The process of emmetropization is the regulation of ocular growth by visual input during postnatal development, to result in the plane of focus of distant objects falling on the retina. This is accomplished by the modulation of the axial elongation so that the final length of the eye matches the focal plane of the front optics (review: 1). In animal models, when spectacle lenses are worn that focus an image in front of (myopic defocus) or behind (hyperopic defocus) the retina, ocular growth slows, in the case of myopic defocus, and increases, in the case of hyperopic defocus, resulting in an emmetropic eye.2,3 A second, faster component of this compensatory process involves changes in choroidal thickness, with the choroid thickening to push the retina forward toward the image plane, in response to myopic defocus, and thinning to pull it back, in response to hyperopic defocus.4,5 The signal sequence involved in emmetropization almost certainly begins at the retina, and continues through the RPE (retinal pigment epithelium) and choroid to effect biosynthetic changes in the sclera resulting in changes in eye length. The molecules involved in this signal cascade are unknown; however, several promising candidates have evolved from animal models. One that is arguably the most promising is the retinal neuromodulator dopamine (review: 6). Retinal dopamine levels fluctuate in a diurnal cycle in most species, being low at night and high during the day, thus mediating the physiological processes involved in light adaptation, such as horizontal cell uncoupling (reviews: 7,8). Dopamine release is also stimulated by increases in light intensity9,10 and by temporal transients (flashing lights11–13).
The initial evidence for the involvement of dopamine in the emmetropization pathway was the finding of lower daytime levels of retinal dopamine in form deprived chick eyes14; this was subsequently found to be true in monkeys as well.15 Furthermore, intravitreal injections of apomorphine (chicks: 14; monkeys: 16) or dopamine (rabbits: 17) prevented the development of deprivation myopia. A D2 receptor-mediated process was supported by the findings that quinpirole, a D2 agonist, prevented myopia in response to form deprivation18 and negative lens wear.19 Together, these results support a role for dopamine in the visual regulation of eye growth. If this is true, a crucial question for developing future therapies is “what is the visual stimulus that drives dopamine release?” A recent study might shed light on this issue: Rose et al20 reported that in children, the amount of time spent out of doors was a protective factor in the development of myopia, and they suggested that the critical factor might be light intensity. Subsequent studies in animal models support this hypothesis. When chickens were raised under high (10,000 lux) versus low (50 lux) luminance levels for 90 days, those raised in low levels became myopic while those raised in high levels became hyperopic.21 A role for dopamine was supported by the result that dihydroxyphenylacetic acid (DOPAC) levels were reduced in these myopic eyes and increased in hyperopic eyes.10 Finally, in chicks22 and monkeys,23 high luminance levels retarded the development of myopia in response to form deprivation, and in chicks, this inhibition was prevented by intravitreal injections of spiperone, a D2 dopamine antagonist.22
If dopamine plays a role in ocular growth inhibition, where in the signal cascade from retina to sclera is it, and what other molecules are involved? Some evidence supports a link between dopamine and the gaseous neurotransmitter nitric oxide (NO). If NO release is inhibited by intravitreal injections of nitric oxide synthase (NOS) inhibitors, the ocular growth inhibition normally effected by myopic defocus is prevented.24,25 NOS inhibitors also prevent the choroidal thickening normally induced by myopic defocus. These results suggest that NO may be involved in defocus-induced ocular growth inhibition and choroidal thickening. In addition, dopamine may cause choroidal thickening, as injections of the agonists apomorphine and quinpirole result in choroidal thickening,19 presumably similar to NO. So which molecule precedes the other? There is evidence in various systems for dopaminergic activation of NO release: First, apomorphine increased the concentration of NO end-products in an in vitro preparation of rat brains.26,27 Second, histological studies showed a close synaptic association between TH-positive axon terminals onto NADPH-positive neurons in rat striatum, suggesting that the activity of NO-positive neurons is regulated by dopamine.28 Third, exogenous dopamine applied to dark-adapted fish retinas in vivo enhanced the production of NO, and inhibiting dopamine synthesis suppressed light-evoked NO release.29
