiasmatic nucleus, which receives input from the intrinsically photosensitive retinal ganglion cells (ipRGCs) through the activation of the photopigment melanopsin. The retina also has an endogenous circadian clock. In laboratory animals developing experimental myopia, oscillations of ocular parameters are perturbed. Retinal signaling is now believed to influence refractive development; dopamine, an important neurotransmitter found in the retina, not only entrains intrinsic retinal rhythms to the light:dark cycle, but it also modulates refractive development. Circadian clocks comprise a transcription/translation feedback control mechanism utilizing so-called clock genes that have now been associated with experimental ametropias. Contemporary clinical research is also reviving ideas first proposed in the nineteenth century that light exposures might impact refraction in children. As a result, properties of ambient lighting are being investigated in refractive development. In other areas of medical science, circadian dysregulation is now thought to impact many non-ocular disorders, likely because the patterns of modern artificial lighting exert adverse physiological effects on circadian pacemakers. How, or if, such modern light exposures and circadian dysregulation contribute to refractive development is not known. Summary The premise of this review is that circadian biology could be a productive area worthy of increased investigation, which might lead to the improved understanding of refractive development and improved therapeutic interventions.

Animal models have shown that myopic defocus is a potent inhibitor of ocular growth: brief (1-2 h) daily periods of defocus are sufficient to counter the effects of much longer periods of hyperopic defocus, or emmetropic vision. While the variables of duration and frequency have been well-documented with regard to effect, we ask whether the efficacy of the exposures might also depend on the time of day that they are given. We also ask whether there are differential effects on the rhythms in axial length or choroidal thickness. 2-week-old chickens were divided into 2 groups: (1) "2-hr lens-wear". Chicks wore monocular +10D lenses for 2 h per day for 5 days at one of 3 times of day: 5:30 a.m. (n = 11), 12 p.m. (n = 8) or 7:30 p.m. (n = 11). (2) "2-hr minus lens-removal". Chicks wore monocular -10D lenses continually for 7 days, except for a 2-hr period when lenses were removed; the removal occurred at one of 2 times: 5:30 a.m. (n = 8) or 7:30 p.m. (n = 8). Both paradigms exposed eyes to brief myopic defocus that differed in its magnitude, and in the visual experience for the rest of the day. High frequency A-scan ultrasonography was done at the start of the experiment; on the last day, it was done at 6-hr intervals, starting at noon, over 24-hr, to assess rhythm parameters. Refractive errors were measured using a Hartinger's refractometer at the end. In both paradigms, myopic defocus in the evening was significantly more effective at inhibiting eye growth than in the morning ("2-hr lens-wear": X-C: -149 vs -83 μm/5d; "2-hr lens-removal": X-C: 91 vs 245 μm/7d; post-hoc Bonferroni test, p

In chicks, the diurnal patterns of retinal dopamine synthesis and release are associated with refractive development. To assess the within-day patterns of dopamine release, we assayed vitreal levels of DOPAC (3,4-dihydroxyphenylacetic acid) using high performance liquid chromatography with electrochemical detection, at 4-hour intervals over 24 hours in eyes with experimental manipulations that change ocular growth rates. Chicks were reared under a 12 hour light/12 hour dark cycle; experiments began at 12 days of age. Output was assessed by modelling using the robust variance structure of Generalized Estimating Equations. CONTINUOUS SPECTACLE LENS-DEFOCUS OR FORM DEPRIVATION: One group experienced non-restricted visual input to both eyes and served as untreated “normal” controls. Three experimental cohorts underwent monocular visual alterations known to alter eye growth and refraction: wearing a diffuser, a negative lens or a positive lens. After one full day of device-wear, chicks were euthanized at 4-hour intervals over 24 hours (8 birds per time/condition). BRIEF HYPEROPIC DEFOCUS: Chicks wore negative lenses for only 2 daily hours either in the morning (starting at ZT 0; n=16) or mid-day (starting at ZT 4; n=8) for 3 days. Vitreal DOPAC was assayed. In chicks with bilateral non-restricted vision, or with continuous defocus or form-deprivation, there was a diurnal variation in vitreal DOPAC levels for all eyes (p

