Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

even without goggles or spectacle lenses. Rearing chicks under constant light stimulates the axial growth of the vitreous chamber but inhibits corneal expansion. The net effect is an enlarged eye, but with a hyperopic refractive error because the marked corneal flattening so reduces corneal power that images are focused behind the retina despite the enlarged eye (Jensen and Matson, 1957; Li et al., 1995; Stone et al., 1995, 2006b). A considerably less robust refractive effect from constant light rearing occurs in young rhesus monkeys: about a third of individuals reared for 6 months show mild myopia or modest anisometropia (Smith et al., 2001). For both chicks and monkeys, the constant light effects present a conceptual contradiction: non-restricted visual images are available to the retina for more than enough daily hours to permit proper emmetropization in either species, based on independent experiments, but somehow the lack of a daily period of darkness disrupts the ordered growth of the eye. If and how these results might apply to children is controversial, as discussed below, but it illustrates that an environmental insult distinct from image quality (i.e., an absent dark period) can override the mechanism governing emmetropization and coordinated eye growth.

B. Some General Results from Eye Growth Models

1. Refractive development and the retina

Using these models, particularly form deprivation and spectacle lens wear, the visual mechanism(s) regulating refractive development has been found to localize largely to the eye itself (Stone, 1997; Wallman, 1993; Wallman and Winawer, 2004). As one example, form deprivation myopia in both monkeys and chicks still develops after optic nerve section to separate the eye from the brain (Raviola and Wiesel, 1985; Troilo et al., 1987; Wildsoet,

2003; Wildsoet and Pettigrew, 1988a). The dual properties of visual (and hence neural) regulation and intrinsic ocular location (as revealed by optic nerve section) identify the retina as a major site integrating refractive development. Many of the neurotransmitters and modulators discussed below localize to subtypes of retinal neurons, often amacrine cells. How retinal signals, including the participation of amacrine cells, control growth of the outer coats of the eye is unknown. No neural connections are known to exist between the retina and either the choroid or sclera. Local control might depend on transport of growth factors across the retinal pigment epithelium to choroid and sclera, for example. Despite such uncertainties, the retinal hypothesis for eye growth control underlies much contemporary myopia pharmacology.

2. Choroidal thickness fluctuations

Eye growth responses to blur or defocus involve not only changes in scleral growth but also changes in choroidal thickness (Figure 9.2). The visual conditions stimulating eye growth (i.e. goggles and minus lens wear) associate with choroidal thinning, and visual conditions inhibiting eye growth (i.e. positive lens wear or myopia recovery) associate with choroidal thickening. It has been hypothesized that these choroidal responses may comprise a novel “focusing mechanism” to move the retinal photoreceptors toward the focal position of distant images. The choroidal responses are more robust in chicks (Wallman et al., 1995) than in the mammals in which they have been studied, rhesus monkeys (Hung et al., 2000), marmosets (Troilo et al., 2000a) and tree shrews (Siegwart and Norton, 1998), perhaps reflecting the slower ocular growth rates of these mammals.

3. Rationale for studying chick

While chicks mature so quickly that refractive studies can be conveniently and accurately completed in days to weeks,

sometimes even in hours, the chick eye has features not seen in mammalian eyes (i.e. scleral ossicles and cartilage to maintain eye shape, striated intraocular muscles and a pecten); and the response of the chick sclera in myopia is only partly representative of mammalian sclera (Marzani and Wallman, 1997; Norton, 1999; Wallman, 1993). Importantly, the fundamental visual and neural systems modulating refractive development show many common features between chicks and mammals (Norton, 1999; Stone, 1997; Stone and Flitcroft, 2004). Normal chicks show precise visually guided emmetropization (Schaeffel et al., 1988; Wallman et al., 1981). Vitreous chamber growth accounts for myopia in chicks (Wallman and Adams, 1987) and mammals (McBrien and Norton, 1992; Raviola and Wiesel, 1985; Troilo and Judge, 1993) and for much human myopia (Curtin, 1985). As just discussed, chicks and mammals adjust eye growth appropriately for defocusing spectacle lenses, develop form deprivation myopia at juvenile and “adolescent” ages and can recover from imposed refractive errors at sufficiently young ages. This remarkable phylogenetic conservation justifies investigating eye growth in chick to formulate initial hypotheses to study refractive development in children. Most myopia pharmacology now addresses the chick, at least initially, and the successful extension of some pharmacology findings in chick to mammalian and human eyes supports continued investigation of this species.