We hypothesize that dopamine precedes NO in the signal cascade mediating ocular growth inhibition. In a pilot study in eyecups, the agonist apomorphine caused choroidal thickening, suggesting that dopamine affects choroidal thickness and acts upstream of the choroid. These choroids also released more NO than controls in plain medium.30 If dopamine acts upstream of NO in the same pathway, then injections of NOS inhibitors concurrent with the dopamine agonist quinpirole should prevent both the ocular growth inhibition18,19 and choroidal thickening19 normally effected by quinpirole. We combined quinpirole with either of two NOS inhibitors, n-ω-propyl-L-arginine (nNOS inhibitor) or L-NIO (more selective for eNOS), in both form-deprived eyes and negative lens–wearing eyes, and asked (1) do the inhibitors prevent the quinpirole-induced ocular growth inhibition and (2) are there differences between the two paradigms? Parts of this manuscript were presented in abstract form.31
Subjects were White Leghorn chickens (Gallus gallus domesticus), hatched in an on-site incubator and raised in temperature-controlled brooders. The light cycle was 12L/12D (8:00 am to 8:00 pm) and the light intensity was approximately 300 lux. Food and water were supplied ad libitum. In all experiments, the right eye was treated and the left eye served as untreated controls. Care and use of the animals conformed to the Association for Research in Vision and Ophthalmology Resolution for the Care and Use of Animals in Research.
Negative lenses (−10 D) or translucent diffusers were mounted on velcro rings and were attached to the matching ring that was glued to the feathers around one eye, in the morning, starting at age 13 days. At around noon on that day, chicks were anesthetized with isoflurane inhalation anesthesia (0.8% in oxygen), and intravitreal injections (20 μL) of the drug dissolved in saline or saline (0.75%) were given at the same time, for 4 days. Drugs (Tocris) used were the relatively D2 selective32 dopamine agonist quinpirole, the potent (EC50 = 57 nM) and selective nNOS inhibitor n-ω-propyl-L-arginine (NPA) (3158-fold selectivity over iNOS; 149-fold selectivity over eNOS33), and the relatively non-selective eNOS/iNOS inhibitor L-NIO (EC50 for eNOS, iNOS, and nNOS, respectively: 0.5, 2.2, and 3.9 μM).34 The groups were as follows: quinpirole alone: 10 nmol in 20 μL, lens: n = 12, diffuser: n = 20; NPA alone: 0.4 μmol, lens: n = 6, diffuser: n = 4; quinpirole + NPA: 10 μL each, at double the above concentration, lens: n = 17, diffuser: n = 19; and quinpirole + L-NIO: 20 μL L-NIO, 300 nmol, lens: n = 12, diffuser: n = 12. Each experiment had a number of diffuser- or lens-wearing saline controls to control for inter-experiment variability; these data were combined (n’s in Table 1), as there were no significant differences between saline controls within the two experimental groups. A 30G needle was inserted (about 5 mm) through the skin of the lids over the superior temporal sclera after removing the feathers and cleaning the skin with alcohol. Care was taken to use the same injection site for subsequent injections. The needle remained in place for 30 seconds before being slowly withdrawn while the skin around the site was held tightly together using a small forceps. The lenses or diffusers were replaced immediately.
In all experiments, axial dimensions were measured using high-frequency A-scan ultrasonography under isoflurane inhalation anesthesia (details in 35) at the start of lens or diffuser wear, and on day 5, immediately prior to the injections, and then again 3 hours later to assess the choroidal response. Axial length is defined as the distance between the front of the cornea to the front of the sclera, and the data are given in rates (micrometers per 4 days). Vitreous chamber depth is defined as the distance from the back of the lens to the front of the retina; these data are also given as rates (micrometers per 4 days). Data on choroidal thickness changes are given as change per 3 hours. Refractive errors were measured using a Hartinger’s refractometer (details in 36) at the end of the experiment. The means, standard errors, and n’s for all data are given in Table 1. The inequality in some categories for n’s within experimental groups are due to the loss of a few data points due to measurement error or unusable ultrasound traces. For the saline groups, refractions only (no ultrasound) were done on a subset in each paradigm and are included in the data (FD, n = 4; lens, n = 9). In all graphs, “fellow” denotes data from all fellow eyes combined within the paradigm (lens wear or form deprivation) as there were no significant differences between drug groups.