It is generally accepted that myopic defocus is a more potent signal to the emmetropization system than hyperopic defocus: one hour per day of myopic defocus cancels out 11 h of hyperopic defocus. However, we have recently shown that the potency of brief episodes of myopic defocus at inhibiting eye growth depends on the time of day of exposure. We here ask if this will also be true of the responses to brief periods of hyperopic defocus: may integration of the signal depend on time of day? If so, are the rhythms in axial length and choroidal thickness altered? Hyperopic defocus: Birds had one eye exposed to hyperopic defocus by the wearing of -10D lenses for 2 or 6 h at one of 3 times of day for 5 days: Morning (7 am - 9 am: n = 13; 7 am - 1 pm: n = 6), Mid-day (12 pm - 2 pm: n = 20; 10 am - 4 pm: n = 8), or Evening (7 pm - 9 pm: n = 12; 2 pm - 8 pm: n = 11). A separate group wore monocular lenses continually as a control (n = 12). Form deprivation: Birds wore a diffuser over one eye for 2 h at one of 3 times of day for 5 days: Morning (n = 12); Mid-day (n = 19) or Evening (n = 6). For all groups, ocular dimensions were measured using high-frequency A-scan ultrasonography at noon on the first day, under inhalation anesthesia. On day 5, eye dimensions were re-measured at 12 pm, and refractive errors were measured using a Hartinger's refractometer. A subset of birds in the 2-h lens group (morning, n = 8; mid-day, n = 8; evening, n = 6), and the deprivation group (n = 6 per time point), were also measured at 6 pm, 12 am, 6 am and 12 pm on the last day of exposure, to obtain the parameters of the diurnal rhythms in axial length and choroidal thickness. The effects of 2 h of defocus depended on time of day of exposure: it stimulated eye growth when exposure was in the morning and inhibited it when it was at mid-day (change in vitreous chamber, X-C; ANOVA p

The muscarinic M4 receptor antagonist MT3 (muscarinic toxin 3) is effective at inhibiting the development of myopia in response to form deprivation, and prevents the deprivation-induced choroidal thinning. We asked if it was equally effective in eyes wearing negative lenses.Chicks wore monocular diffusers or -15 D lenses for 7 days. Intravitreal injections of MT3 (90 nmoles) were given on days 2, 4 and 6 (diffusers: n = 13; lenses: n = 12); saline was used as injection controls (diffusers: n = 11; lenses: n = 13). Ocular dimensions were measured with A-scan ultrasound on days 1 and 7. Refractions were measured using a Hartinger's refractometer. A third group of "normal" chicks received monocular injections of drug (n = 7) or saline (n = 7), and eyes were measured 3 and 72 h later.MT3 inhibited the myopia in response to form deprivation, but did not affect the compensation to negative lenses (drug versus saline: FD: -3.2 versus -7.4 D; p 0.001; Lenses: -4.5 versus -4.9 D). The myopia inhibition in deprived eyes was due to inhibition of axial growth (610 µm versus 827 µm; p 0.005); lens-wearing eyes grew similar to saline controls (747 µm versus 743 µm). There was no effect of the drug on the choroidal thinning in either condition. Unexpectedly, MT3 produced choroidal thinning in normal eyes (drug versus saline: -45 versus 16 µm/3 h; p 0.05), but had no effect on refractions or ocular growth.MT3 does not inhibit the development of myopia in response to hyperopic defocus. It also causes choroidal thinning, an anomalous effect for a muscarinic receptor antagonist. These results support the existence of different muscarinic mechanisms in the excessive eye growth resulting from the open-loop condition of form deprivation, versus that of hyperopic defocus, a closed-loop condition.

Many ocular processes show diurnal oscillations that optimize retinal function under the different conditions of ambient illumination encountered over the course of the 24 h light/dark cycle. Abolishing the diurnal cues by the use of constant darkness or constant light results in excessive ocular elongation, corneal flattening, and attendant refractive errors. A prevailing hypothesis is that the absence of the Zeitgeber of light and dark alters ocular circadian rhythms in some manner, and results in an inability of the eye to regulate its growth in order to achieve emmetropia, the matching of the front optics to eye length. Another visual manipulation that results in the eye growth system going into a "default" mode of excessive growth is form deprivation, in which a translucent diffuser deprives the eye of visual transients (spatial or temporal) while not significantly reducing light levels; these eyes rapidly elongate and become myopic. It has been hypothesized that form deprivation might constitute a type of "constant condition" whereby the absence of visual transients drives the eye into a similar default mode as that in response to constant light or dark. Interest in the potential influence of light cycles and ambient lighting in human myopia development has been spurred by a recent study showing a positive association between the amount of time that children spent outdoors and a reduced prevalence of myopia. The growing eyes of chickens and monkeys show a diurnal rhythm in axial length: Eyes elongate more during the day than during the night. There is also a rhythm in choroidal thickness that is in approximate anti-phase to the rhythm in eye length. The phases are altered in eyes growing too fast, in response to form deprivation or negative lenses, or too slowly, in response to myopic defocus, suggesting an influence of phase on the emmetropization system. Other potential rhythmic influences include dopamine and melatonin, which form a reciprocal feedback loop, and signal "day" and "night" respectively. Retinal dopamine is reduced during the day in form deprived myopic eyes, and dopamine D2 agonists inhibit ocular growth in animal models. Rhythms in intraocular pressure as well, may influence eye growth, perhaps as a mechanical stimulus triggering changes in scleral extracellular matrix synthesis. Finally, evidence shows varying influences of environmental lighting parameters on the emmetropization system, such as high intensity light being protective against myopia in chickens. This review will cover the evidence for the possible influence of these various factors on ocular growth. The recognition that ocular rhythms may play a role in emmetropization is a first step toward understanding how they may be manipulated in treatment therapies to prevent myopia in humans.