V. PHARMACOLOGY AS AN

APPROACH TO MYOPIA

PATHOGENESIS

Laboratory methods, including pharmacology, are now addressing eye growth regulation and myopia pathogenesis using these experimental models of eye growth, with initial follow-on clinical studies. The laboratory approaches have either tested the effects of drugs, chosen empirically

based on hypothesized mechanisms, or have assayed the retina and/or related tissues seeking to implicate a receptor system in eye growth control. Assay methods have included histochemistry, immunohistochemistry, biochemistry and molecular biology. The immunohistochemical approaches have included not only comparisons between tissues of experimental and control eyes, but also the identification of transmitters/modulators in neurons expressing immediate-early genes shortly after perturbing the visual image. Molecular biology methods have included assaying the expression of candidate genes or more general screening methods, such as differential display or microarrays. Because eye growth models are generated by perturbing the visual image, evidence is needed that an implicated signaling system actually participates in refractive development and that the experimental results are not merely a consequence of image perturbation unrelated to eye growth control. A signaling system is usually implicated by demonstrating ocular growth and/or refractive effects in response to specific drugs.

Several cautions are needed in interpreting the effects of drugs on refractive development. Drug-specific issues, such as penetration, distribution and pharmacokinetics, are pertinent to agents given to the eye. Experiments with chronically administered drugs permit delayed, secondary, compensatory or indirect drug actions, and multiple sites of action are possible. Most contemporary pharmacology of eye development is conducted in chicks. Because receptor identities and their properties can vary between chicks and mammals, and because available drugs may not be well characterized against chick receptors, interpreting both positive and negative drug effects at the receptor level requires appropriate caution. Understanding refractive mechanisms is further complicated by the possibility, some would suggest the likelihood, that multiple, redundant, interacting

TABLE 9.1 Signaling molecules potentially involved in myopia and/or emmetropization

For references, see text.

and/or independent pathways regulate so essential a physiologic need as refraction.

VI. PHARMACOLOGY OF

MYOPIA

A substantial number of signaling molecules (Table 9.1) and corresponding receptor classes (Table 9.2) have been implicated in studies of experimental myopia or emmetropization. Some of these signaling systems have been investigated more intensely than others; for some, the currently available data are quite fragmentary. The pharmacology will be reviewed here in terms of specific signaling molecules. Arguably because of direct or indirect extensions to children, the greatest advances so far have been made in cholinergic and dopaminergic pharmacology.

A. Acetylcholine

1. Muscarinic acetylcholine receptors

Acetylcholine is expressed by a subset of retinal amacrine cells and in parasympathetic peripheral nerve fibers innervating non-retinal eye tissues. Muscarinic acetylcholine receptors, one of many G-protein coupled receptor classes, are widely

TABLE 9.2 Receptor classes potentially involved in myopia and/or emmetropization

expressed in ocular tissues, including the mammalian ciliary and iris sphincter muscles (Nietgen et al., 1999). Based on a hypothesized role of accommodation in refractive development, the muscarinic receptor antagonist atropine has been studied, and sometimes used to slow myopia progression, for almost two centuries (Bedrossian, 1979; Curtin, 1985; Kennedy, 1995). Numerous study design problems, including lack of adequate controls and patient dropout, mostly from the side effects of mydriasis and cycloplegia, have hampered interpreting this literature. On balance, a favorable therapeutic response to atropine seems present in children.