The Shapiro-Wilk normality test showed no evidence against normality for any parameter in any of the experimental groups, so we applied the classical one-way ANOVA to test significance between drug and saline-injected groups. Post hoc Bonferroni tests were used for comparisons between treatment groups and the saline group when the overall ANOVA showed significant differences across groups (Table 1). Tests between untreated fellow eyes (combined from all groups within either the lens or diffuser paradigms) and experimental eyes used two-tailed Student t tests.
As expected, quinpirole prevented the development of both lens-induced19 and form deprivation–induced (FD) myopia18 compared to saline controls (Fig. 1: FD ANOVA, p = 0.001: −3.4 vs. −6.2 D, p = 0.0008; lens ANOVA, p = 0.0007: −0.4 D vs. −4.8 D, p = 0.0004). However, if quinpirole is combined with either NOS inhibitor NPA or L-NIO, this inhibitory effect is eradicated in form-deprived eyes (quinpirole vs. combined with NPA and L-NIO, respectively: −3.4 D vs. −5.9 and −5.8 D; p = 0.0095; p = 0.0481). In form-deprived eyes, the refractive errors of neither of the NOS inhibitor–combined groups differed from those of saline controls (FD: −5.8 D and −5.7 D vs. −6.2 D). In lens-wearing eyes, NPA was effective at countering the effect of quinpirole (−0.4 D vs. −3.5 D; p = 0.0198), but this effect was not significant for L-NIO (−0.4 D vs. −2.4 D; p = 0.6973).
Ocular Growth: Vitreous Chamber Depth
As shown before,18,19 quinpirole inhibits myopia by inhibiting growth of the back of the eye, as measured by the change in vitreous chamber depth compared to saline controls (Fig. 2A) (FD ANOVA, p = 0.0042, 275 μm vs. 384 μm/4 days, p = 0.0847; lens ANOVA, p = 0.0044, 253 μm vs. 378 μm/4 days, p = 0.0584). The vitreous chamber elongation in quinpirole-injected eyes did not differ from those of untreated fellow controls in either paradigm (FD: 275 vs. 211 μm, p > 0.05; lens: 253 vs. 224 μm, p > 0.05). For axial elongation (Fig. 2B), while there was a tendency for the same effect, the differences did not reach statistical significance (FD ANOVA, p = 0.0064, 330 vs. 444 μm/4 days, p = 0.2855; lens ANOVA, p = 0.0861, 315 vs. 469 μm, p = 0.126).
The myopia in eyes injected with the combination of quinpirole and either NOS inhibitor was the result of a disinhibition of vitreous chamber elongation; in both paradigms, these eyes grew significantly faster than eyes injected with quinpirole alone (Fig. 2A: quinpirole + NPA—FD: 401 vs. 275 μm/4 days, p = 0.0341; lens: 407 vs. 253 μm/4 days, p = 0.0056; quinpirole + L-NIO—FD: 440 vs. 275 μm/4 days, p = 0.0071; lens: 403 vs. 253 μm/4 days, p = 0.0162). A similar trend was seen for axial length (Fig. 2B: quinpirole + NPA—FD: 492 vs. 330 μm/4 days, p = 0.0232; lens: 457 vs. 315 μm/4 days, p = 0.1825; quinpirole + L-NIO—FD: 530 vs. 330 μm/4 days, p = 0.0111; lens: 451 vs. 315 μm/4 days, p = 0.2798) despite not reaching statistical significance in the lens paradigm. Within both paradigms, eye growth as measured by axial or vitreous chamber elongation did not differ from that in the respective saline-injected control eyes (Table 1). To summarize, combining quinpirole with either of the NOS inhibitors countered the refractive and ocular growth inhibition normally effected by quinpirole. The ocular growth inhibition was more apparent when measuring the growth at the back of the eye as reflected by vitreous chamber elongation, mainly because of the increased variability in the axial length data due to an “injection effect”, which commonly causes a decrease in anterior chamber growth (see Table 1; asterisks indicate groups in which anterior chamber growth was significantly decreased relative to uninjected fellow controls; note, however, that anterior chamber growth in all experimental groups was less than that in fellow eyes).