Changes in ocular growth that lead to myopia or hyperopia are associated with alterations in the circadian rhythms in eye growth, choroidal thickness and intraocular pressure in animal models of emmetropization. Recent studies have shown that light at night has deleterious effects on human health, acting via “circadian disruptions” of various diurnal rhythms, including changes in phase or amplitude. The purpose of this study was to determine the effects of brief, 2-h episodes of light in the middle of the night on the rhythms in axial length and choroidal thickness, and whether these alter eye growth and refractive error in the chick model of myopia. Starting at 2 weeks of age, birds received 2 h of light between 12:00 am and 2:00 am for 7 days (n = 12; total hours of light: 14 h). Age-matched controls had a continuous dark night (n = 14; 14L/10D). Ocular dimensions were measured using high-frequency A-scan ultrasonography on the first day of the experiment, and again on day 7, at 6-h intervals, starting at noon (12 pm, 6 pm, 12 am, 6 am, 12 pm). Measurements during the night were done under a photographic safe-light. These data were used to determine rhythm parameters of phase and amplitude. 2 groups of birds, both experimental (light at night) and control, were measured with ultrasound at various intervals over the course of 4 weeks to determine growth rates. Refractive errors were measured in 6 experimental and 6 control birds at the end of 2 weeks. Eyes of birds in a normal L/D cycle showed sinusoidal 24-h period diurnal rhythms in axial length and choroid thickness. Light in the middle of the night caused changes in both the rhythms in axial length and choroidal thickness, such that neither could be fit to a sine function having a period of 24 h. Light caused an acute, transient stimulation in ocular growth rate in the subsequent 6-h period (12 am–6 am), that may be responsible for the increased growth rate seen 4 weeks later, and the more myopic refractive error. It also abolished the increase in choroidal thickness that normally occurs between 6 pm and 12 am. We conclude that light at night alters the rhythms in axial length and choroidal thickness in an animal model of eye growth, and that these circadian disruptions might lead to the development of ametropias. These results have implications for the use of light during the night in children.

Ciliary ganglionectomy inhibits the development of myopia in chicks (Schmid et al., 1999), but has no effect on the compensatory responses to spectacle lenses (Schmid and Wildsoet, 1996). This study was done to assess the potential influence of the other parasympathetic input to the choroid, the pterygopalatine ganglia, on the choroidal and axial responses to retinal defocus, and to form deprivation. 4-5 week-old chicks had one of the following surgeries to one eye: (1) Section (X) of the autonomic part of cranial N VII (input to the pterygopalatine ganglia) (PPGX, n = 16), (2) PPGX plus ciliary ganglionectomy (PPG/CGX, n = 23) or (3) PPGX plus superior cervical ganglionectomy (PPG/SCGX, n = 10). Experimental eyes were fitted with positive or negative lenses, or diffusers, several days after surgery. In one group of PPG/CGX, eyes did not wear any devices (n = 8). Intact (no surgery) controls were done for all visual manipulations (lenses or diffusers). Sham surgeries were done for the PPG/CGX condition (n = 4). Ocular dimensions were measured using A-scan ultrasonography prior to the surgery, 5 days later when visual devices were placed on the eyes, at the end of lens- or diffuser-wear, and in the case of diffusers, 4 days after diffuser removal to look at "recovery". Refractive errors were measured using a Hartinger's refractometer. IOP was measured in 7 PPG/CGX birds 7d after surgery. PPGX/CGX resulted in choroidal thickening (125 μm) and a decrease in IOP over one week post-surgery. It also prevented the development of myopia in response to form deprivation (X vs intact: 0.2 D vs -4.1 D; p 0.005), by preventing the increase in axial elongation (250 μm vs 670 μm/5d; p 0.005). In fact, growth rate slowed below normal (X vs fellow eyes: 250 μm vs 489 μm/5d; p = 0.002). By contrast, there were no effects of this lesion on the development of myopia in response to negative lenses (X vs intact: -5.4 D vs -5.3 D). All three lesions inhibited the compensatory choroidal thickening in response to myopic defocus (ANOVA, p = 0.0008), but had no effect on the thinning response to hyperopic defocus. These results argue for different underlying mechanisms for the growth responses to form deprivation vs negative lens wear. They also imply that choroidal thickening and thinning are not opposing elements of a single mechanism.