Besides its apparent efficacy in slowing myopia in children, atropine inhibits myopia development not only in tree shrew and monkey myopia models (McKanna and Casagrande, 1981; Raviola and Wiesel, 1985; Tigges et al., 1999), but it also blocks form deprivation myopia or lens induced myopia in chicks (Schmid and Wildsoet, 2004; Stone et al., 1991). Unlike the mammalian eye, the avian eye contains striated intraocular muscles, and atropine has been long known to have neither mydriatic nor cycloplegic activity in birds (Glasser

and Howland, 1996), indicating a nonaccommodative mechanism for the antimyopia activity of atropine in chick (Stone et al., 1991). Modern molecular pharmacology has identified four subtypes of muscarinic acetylcholine receptors in chick (Fischer et al., 1998a) and five subtypes in mammals (Caulfield, 1993). For the primate and human ciliary and iris sphincter muscles, the predominant muscarinic receptor is the m3 subtype (Gil et al., 1997; Poyer et al., 1994). Atropine is a high affinity, non-selective antagonist at all muscarinic receptor subtypes. Among the few available subtype selective muscarinic antagonists, the relatively m1-subtype-selective antagonist pirenzepine and the m4-subtype-selective antagonist himbacine each inhibit myopia development in chick, but the m3 subtype receptor antagonist 4-DAMP is ineffective (Cottriall et al., 2001b; Stone et al., 1991). While conducted in birds, these experiments effectively dissociate the anti-myopia from the cycloplegic/mydriatic actions of muscarinic antagonists. Pirenzepine also reduces experimental myopia in tree shrews (Cottriall and McBrien, 1996) and rhesus monkey (Tigges et al., 1999).

Pirenzepine was initially hypothesized to inhibit chick myopia by acting at the retina because of its muscarinic receptor subtype profile and the evidence for the retinal control of eye growth (Stone et al., 1991), but subsequent findings have questioned this initial mechanistic hypothesis. Muscarinic acetylcholine receptors are expressed in many regions of the chick eye (Fischer et al., 1998a), the retinal activity of the biosynthetic enzyme choline acetyltransferase is minimally affected in various eye growth models in chick (Pendrak et al., 1995), and a somewhat non-selective neurotoxin ablates most retinal cholinergic neurons without preventing experimental myopia in the chick or inhibiting the anti-myopia activity of atropine (Fischer et al., 1998b; McGurk et al., 1987). It has been suggested that muscarinic antagonists may act as non-muscarinic receptors to

inhibit myopia, based on variable effects of a series of muscarinic receptor antagonists (Bitzer et al., 2006; Luft et al., 2003). Because of possible compensatory developmental changes, the complex pharmacokinetics of drug distribution in the eye and the inherent differences between avian and mammalian muscarinic receptors, none of these experiments establish either the site or mechanism of these anti-myopia effects at the cellular or receptor level.

Regardless of the mechanistic uncertainties, the anti-myopia activity of pirenzepine in laboratory animals has stimulated its study in children. Pirenzepine had a long clinical history of systemic use in Europe and Asia as a gastrointestinal medication, and has a remarkably favorable safety profile. Thus, topical 2% pirenzepine gel was assessed in children for anti-myopia activity in two Phase II clinical trials, each of one year duration (Figure 9.3). In these United Statesand Asian-based trials, pirenzepine reduced myopia progression in children by approximately 50% and 44%, respectively, relative to control groups, with side effects judged as mild to moderate and generally well tolerated (Siatkowski et al., 2004; Tan et al., 2005). At present, development of pirenzepine as an anti-myopia therapeutic has ceased because the United States Food and Drug Administration (FDA) insisted on a clinically impractical Phase III plan. Others can debate whether the FDA’s position was appropriate safety monitoring for children, or instead an example of regulatory error (Carpenter and Ting, 2005) that denies children and hence adults the possibility of ameliorating a complex and significant ocular disorder. Introducing a novel clinical drug to retard myopia will be difficult, though, unless the FDA shifts its position to permit clinically practical development plans.

Despite the termination of pirenzepine’s development program, several important conclusions emerge. Originally identified in form deprivation myopia of chick, pirenzepine’s clinical efficacy demonstrates

178

Mean spherical equivalent (D)

(a)

the potential relevance of laboratory animal pharmacology to common human myopia. Like the anti-myopia activity of muscarinic antagonists in chick, the clinical results confirm that cycloplegia is not necessary for the anti-myopia activity of muscarinic antagonists in children, and suggest that mechanisms besides accommodation are operative in human myopia progression.