Transient Choroidal Thickening
It has previously been shown that quinpirole causes a transient increase in choroidal thickness within 3 hours of the injection into negative lens–wearing eyes.19 We here find a similar effect in lens-wearing eyes (Fig. 3: quinpirole vs. saline: ANOVA, p = 0.0118, 31 vs. −9 μm/3 hours, p = 0.0147). Because NPA was shown to prevent the transient choroidal thickening in response to positive lens–induced myopic defocus,25 if it is so that quinpirole mediates the effects of myopic defocus as manifested in brief daily occluder removal,18 then it should similarly inhibit the thickening when combined with quinpirole, and in fact, there was a tendency for this, but in lens-wearing eyes only (0 μm vs. 31 μm/3 hours; p = 0.0798). L-NIO had no effect (Fig. 3) on lens-wearing eyes, and neither NPA nor L-NIO inhibited thickening in form-deprived eyes (ANOVA p = 0.0795).
Injections of the nNOS inhibitor n-ω-propyl-L-arginine alone had no effect on the development of form deprivation–induced myopia (vs. saline: −4.2 D vs. −6.2 D; p = 0.15) or lens-induced myopia (−5.1 D vs. −4.8 D; p = 0.5; Table 1). In keeping with the lack of a refractive effect, the drug-injected form-deprived eyes grew similar to saline controls (vitreous chamber: 312 vs. 384 μm/4 days, p = 0.15; axial: 422 vs. 444 μm/4 days, p = 0.53). In the lens-wearing group, however, drug-injected eyes grew significantly faster (vitreous chamber: 530 vs. 378 μm, p = 0.008; axial: 604 vs. 469 μm, p = 0.036) despite the absence of a refractive effect. We did not test the effects of L-NIO alone in either paradigm.
Our results show that combining either the nNOS inhibitor n-ω-propyl-L-arginine (NPA) or the non-specific NOS inhibitor L-NIO with quinpirole prevents the ocular growth inhibition that normally is conferred by quinpirole. This is true for both paradigms of myopia induction, form deprivation, and negative lens wear, supporting a similar mechanism of action for these two molecules in the two paradigms, despite some evidence suggestive of different dopaminergic mechanisms (review: 6,37). These results support the hypothesis that dopamine acts upstream of nitric oxide in the signal cascade mediating ocular growth inhibition, and furthermore, that the release of NO is essential to the ocular growth inhibition mediated by quinpirole in negative lens–wearing and form-deprived eyes. NPA also prevents the transient choroidal thickening normally occurring in response to quinpirole in the lens-wear paradigm only, similar to its effect in eyes exposed to myopic defocus by positive lenses.25 If both dopamine and NO are indeed part of the signal cascade mediating ocular growth inhibition, two crucial questions remaining are as follows: first, from which ocular tissues are they released, and second, on what tissues do each exert its effect?
Dopamine and NO
It is generally believed that the dopamine involved in the signal cascade in eye growth regulation is released from dopaminergic amacrine cells in the retina.6 Both form deprivation9 and negative lens wear38,39 result in rapid reductions in vitreal levels of the dopamine metabolite DOPAC, considered to be the best indicator of dopamine release.40 In the retina as well, DOPAC levels are decreased in eyes wearing negative lenses and increased in eyes wearing positive lenses,39 or recovering from form-deprivation myopia.41 Finally, there is a steep gradient in dopamine concentration from the retina in both inner and outer directions: to the vitreous, where it is reduced to 1/10th that of retinal levels, and to the choroid and sclera, where it is reduced to 1/3 and 1/20th, respectively.38 Because the defocus-related effects of the lenses alter the DOPAC levels in both retina and vitreous, but have no effect on levels in the choroid or sclera,38 it is most likely that the tissue of origin of this visually influenced molecule is in fact the retina, as opposed to the RPE or the choroid, neither of which are allegedly dopaminergic (although there is some evidence for dopaminergic innervation of the choroid42). Our evidence that NPA prevented the quinpirole-induced choroidal thickening in lens-wearing eyes is also consistent with the hypothesis that dopamine is released upstream of the choroid, in either the retina or RPE.