In eyes wearing negative lenses, the D2 dopamine antagonist spiperone was only partly effective in preventing the ameliorative effects of brief periods of vision (Nickla et al., 2010), in contrast to reports from studies using form-deprivation. The present study was done to directly compare the effects of spiperone, and the D1 antagonist SCH-23390, on the two different myopiagenic paradigms. 12-day old chickens wore monocular diffusers (form-deprivation) or -10 D lenses attached to the feathers with matching rings of Velcro. Each day for 4 days, 10 μl intravitreal injections of the dopamine D2/D4 antagonist spiperone (5 nmoles) or the D1 antagonist SCH-23390, were given under isoflurane anesthesia, and the diffusers (n = 16; n = 5, respectively) or lenses (n = 20; n = 6) were removed for 2 h immediately after. Saline injections prior to vision were done as controls (form-deprivation: n = 11; lenses: n = 10). Two other saline-injected groups wore the lenses (n = 12) or diffusers (n = 4) continuously. Axial dimensions were measured by high frequency A-scan ultrasonography at the start, and on the last day immediately prior to, and 3 h after the injection. Refractive errors were measured at the end of the experiment using a Hartinger's refractometer. In form-deprived eyes, spiperone, but not SCH-23390, prevented the ocular growth inhibition normally effected by the brief periods of vision (change in vitreous chamber depth, spiperone vs saline: 322 vs 211 μm; p = 0.01). By contrast, neither had any effect on negative lens-wearing eyes given similar unrestricted vision (210 and 234 μm respectively, vs 264 μm). The increased elongation in the spiperone-injected form-deprived eyes did not, however, result in a myopic shift, probably due to the inhibitory effect of the drug on anterior chamber growth (drug vs saline: 96 vs 160 μm; p < 0.01). Finally, spiperone inhibited the vision-induced transient choroidal thickening in form-deprived eyes, while SCH-23390 did not. These results indicate that the dopaminergic mechanisms mediating the protective effects of brief periods of unrestricted vision differ for form-deprivation versus negative lens-wear, which may imply different growth control mechanisms between the two.

The dopaminergic system has been implicated in ocular growth regulation in chicks and monkeys. In both, dopamine D2 agonists inhibit the development of myopia in response to form deprivation, and in chicks, to negative lenses as well. Because there is mounting evidence that the choroidal response to defocus plays a role in ocular growth regulation, we asked whether the effective agonists also elicit transient thickening of the choroid concomitant with the growth inhibition. Negative lenses mounted on velcro rings were worn on one eye starting at age 8-12 days. Intravitreal injections (20 μl; dose = 10 nmole) of the agonist (dissolved in saline) or saline, were given through the superior temporal sclera using a 30G needle. Eyes were injected daily at noon, for 4 days, and the lenses immediately replaced. Agonists used were apomorphine (non-specific; n = 17), quinpirole (D2; n = 10), SKF-38393 (D1; n = 9), and saline controls (n = 22). For the antagonists, the same protocol was used, but on each day, the lenses were removed for 2 h. Immediately prior to lens-removal, the antagonist was injected (20 μl; dose = 5 nmole). Antagonists used were methylergonovine (non-specific; n = 12), spiperone (D2; n = 20), SCH-23390 (D1; n = 6) and saline controls (n = 27). Comparisons to saline (continuous lens wear) controls were from the agonist experiment. Axial dimensions were measured using high frequency A-scan ultrasonography at the start of lens wear, and on day 4 prior to the injections, and then again 3 h later. Refractive errors were measured using a Hartinger's refractometer at the end of the experiment. Apomorphine and quinpirole inhibited the refractive response to the hyperopic defocus induced by the negative lenses (drug vs saline controls: -1.3 and 1.2 D vs -5.6 D; p < 0.005 for both). This effect was axial: both drugs prevented the excessive ocular elongation (change in axial length: 233 and 205 μm vs 417 μm; p < 0.01 for both). Both drugs were also associated with a transient thickening of the choroid over 3 h (41 and 32 μm vs -1 μm; p < 0.01; p = 0.059 respectively) that did not summate: choroids thinned significantly over the 4 day period in all lens-wearing eyes. Two daily hours of unrestricted vision during negative lens wear normally prevents the development of myopia. Spiperone and SCH-23390 inhibited the ameliorating effects of periods of vision on lens-induced refractive error (-2.9 and -2.8 D vs 0.6 D; p < 0.0001), however, the effects on neither axial length nor choroidal thickness were significant. These data support a role for both D1 and D2 receptors in the ocular growth responses.