2. Nicotinic acetylcholine receptors

Acetylcholine gated ion channels, commonly called nicotinic acetylcholine receptors, comprise the other large class of acetylcholine receptors. Of the few available laboratory reports, the effects of several neural nicotinic acetylcholine receptors against form deprivation myopia in chick provide the strongest evidence implicating this other cholinergic receptor class in refractive development (Stone et al., 2001). The drugs studied in this report each have low affinity at nicotinic receptors in striated muscle, as occurs in the intraocular muscles of the bird, and their activity conforms to a

presumed neural site of action. The drugs with greatest activity were non-selective nicotinic acetylcholine receptor antagonists (Figure 9.4). Two of these drugs altered the refraction and growth of form deprived eyes with multiphasic dose responses, slightly exaggerating the myopic growth response at lower doses and inhibiting it at higher doses. A detailed mechanistic interpretation of these results in chick myopia is not now possible, because the multiphasic dose response curves potentially implicate multiple nicotinic acetylcholine receptor subtypes.

To learn whether these laboratory findings might be pertinent to refractive development in children and hence worth follow-up investigations, surveys of the association of refraction with passive exposure to cigarette smoke have been conducted in two pediatric populations, a tertiary care ophthalmology clinic in the United States and a group of young schoolchildren in Singapore. In the US survey (Figure 9.5), a strong association of reduced myopia prevalence with passive smoke exposure

Differences in refraction (goggle-open eye,in diopters)

(a)

VI. PHARMACOLOGY OF MYOPIA

179

Axial length

from either parent was observed, and an overall shift towards hyperopic refractions in the children exposed to tobacco smoke accounted for this effect (Stone et al., 2006c). In the Singapore study, a lower prevalence of myopia was observed among children exposed to cigarette smoke from their mothers, but not from their fathers (Saw et al., 2004). Questionnaire-based surveys have a variety of limitations, tobacco smoke contains many constituents besides nicotine, and the pharmacologic effects of cigarette smoke in the brain are known to be quite complex. Nonetheless, these results suggest a number of hypotheses for future research (Stone et al., 2006c), support the potential applicability of chick eye growth models to human refractive development, and justify further research into the possibility that nicotinic acetylcholine

receptors might modulate eye growth not only in chicks but also in children.

3. Acetylcholine esterase inhibition

Reports from Japan many years ago implicated exposure to organophosphate insecticides that inhibit acetylcholine esterase as a potential cause for myopia (Dementi, 1994; Ishikawa and Miyata, 1980). In chicks, the opposite effect has been observed. A single systemic dose of chlorpyrifos (an acetylcholine esterase inhibitor and organophosphate insecticide) or an intravitreal injection of diisopropylfluorophosphate (DFP; an acetylcholine esterase inhibitor) each reduced form deprivation myopia but did not affect the refractions of eyes with non-restricted vision (Cottriall et al., 2001a; Geller et al., 1998). Inhibiting acetylcholine esterase increases local

180

Percentage of children

9. MYOPIA PHARMACOLOGY: ETIOLOGIC CLUES, THERAPEUTIC POTENTIAL

High hyperopia

100

80

60

40

20

0

Parental smoking

acetylcholine levels. Thus, it is unclear how to reconcile the clinical and laboratory findings with acetylcholine esterase inhibition with each other, or with the laboratory and/or clinical findings that muscarinic or nicotinic antagonists can inhibit myopia. Increased retinal levels of both acetylcholine and dopamine develop in DFP-treated eyes, and the anti-myopia protective effects of DFP were partially reduced by co-administration of a dopamine receptor antagonist. It thus has been hypothesized that organophosphates might exert protective effects against form deprivation myopia indirectly through

effects on retinal dopamine metabolism (Cottriall et al., 2001a). Possible involvement of multiple cholinergic receptors, with different actions and/or affinities to acetylcholine, might also reconcile these apparently contradictory results.

B. Dopamine

Dopamine, a neurotransmitter in a subset of wide-field amacrine cells, normally undergoes a diurnal rhythm, with its retinal content and release higher during the day than at night. In both chicks and monkeys, visual deprivations that produce