As already discussed, there is evidence for dopaminergic modulation of NO release in other systems.26–29 In chick eyes, the neuronal NOS inhibitor NPA transiently disinhibited ocular growth and prevented the defocus-induced transient choroidal thickening in eyes wearing positive lenses, supporting a link between NO, choroidal thickening, and ocular growth inhibition.25 The growth-inhibiting dopamine agonists apomorphine and quinpirole also induce transient choroidal thickening,19 linking dopamine to this sequence of events that lead to growth inhibition and supporting the hypothesis that dopamine acts upstream of the choroid. Our current findings that two NOS inhibitors prevent the growth-inhibiting effect of quinpirole (and that NPA prevents the quinpirole-induced choroidal thickening in lens-wearing eyes) strengthen this association. If dopamine is released by retinal amacrine cells, is there a direct link between dopamine and NO, or is there an intermediary signal molecule?
If we assume that dopamine is released by amacrine cells in response to the relevant visual stimuli (myopic defocus?), it could act on other retinal neurons, or on the RPE, the apical aspect of which contains D2/D4 receptors.43 NO is ubiquitous in ocular tissues, being found in retina,44–47 RPE and choroid,44,48,49 and in axon terminals of parasympathetic origin,50,51 thus any of these could be downhill sources of NO. If we assume that NO directly acts on the choroid to mediate thickening, as has been suggested,24,25 then it is unlikely that the amacrine cell–released dopamine acts on nitrergic retinal cells to release NO, as the NO would have to traverse the RPE, Bruch’s, and the high-volume blood flow of the choriocapillaris to exert its effect. For this same reason, it is unlikely that dopamine released from the retina crosses the RPE and choriocapillaris to act on nitrergic choroidal neurons. Alternative possibilities are that (1) dopamine binds receptors in the RPE that releases a signal molecule which effects the release of NO from a choroidal source, such as the ICNs or the axon terminals of the parasympathetic inputs to the choroid, or (2) the RPE is the source of NO, which diffuses through Bruch’s and the choriocapillaris to reach the choroid. Further research is required to answer this important question.
Why Did Both NOS Inhibitors Inhibit Quinpirole, But Only One Inhibit the Effects of Lens-Induced Myopic Defocus?
In previous work, we found that of three tested NOS inhibitors, only NPA had similar effects as those of the non-specific inhibitor L-NAME on eyes exposed to myopic defocus. From this, we inferred that the neuronal isoform was the one that was part of the inhibitory signal cascade activated by myopic defocus and leading to growth inhibition.25 In this study, we used both NPA and L-NIO, which is relatively non-selective, in combination with quinpirole and found that both drugs prevented the inhibitory effects of quinpirole on eye growth in both paradigms. What are the implications? If we assume that the inhibitors were acting on separate isoforms (NPA on nNOS; L-NIO on iNOS or eNOS), then (1) if quinpirole (dopamine) was effective on both isoforms to effect ocular growth inhibition, then neither inhibitor alone should have prevented quinpirole’s effect; (2) if quinpirole acted only on nNOS, then NPA should have prevented quinpirole’s effect, but L-NIO should have been ineffective; (3) if quinpirole acted only on eNOS (or iNOS), then L-NIO should have prevented quinpirole’s effect, but NPA should have been ineffective. Because both inhibitors were effective at preventing the quinpirole-induced growth inhibition suggests that the L-NIO was exerting a “cross-over” effect on nNOS, perhaps due to the multiple injection protocol over the 4 day period in this paradigm, as opposed to the single-injection one over a 1 day period in the former study.25 It should be noted that NPA alone showed a significant affect on eye growth only in the lens-wearing eyes (which was not reflected in refractive error): eyes grew faster than saline controls. If this is a real effect, it would suggest that loss of nitric oxide alone might lead to ocular growth stimulation. This would have to be tested explicitly.