The choroid of the eye is primarily a vascular structure supplying the outer retina. It has several unusual features: It contains large membrane-lined lacunae, which, at least in birds, function as part of the lymphatic drainage of the eye and which can change their volume dramatically, thereby changing the thickness of the choroid as much as four-fold over a few days (much less in primates). It contains non-vascular smooth muscle cells, especially behind the fovea, the contraction of which may thin the choroid, thereby opposing the thickening caused by expansion of the lacunae. It has intrinsic choroidal neurons, also mostly behind the central retina, which may control these muscles and may modulate choroidal blood flow as well. These neurons receive sympathetic, parasympathetic and nitrergic innervation. The choroid has several functions: Its vasculature is the major supply for the outer retina; impairment of the flow of oxygen from choroid to retina may cause Age-Related Macular Degeneration. The choroidal blood flow, which is as great as in any other organ, may also cool and warm the retina. In addition to its vascular functions, the choroid contains secretory cells, probably involved in modulation of vascularization and in growth of the sclera. Finally, the dramatic changes in choroidal thickness move the retina forward and back, bringing the photoreceptors into the plane of focus, a function demonstrated by the thinning of the choroid that occurs when the focal plane is moved back by the wearing of negative lenses, and, conversely, by the thickening that occurs when positive lenses are worn. In addition to focusing the eye, more slowly than accommodation and more quickly than emmetropization, we argue that the choroidal thickness changes also are correlated with changes in the growth of the sclera, and hence of the eye. Because transient increases in choroidal thickness are followed by a prolonged decrease in synthesis of extracellular matrix molecules and a slowing of ocular elongation, and attempts to decouple the choroidal and scleral changes have largely failed, it seems that the thickening of the choroid may be mechanistically linked to the scleral synthesis of macromolecules, and thus may play an important role in the homeostatic control of eye growth, and, consequently, in the etiology of myopia and hyperopia.

It is generally accepted that the increase in choroidal thickness in response to myopic defocus in chicks acts to move the retina towards the image plane. It may also constitute part of the signal cascade in the visual regulation of eye growth. To test this, we used the nitric oxide synthase inhibitor l-NAME to inhibit the defocus induced choroidal thickening under two different visual conditions, and looked at the effects on ocular growth rate. Exp. 1: Deprivation/Vision: chicks were monocularly deprived of form vision with translucent diffusers from day 6 to day 9. In the middle of each day the diffusers were removed for 2 h. One group received an intravitreal injection of 30 microl l-NAME (16 micromole; n=12) prior to the vision, a second group received injections of physiological saline (n=11). Exp. 2: Recovery/Vision: chicks were made myopic by form deprivation from day 6 to day 10. On days 11 to 14 the diffusers were removed for 2 h per day for 4 days to allow eyes to "recover" from the myopia. One group received an injection of l-NAME prior to vision (n=8), the other saline (n=6). Refractive errors were measured with a refractometer at the start (days 6 and 11) and end (days 10 and 15, respectively) of both experiments. Ocular dimensions were measured with high frequency A-scan ultrasonography at the start and end, and on the third experimental day immediately before and after the period of vision. Choroidal retinoic acid synthesis was measured by HPLC. Finally, NO production and scleral proteoglycan synthesis were measured in eyes wearing positive lenses 6 and 24h after an injection of l-NAME. l-NAME prevented the transient vision-induced choroidal thickening in both experiments. Furthermore, l-NAME inhibited the protective effect of brief daily vision: eyes became significantly more myopic than saline controls (exp. 1: -9 D vs -2.7D; exp. 2: -0.9 D vs +4.3 D; p

Animal models have been crucial in shaping our understanding of emmetropisation as an active, visually-guided mechanism for overcoming innate refractive errors. For instance, when a ‘minus’ power spectacle lens is mounted in front of the eye of a juvenile animal, the eye is cued to thin its choroid and accelerate its rate of axial elongation. Such altered ocular growth responses serve to attenuate the imposed defocus and so continue over a period of days, weeks or months (depending on the rate of emmetropisation of the species concerned) until the eye attains its optimal refraction. Notably, refracted without the minus lens in place, the eye of an animal emmetropising to a minus lens is myopic (its axial length now being excessive for its anterior segment optics). Similar ocular growth changes are seen with form deprivation, typically achieved by covering the eye with a frosted diffuser, and preventing the eye from receiving clear vision. However while both minus lens wear and form deprivation induce axial myopia, the absence of meaningful visual feedback allows growth to continue in an ‘open-loop’ manner in the latter case, limited mostly by the duration of treatment. Here, Ian Morgan and Regan Ashby present the evidence that form deprivation and lens-induced myopia share the same physiological mechanisms, while Debora Nickla presents the opposing evidence that the underlying mechanisms are different.