ng/mg protein

1.2

0.8

0.4

0

Open eye

Translucent goggle

myopia reduce both the retinal content of dopamine and its release during the light phase, eliminating the normal day: night differences (Figure 9.6) (Iuvone et al., 1989; Stone et al., 1989). While the specific dopamine receptor subtype involved remains somewhat uncertain, local application of dopamine agonists inhibits experimental myopia in both chicks and monkeys, and provides evidence for the potential involvement of dopaminergic amacrine cells in the retinal pathway linking vision to the regulation of eye growth (Iuvone et al., 1991; Rohrer et al., 1993; Stone et al., 1989, 1990). Recovery from form deprivation myopia is also accompanied by rapid normalization of retinal dopamine levels (Pendrak et al., 1997). Parallel evidence suggests that retinal dopamine also participates in visually guided growth in chicks. Negative spectacle lenses that stimulate eye growth decrease indices of retinal dopamine turnover and positive lens wear causes the opposite (Guo et al., 1995; Schaeffel et al., 1995). Local therapy with dopamine agonists also inhibits the excess eye growth and myopia following negative lens wear, and augments the inhibition of

eye growth and hyperopia from positive lens wear (Schmid and Wildsoet, 2004).

While the altered dopamine release and metabolism with visual conditions influencing refractive development and the effects of dopamine agonist drugs are well established, the detailed biological mechanisms relating retinal dopamine metabolism to eye development are inadequately defined. It is uncertain if or how dopaminergic amacrine cells respond to the spatial and temporal stimuli involved in emmetropization (Luft et al., 2004). It also seems that dopamine may associate more with changes in eye growth rather than refraction per se, because retinal dopamine is reduced in specialized visual deprivations that induce generalized vitreous cavity expansion, but also permit emmetropization (Stone et al., 2006a). In comparing lens induced and form deprivation myopia, the similar changes in retinal dopamine content and inhibitory actions of dopamine agonists indicate at least some parallels in the signaling pathways in these two eye growth models, despite the differences described above. The similar inhibitory action of muscarinic antagonists

in both models also attests to some parallels in the signaling pathways. Whether there are other biochemical similarities of the growth signaling pathways in the two models is unknown, as is the molecular bases for their differences.

The effects of dopamine agonist drugs on myopia in children have not been studied directly because of concerns about potential systemic side effects. Two indirect and somewhat distinct approaches, however, hint that the dopamine signaling might contribute to refractive development in children as in experimental animals. Retinal dopamine metabolism responds to light and dark (Parkinson and Rando, 1983), and retinal dopamine rhythms modulate a variety of rhythms in the retina (Cahill and Besharse, 1995; Iuvone et al., 2005; Manglapus et al., 1999). Available studies pertinent to a potential role of retinal dopamine in human refraction draw from the dual influences of retinal dopamine on various endogenous retinal rhythms and on refractive development.

The first approach assesses whether modifying the daily light–dark cycle influences eye development, as might happen if ocular rhythms essential for emmetropization are masked (Doi et al., 2006; Mrosovsky, 1999) or otherwise disrupted with the absence of a suitable dark period. Because refractive development of laboratory animals can be influenced by altering photoperiod, several population surveys have assessed whether features of human refractive development are associated with a history of disrupting the dark phase of the daily light:dark cycle with artificial lighting at night. Some surveys have suggested that exposure to nighttime ambient lighting is associated with reduced myopia or eye length (Chapell et al., 2001; Czepita et al., 2004, 2005; Loman et al., 2002; Quinn et al., 1999; Saw et al., 2002c), but similar studies in other populations have not found such associations (Guggenheim et al., 2003; Gwiazda et al., 2000; Saw et al., 2001; Stone et al., 2006c; Zadnik et al., 2000).

Explanations for these different results are purely speculative, but may relate to population differences, inherent limitations in questionnaire-based surveys, or even reporting bias or behavioral modifications in response to the media attention to these reports. A high prevalence of myopia in army conscripts from northern Finland, compared to those from southern Finland, also suggests some association of lighting and refractive development (Vannas et al., 2003). Despite the disparate results in available clinical studies, altering artificial light exposures certainly is a feature of the modern environment more amenable to modification than most other putative myopia risk factors, and would seemingly warrant clinical study with more definitive experimental designs.

