Atropine reduces experimental myopia and eye enlargement via a ...

Atropine Reduces Experimental Myopia and EyeEnlargement Via a Nonaccommodative MechanismNeville A. McBrien, Hadi 0. Moghaddam, and Anne P. Reeder

Purpose. To determine whether the muscarinic antagonist atropine effectively reduces or pre-vents experimentally induced myopia via a nonaccommodative mechanism.

Methods. Chicks were monocularly deprived (MD) of pattern vision by placement of a translu-cent occluder over the left eye. In two of the three MD groups, chicks received a series ofintravitreal injections of atropine (n = 8) or saline vehicle (n = 8) with MD. Control groups (n= 8) of chicks were employed to assess the effects of MD, intravitreal injections, and drugeffects.

Results. In sham-injected or saline-injected MD chicks, 8 days of MD produced —18.5 D and—20.9 D of experimental myopia, respectively. In atropine-injected MD chicks, 8 days of MDproduced only —2.8 D of experimental myopia. This significant reduction in experimentallyinduced myopia in atropine-injected MD chicks was associated with a marked reduction in therelative axial elongation of the deprived eye (0.21 mm) when compared to saline-injected orsham-injected MD chicks (1.04 mm and 1.00 mm). This reduction in axial length in atropine-injected MD chicks was predominantly the result of a reduction in vitreous chamber elonga-tion, although a reduction in anterior segment depth also was observed. Mean equatorialdiameter was significantly reduced in atropine-injected MD chicks compared to saline-injectedand sham-injected MD chicks, although to a lesser extent. Control experiments demonstratedthat intravitreally injected atropine did not reduce carbachol-induced accommodation orlight-induced pupil constriction in the skeletal intraocular muscles of the chick eye.

Conclusions. These findings demonstrate that chronic administration of the muscarinic antago-nist atropine prevents experimentally induced myopia in chick via a nonaccommodative mecha-nism. Invest Ophthalmol Vis Sci. 1993;34:205-215.

Ueprivation of pattern vision in human infants (eg,ptosis, hemangioma of the lid) has been found to causean axial elongation of the deprived eye and high de-grees of myopia.1"4 It also has been demonstrated inseveral animal species that monocular deprivation

From the Department of Optometry & Vision Sciences, University ofWales, College of Cardiff, Cardiff, United Kingdom.Supported in part by a grant from Royal National Institute for the Blind(UK).Presented in part at the Annual Meeting of the Association for Researchin Vision and Ophthalmology, Sarasota, Florida, May 3, 1991.Submitted for publication: February 28, 1992; accepted July 30, 1992.Proprietary interest category: N.Reprint requests: Neville A. McBrien, Department of Optometry &Vision Sciences, University of Wales, College of Cardiff', King EdwardVII Avenue, Cardiff, CF1 3XF, United Kingdom.

(MD) of pattern vision in neonates produces an axialelongation of the eye, resulting in high degrees of myo-pia. These results have been found to occur inchicks,5"7 tree shrews,8'9 cats,10 gray squirrels,11 mar-mosets,12 and monkeys.13*14

One mechanism that has been proposed as a caus-ative factor in myopia development in humans and inanimal models is that of accommodation.15"18 Supportfor this suggestion comes from results of studies thathave employed cycloplegic agents such as atropine toretard the development of myopia. In adolescent hu-mans, daily administration of 1% atropine was foundto prevent the progression of juvenile myopia in thetreated eye,19-20 although less conclusive results alsohave been observed, especially with weaker cyclople-gics.21 In animal models, atropine administration also

Investigative Ophthalmology & Visual Science, January 1993, Vol. 34, No.Copyright © Association for Research in Vision and Ophthalmology 205

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206 Investigative Ophthalmology & Visual Science, January 1993, Vol. 34, No. 1

has been found to prevent or reduce the developmentof experimentally induced myopia. Young2216 foundthat monkeys {Macaca nemestrina) restricted to a nearviewing environment reliably developed myopia thatcould be halted if animals were treated with topical 1%atropine eye drops three times a day.

Raviola and Wiesel18 found that chronic atropineadministration to lid-sutured stumptail Macaque mon-key eyes prevented the development of experimentalmyopia, although they also found that atropine did notprevent myopia in a different species of monkey (rhe-sus). In lid-sutured tree shrews, chronic administra-tion of atropine was found to prevent the develop-ment of axial myopia,17 and although a recent study23

found topical atropine did not block myopia in treeshrews, results in our laboratory using intravitreallyinjected atropine in tree shrew did block myopia(McBrien and Reeder, unpublished observations),suggesting a dose-dependent effect. In all the studieswith positive findings, it was concluded that atropineeffectively prevented or retarded the progression ofthe myopia because of its cycloplegic effect on smoothciliary muscle, which blocked the accommodativefunction of the eye. This has led to the development offeedback models of refractive development that incor-porate a pivotal role for accommodation.1517

In recent years, several investigations have furtherelucidated the possible underlying mechanisms re-sponsible for altering coordinated ocular growth inthe absence of form vision. Studies have shown thatblocking communication between the eye and highercenters—by optic nerve section in chicks24'25 andmonkeys18 or blockade of retinal ganglion cell actionpotentials in tree shrews26 and chicks27—does not pre-vent the development of experimentally induced myo-pia. Other studies have shown that deprivation of onlypart of the visual field results in local areas of elonga-tion and myopia in the eye.2829 It also has been demon-strated that experimental myopia can be induced in aspecies that does not possess a functional accommoda-tive system11 or where accommodation has beenblocked by bilateral lesioningof the Edinger-Westphalnucleus.30 Recently, it has been shown in chick thatexperimentally induced myopia is associated with in-creased synthesis of scleral protein,31'32 DNA,31'32 andproteoglycans3233 and with increases in total scleralcollagen content.32 This indicates that active growth ofthe sclera, as opposed to passive stretching, is takingplace. The above findings, which point toward localocular control of postnatal eye growth, question therole of accommodation as a major causative factor ofexperimental myopia and suggest that the mechanismby which atropine prevents or reduces myopia may benonaccommodative.