Purpose. Laser Doppler interferometry (LDI) permits the measurement of intraocular distances to a precision of better than 20 microm. The signal complex from the posterior segment of the eye consists of four peaks in the chick, an animal frequently used in ocular development studies. The present study sought to identify anatomical landmarks corresponding to these LDI peaks. Methods. Distances obtained with LDI at the posterior pole were compared to axial length components measured with three independent methods: vernier calipers, tissue sections and high frequency A-scan ultrasound. Results. LDI reflections appear to originate from the retinal inner limiting membrane, Bruch's membrane and the inner and outer scleral surfaces. Conclusions. The non-invasive and highly precise nature of LDI measurements enables repetitive and accurate assessment of intraocular distances. Such measurements should prove particularly useful for the assessment of short-term cyclic variations in intraocular distances as well as post-natal eye growth.

The chick eye is extensively used as a model in the study of myopia and its progression; however, analysis of the photoreceptor mosaic has required the use of excised retina due to the uncorrected optical aberrations in the lens and cornea. This study implemented high resolution adaptive optics (AO) retinal imaging to visualize the chick cone mosaic in vivo.The New England College of Optometry (NECO) AO fundus camera was modified to allow high resolution in vivo imaging on two 6-week-old White Leghorn chicks (Gallus gallus domesticus)-labeled chick A and chick B. Multiple, adjacent images, each with a 2.5(o) field of view, were taken and subsequently montaged together. This process was repeated at varying retinal locations measured from the tip of the pecten. Automated software was used to determine the cone spacing and density at each location. Voronoi analysis was applied to determine the packing arrangement of the cones.In both chicks, cone photoreceptors were clearly visible at all retinal locations imaged. Cone densities measured at 36(o) nasal-12(o) superior retina from the pecten tip for chick A and 40(o) nasal-12(o) superior retina for chick B were 21,714 ± 543 and 26,105 ± 653 cones/mm(2) respectively. For chick B, a further 11 locations immediately surrounding the pecten were imaged, with cone densities ranging from 20,980 ± 524 to 25,148 ± 629 cones/mm(2).In vivo analysis of the cone density and its packing characteristics are now possible in the chick eye through AO imaging, which has important implications for future studies of myopia and ocular disease research.

In birds, the choroid plays a role in the visual regulation of eye growth, thickening in response to myopic defocus, and thinning in response to hyperopic defocus, in both cases moving the retina towards the image plane. This response is rapid, occurring within hours of the defocus stimulus. These changes are consistently associated with slower changes in the sclera, that result in the appropriate changes in axial elongation, decreasing growth in response to myopic defocus and increasing it in response to hyperopic defocus. The molecular mechanisms underlying the scleral response involve changes in the synthesis of extracellular matrix molecules, however, those underlying the changes in choroidal thickness are not known. However, evidence suggests that it may involve the gaseous signal molecule nitric oxide, as nitric oxide is a potent smooth muscle relaxant, and injections of the non-specific nitric oxide synthase inhibitor L-NAME transiently inhibits the thickening response. Interestingly, it also dis-inhibits ocular growth, in accordance with a mechanistic link between the two responses. If nitric oxide is part of the signal cascade underlying the visual regulation of eye growth, it would be important to ascertain the source of the molecule. As a first step towards doing so, we used various more specific NOS inhibitors and studied their effects on the choroidal and growth responses. Birds (7-12 days old) were fitted with +10 D lenses on one eye. On that day, single intravitreal injections (30 microl) of the following inhibitors were used: nNOS inhibitor N(omega)-propyl-L-arginine (n=12), iNOS inhibitor L-NIL (n=16), eNOS/iNOS inhibitor L-NIO (n=15), non-specific inhibitor L-NMMA (n=30) or physiological saline (n=18). Ocular dimensions were measured using high-frequency A-scan ultrasonography at the start of the experiment, and at 7, 24 and 48 h after. We found that the nNOS inhibitor N(omega)-propyl-L-arginine had the same inhibitory effects on the choroidal response, and dis-inhibition of the growth response, as did L-NAME; neither of the other inhibitors had any effect except L-NMMA. We conclude that the choroidal compensatory response is influenced by nNOS, possibly from the intrinsic choroidal neurons, or the parasympathetic innervation from the ciliary and/or pterygopalatine ganglia.