The second approach extends laboratory work originally stimulated by the apparent role of altered dopamine rhythms in experimental myopia. The axial dimensions of the eyes of chicks, rabbits and marmosets with non-restricted vision have been found to fluctuate in amplitude during the day (Liu and Farid, 1998; Nickla et al., 2002, 1998a,b; Papastergiou et al., 1998b; Weiss and Schaeffel, 1993). These fluctuations involve diurnal changes in axial length, vitreous chamber, choroidal thickness and, at least in rabbit, anterior chamber depth. The net effect of these oscillations is that normally developing eyes of chicks grow chiefly during the day and not at night (Nickla et al., 1998b; Papastergiou et al., 1998b; Weiss and Schaeffel, 1993). In chicks with form deprivation myopia, the growth rhythms are shifted, resulting not only in accelerated growth, but also in more comparable growth during the day and night. Thus, both dopamine rhythms and growth rhythms are perturbed in chick myopia.

High resolution interferometry techniques have now revealed comparable diurnal fluctuations in human eye size as well (Stone et al., 2004), but no data are as yet available on children developing refractive errors. In both chicks and in humans,

eye length fluctuations appear to be a novel diurnal rhythm, not merely passive expansion and contraction of the globe in response to diurnal changes in intraocular pressure (Schmid et al., 1999; Wilson et al., 2006). Though not developing frank refractive errors during rearing periods of 3 weeks, chicks reared under certain continuous but non-constant light (i.e. light-dim) cycles also demonstrate disrupted daily rhythms in both dopamine metabolism and in the daily fluctuations of intraocular dimensions (Liu et al., 2004). While much work is needed to achieve clinical insights, these data suggest that study of the diurnal rhythms in ocular dimensions could be a productive research approach applicable to children.

C. GABA (γ-Aminobutyric Acid)

GABA, an amino acid neurotransmitter, is expressed by many subtypes of retinal neurons, including some amacrine cells that co-express dopamine or acetylcholine. GABA has so far not been identified in peripheral nerves to the eye or in other non-retinal ocular tissues. Locally adminis-

tered drugs selective for GABAA, GABAA0r or GABAB receptor subtypes modulate eye

growth and refractive development in chick (Stone et al., 2003). Inhibitors of all three receptor subtypes, for example, reduce form deprivation myopia. Goggled chick eyes have slightly reduced retinal levels of GABA compared to contralateral control eyes (Stone et al., 2003). Based on the known distribution of GABA containing cells, these findings are consistent with a retinal site of action for GABA drugs in altering eye growth. In infant rhesus monkeys, image defocus influences the transcription of immediateearly genes in many retinal neurons, including a subset of GABA-containing amacrine cells (Zhong et al., 2004).

D. Retinoic Acid

Retinoic acid, a vitamin A derivative, is an endogenous signaling molecule that

modulates many developmental processes, such as the overall somatic organization, central nervous system development, and the patterning of specific organs. It also has been suggested as a potential signaling molecule regulating refractive development, perhaps even controlling the direction of eye growth (McFadden et al., 2004; Mertz and Wallman, 2000; Troilo et al., 2006).

Retinal levels of retinoic acid and the mRNA for retinoic acid receptor-α are slightly increased in form deprived chick eyes; and sclera from myopic eyes demonstrates increased expression of mRNA for the retinoic acid receptor-β (Morgan et al., 2004; Seko et al., 1998, 1996). In contrast to the increased retinoic acid concentration in the retina of myopic chick eyes, retinoic acid synthesis is decreased in the choroid with goggle and minus lens wear, conditions that accelerate eye growth; conversely, choroidal retinoic acid synthesis is increased by plus lens wear and myopia recovery, conditions with reduced growth rates. In chick retina, negative lens wear upregulates the mRNA level for retinaldehyde dehydrogenase-2, an enzyme involved in retinoic acid synthesis; positive lens wear upregulates the retinal mRNA for retinoic acid receptor-β; and an inhibitor of retinoic acid synthesis partially reduces form deprivation myopia, but does not influence the responses to minus or plus spectacle lens wear (Bitzer et al., 2000). Pertinent localizations of retinoid binding proteins, retinoid receptors and retinaldehyde dehydrogenase have been reported for the chick retina and supportive tissues, consistent with potential signaling roles in refractive development (Fischer et al., 1999c).

The retinoic acid levels of mammalian eyes also respond to visual conditions that influence eye growth, although the details of the patterns differ from chick. In guinea pig eyes, eyes with stimulated growth rates, from either goggles or minus lens wear, develop increased retinal and choroid/scleral levels of retinoic acid; eyes with reduced growth rates during myopia