An alternative explanation for the lack of effect of L-NIO in preventing the positive lens–induced ocular growth inhibition,25 in the prior study versus its effectiveness in preventing quinpirole’s effect in the present study, is that dopamine (quinpirole) does not, in fact, mediate the effects of myopic defocus. This supposition, however, is not supported by existing data: quinpirole prevented the myopiagenic effects of form deprivation, and injecting spiperone, a D2 antagonist, prior to giving these eyes brief periods of daily vision prevented the vision-related (presumably small amounts of myopic defocus) effects.18 These results support the hypothesis that dopamine mediates the effects of form vision (or myopic defocus). Quinpirole also prevented the myopiagenic effects of negative lens wear in a similar manner,19 further supporting the above hypothesis.
It was previously shown that the growth inhibition elicited by quinpirole in eyes wearing negative lenses was associated with transient choroidal thickening,19 possibly indicating that the two are in the same pathway. On the other hand, in negative lens–wearing and form-deprived eyes, ocular growth stimulation was associated with choroidal thinning, possibly linking these in the same pathway.52 We here show that co-injections of quinpirole with the nNOS inhibitor NPA inhibited the choroidal thickening response in lens-wearing eyes, as would be expected if a thinner choroid was an integral part of the growth stimulatory cascade; however, L-NIO was ineffective: choroids thickened presumably in response to the quinpirole. Because both inhibitors prevented the quinpirole-induced ocular growth inhibition, but only one prevented choroidal thickening, suggests that choroidal thinning is not an integral part of the growth-stimulating pathway. This idea is supported by several studies: first, double parasympathectomy inhibited (rather than stimulated) eye growth in form-deprived eyes, but it did not prevent the choroidal thinning response.53 Second, muscarinic agonists that caused choroidal thinning in normal eyes had no effect on eye growth.54 Alternatively, the apparent lack of effect of L-NIO in the former study may be reflective of a difference in the time course for the effects between the two drugs, as we only measured 3 hours after the injection, or of a different efficacy of the inhibitor’s effect on NO and on the choroid.
Another interesting result was that neither inhibitor was effective at preventing choroidal thickening in form-deprived eyes. This might reflect a deprivation-related difference in the physiological state of choroids versus those in lens-wearing eyes. This idea is supported by evidence indicating pathological changes in the RPE of form-deprived eyes that were indicative of hypo-osmolarity of the choroids.55 Another finding supporting physiological differences in choroids of deprived eyes versus those of lens-wearing eyes is that in double-parasympathectomized form–deprived eyes, the choroids that thinned in response to the deprivation (without attendant myopia development) thickened upon removal of the diffuser despite the absence of myopic defocus.53 It follows that the compensatory choroidal thinning induced by negative lens wear is fundamentally different from that (non-compensatory) thinning induced by form deprivation. By extension, the lack of effect of the inhibitors on the choroidal thickening in these form-deprived eyes presumably implies that this thickening is mediated by a non-nitrergic mechanism.
In conclusion, our results suggest that dopamine acts upstream of NO and the choroidal response in the signal cascade mediating ocular growth inhibition in both form deprivation and negative lens wear. That NPA inhibited the quinpirole-induced choroidal thickening in lens-wearing eyes but not in form-deprived eyes suggests that choroidal thinning is not required for ocular growth stimulation. That neither inhibited choroidal thickening in FD eyes suggests that the choroidal mechanisms differ in the two paradigms.
Debora L. Nickla
Department of Biosciences
The New England College of Optometry
424 Beacon St.
Boston, MA 02115
This work was supported by NEI-NIH-013636.
Received April 12, 2013; accepted June 23, 2013.
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