The sclera in myopic regions of chick eyes was studied histologically and compared to the sclera in corresponding regions of normal fellow eyes. Chicks had been monocularly deprived of form vision in the nasal half of the retina from hatching. The fellow control eye and the temporal retina of the deprived eye had normal vision. With this treatment, the resulting form-deprivation myopia and eye enlargement are restricted to the retinal region that had been form deprived. We found that the cartilaginous sclera in the myopic nasal region exhibited several differences from that in the corresponding non-myopic region: it was thicker, its cell density was lower, and the number of chondrocytes and binucleate cells was higher. In contrast, the fibrous sclera was thinner. These changes suggest that form-deprivation myopia causes an increased production of extracellular matrix and an increased level of mitotic activity in the cartilaginous sclera. As expected, the non-myopic temporal regions of experimental and control eyes did not differ in any of these parameters. The findings of the present study suggest that the eye enlargement accompanying form-deprivation myopia is not the consequence of scleral stretching but of abnormal growth.

In chickens, transient changes in choroidal thickness are found in conditions in which the eye is slowing its growth in response to visual episodes that prevent excessive elongation. To test the hypothesis that the choroidal and ocular growth responses are linked, we used a variety of "brief daily" stimuli known to ameliorate the development of myopia and assessed the concurrence of the responses. If the hypothesis is true, they should always be correlated. Form deprivation w/vision or strobe. Diffusers were worn for 5 days and removed for 2h of "vision" each day in: (a) one block of 2-h (n=16); or (b) two 1-h periods (n=10). Strobe. Birds were given 0.5h episodes of 12 Hz strobe at dawn and dusk (12h apart, n=11). Negative lenses w/vision or strobe. Lenses (-10D) were worn for 5 days and removed for 2h of vision each day (n=14). Strobe. Same as above (n=11). Darkness/brief vision or myopic defocus. Birds in constant darkness were given 2 daily 0.5h episodes of light 12h apart (n=6) or one daily 0.5h episode of +10D myopic defocus (n=6) for 4 days. Darkness/"frequent" or "infrequent" myopic defocus. Birds in constant darkness were given frequent (2 min x 14) or infrequent (1 min x 7) episodes of +10D myopic defocus for 4 days. In all experiments a control group had the myopia-inducing treatment but did not receive the visual stimulation. High frequency ultrasonography was done at the start and end of the experiment, and on the last day immediately prior to and 1h after the period of stimulation. Refractive errors were measured using a Hartinger's refractometer at the end of the experiment. We found that in 7 of the 8 conditions the development of myopia was inhibited. Form deprivation: vision or strobe vs control: -1.2 and -1.8 vs -9.8D. Negative lenses: vision or strobe vs control: -1.2 and -4.3 vs -8D. Constant dark: vision or myopic defocus vs control: -0.7 and 1.8 vs -1.8D. Constant dark: frequent myopic defocus vs control: 4.8 vs -0.4D (p

Recent investigations have raised the possibility that ocular diurnal rhythms might be involved in the regulation of eye growth. Specifically, the chick eye elongates with a daily rhythm, said to be absent in form-deprived eyes. The present study asks: (1) Which components of the eye have daily rhythms—only the overall eye size, or also choroidal thickness or anterior chamber depth? (2) Does the phase or amplitude of these rhythms differ in eyes growing either faster than normal (form-deprived eyes) or slower than normal (eyes recovering from form-deprivation myopia)? Using high-frequency A-scan ultrasonography that allowed fine (8–20 μm) resolution of anterior chamber depth, vitreous chamber depth, choroidal thickness and axial length, we measured normal eyes, form-deprived eyes and eyes recovering from form-deprivation myopia at 6 hour intervals for 5 days and 4 nights. All eyes showed daily rhythms in axial elongation and choroidal thickness. In both normal and form-deprived eyes, the axial length was greatest in the afternoon when the choroid was thinnest, and hence, these rhythms were approximately in anti-phase to one another; in addition, there is some evidence that the axial length rhythm in form-deprived eyes is phase-advanced relative to that of their fellow control eyes. The amplitude of the rhythm in choroidal thickness in form-deprived eyes was significantly larger than in normal eyes. In recovering eyes in which elongation is slowed, the rhythm in axial length was significantly phase-delayed relative to normal eyes (peak at 8 pm) and the rhythm in choroidal thickness was phase-advanced (peak at 8 pm); thus in these eyes, the two rhythms are in phase. In these eyes, the choroids were thickening by approximately 100 μm/day. In all three groups, the rhythm in anterior chamber depth appears to differ in phase from the rhythm in axial length (and hence from the rhythm at the posterior wall of the eye). We propose that the phase relationship between these choroidal and eye length rhythms influence the rate of growth of the eye, and conclude that diurnal ocular rhythms may be important in eye growth regulation.

It has been demonstrated that chick eye growth compensates for defocus imposed by spectacle lenses: the eye elongates in response to hyperopic defocus imposed by negative lenses and slows its elongation in response to myopic defocus imposed by positive lenses. We ask whether the synthesis of scleral extracellular matrix, specifically glycosaminoglycans, changes in parallel with the changes in ocular elongation. In addition, there is a choroidal component to compensation for spectacle lenses; the choroid thickens in response to myopic defocus and thins in response to hyperopic defocus. We ask whether choroidal glycosaminoglycan synthesis changes in parallel with changes in choroidal thickness.Chicks wore either a +15 diopter (D) or -15 D spectacle lens over one eye, or they wore one lens of each power over each eye for 5 days. At the end of this period, we measured refractive errors and ocular dimensions by refractometry and A-scan ultrasonography, respectively. Pieces of the scleras and choroids from these eyes were put into culture and the synthesis of glycosaminoglycans was assessed by measuring the incorporation of radioactive inorganic sulfur.We here report that the compensatory modulation of the length of the eye involves changes in the synthesis of glycosaminoglycans in the sclera, with synthesis increasing in eyes wearing -15 D spectacles lenses and decreasing in eyes wearing +15 D lenses. In addition, changes in the synthesis of glycosaminoglycans in the choroid are correlated with changes in choroidal thickness: eyes wearing +15 D lenses develop thicker choroids and these choroids synthesize more glycosaminoglycans than choroids from eyes wearing -15 D lenses.Changes in scleral glycosaminoglycan synthesis accompany lens-induced changes in the length of the eye. Furthermore, changes in the thickness of the choroid are also associated with changes in the synthesis of glycosaminoglycans. These results are consistent with the regulation of the growth of the eye being bidirectional, and with the retina being able to sense the sign of defocus.

The chick eye is able to change its refractive state by as much as 7 D by pushing the retina forward or pulling it back; this is effected by changes in the thickness of the choroid, the vascular tissue behind the retina and pigment epithelium. Chick eyes first made myopic by wearing diffusers and then permitted unrestricted vision developed choroids several times thicker than normal within days, thereby speeding recovery from deprivation myopia. Choroidal expansion does not occur when visual cues are reduced by dim illumination during the period of unrestricted vision. Furthermore, in chick eyes presented with myopic or hyperopic defocus by means of spectacle lenses, the choroid expands or thins, respectively, in compensation for the specific defocus imposed. Consequently, when the lenses are removed, the eye finds its refractive error suddenly of opposite sign, and the choroidal thickness again compensates by changing in the opposite direction. If a local region of the eye is made myopic by a partial diffuser and then given unrestricted vision, the choroid expands only in the myopic region. Although the mechanism of choroidal expansion is unknown, it might involve either a increased routing of aqueous humor into the uveoscleral outflow or osmotically generated water movement into the choroid. The latter is compatible with the increased choroidal proteoglycan synthesis either when eyes wear positive lenses or after diffuser removal.

In birds, neurons of the isthmo-optic nucleus (ION), as well as “ectopic” neurons, send axons to the retina, where they synapse on cells in the inner nuclear layer (INL). Previous work has shown that centrifugal axons can be divided into two anatomically distinct types depending on their mode of termination: either “convergent” or “divergent” (Ramon y Cajal, 1889; Maturana & Frenk, 1965). We show that cytochrome-oxidase histochemistry specifically labels “convergent” centrifugal axons and target neurons which appear to be amacrine cells, as well as three “types” of ganglion cells: two types found in the INL (displaced ganglion cells) and one in the ganglion cell layer. Labeled target amacrine cells have distinct darkly labeled “nests” of boutons enveloping the somas, are associated with labeled centrifugal fibers, and are confined to central retina. Lesions of the isthmo-optic tract abolish the cytochrome-oxidase labeling in the centrifugal axons and in the target amacrine cells but not in the ganglion cells. Cytochrome-oxidase-labeled ganglion cells in the INL are large; one type is oval and similar to the classical displaced ganglion cells of Dogiel, which have been reported to receive centrifugal input; the other type is rounder. Rhodamine beads injected into the accessory optic system results in retrograde label in both types of cells, showing that two distinct types of displaced ganglion cells project to the accessory optic system in chickens. The ganglion cells in the ganglion cell layer that label for cytochrome oxidase also project to the accessory optic system. These have proximal dendrites that ramify in the outer inner plexiform layer. Neither the target amacrine cells nor either of the displaced ganglion cells are immunoreactive for the inhibitory transmitter gamma aminobutyric acid. At least some of the target amacrine cells may, however, be cholinoceptive: we found that the antibody to the alpha-7 subunit of the nicotinic ACh receptor labels a population of cells in the INL that are similar in location, size, and the presence of labeled bouton-like structures to those we find labeled with cytochrome oxidase. This antibody also labels neurons in the ION proper but not ectopic cells. In conclusion, it appears that cytochrome oxidase may be a marker for “convergent” centrifugal axons and at least one of their target cells in the INL.