In primate and mammalian models of experimen-tal myopia, atropine invariably blocks the accommoda-

tive function of the eye, because of its effect at musca-rinic receptors in the ciliary muscle.34 As a result, inthese species it has been difficult to address whetheratropine has its effect on myopia development via anonaccommodative route. However, because the intra-ocular muscles of the chick eye are striated and con-tain predominantly nicotinic receptors,35-36 atropine (amuscarinic receptor antagonist) should not producecycloplegia or pupil dilation. The present study soughtto determine whether atropine effectively reduces orprevents experimentally induced myopia via a nonac-commodative mechanism in chick.

MATERIALS and METHODS

Animals

One-day-old hatched chicks (Rhode Island cross) weredelivered and kept on a 12 hr light/12 hr dark cycle ina temperature-controlled environment. Food andwater were available ad libitum. Chicks were allocatedto one of six experimental groups on the basis ofwhether they were monocularly occluded (MD) andwhether they received intravitreal injections of eitheratropine or saline. Each of the six groups containedeight chicks. All investigations concerning animals ad-hered to the ARVO Statement for the Use of Animalsin Ophthalmic and Vision Research.

Treatment Protocols

Initial pilot experiments indicated that topical applica-tion of 1% atropine sulfate drops daily produced nosignificant difference in experimentally induced vitre-ous chamber elongation and myopia for atropine-treated (n = 6) compared to saline-treated (n = 6) MDchicks. Because of this finding, it was decided to applyatropine via intravitreal injection to enhance the possi-bility that an effective dose would reach potential tar-get sites. On day 6 after hatching, chicks were given anintravitreal injection of 7 fi\ of phosphate bufferedatropine (calculated concentration at retina, 250— 300 jumol/1, equivalent to 0.01%) or phosphate buff-ered saline under halothane (2-3.5%) anesthesia. In-travitreal injections were carried out by surgically wid-ening the palpebral aperture on the temporal side ofthe left eye, inserting a sterile 30 G needle (connectedto a microsyringe via sterile tubing), and injecting thedrug or vehicle control into the vitreous. The temporalopening then was closed with a single surgical suture,and genticin ointment was applied to the injected areato prevent infection. Chicks in the MD groups thenhad a translucent hemispheric occluder placed overthe injected eye. This initial injection procedure wasrepeated every 48 hr over 8 days, resulting in fourintravitreal injections per animal. It was nearly alwayspossible to find the same injection site on subsequent


Atropine Reduces Experimental Myopia 207

sessions. The occluders were renewed after every in-jection for MD animals and were never off for morethan 15 min, during which time the animal was anes-thetized.

As a control for the effect of intravitreal injection,one group of chicks (sham-injected) underwent halo-thane anesthesia, opening of the palpebral aperture,and mechanical pressure from, but without insertionof, the needle on the sclera, on exactly the same sched-ule as MD chicks who had intravitreal injections. Twogroups of chicks had intravitreal injections of atropineor saline without MD to control for drug and injectioneffects, respectively. One group of chicks underwentneither intravitreal injection nor monocular occlusionbut were housed in identical conditions and had opti-cal and structural measures taken. Thus, the sixgroups were sham-injected MD, saline-injected MD,atropine-injected MD, atropine-open, saline-open,and normal-open, with eight chicks in each group.

In Vivo Optical and Structural Measures

Optical and structural measures were taken 8 daysafter the first injection. Chicks were anesthetized withketamine (50 mg/kg) and xylazine (3.5 mg/kg), andbody temperature was maintained (at 37°C) via a heat-ing pad. Supplementary doses of anesthetic were givenas required. All refractive and structural measureswere taken after cycloplegia. Because of the predomi-nantly striated nature of chick intraocular muscles,37

cycloplegia was achieved using five 25 ix\ drops of ver-curonium bromide (2 mg/ml) spaced at 1 min inter-vals. The cornea was pre-treated with topical proxy-metacaine HC1 (0.5%) to enhance penetration of thedrug. Measurements were begun 30 min after the finaldrop, at which time there was full pupil dilation and noevidence of pupil reactions. Corneal curvature wasmeasured using a modified one-position keratometerfitted with a brighter light source and a +8 diopterlens to extend the scale. Eight readings were taken ofboth the horizontal and vertical meridians of the cor-nea for each eye. Readings were converted to cornealradius (mm) using a calibration equation derived bymeasuring ball bearings of known radii. Refractionwas measured to the nearest 0.5 D by streak retino-scopy on the horizontal and vertical meridians at aworking distance of 33 cm. Values were converted intomeasures of ocular refraction at the corneal plane bycorrecting for the effectivity of the correcting lens(held 5 mm from the cornea) and the working dis-tance. Horizontal and vertical measures were aver-aged to obtain the spherical equivalent refraction. Nocorrection was made for the effect of eye size on retin-oscopy measures.38

In vivo measurements of the axial dimensions ofthe structural components of the eye, along the opticaxis, were obtained by A-scan ultrasonography. A 10

MHz, 6.35 mm diameter ultrasound transducer fo-cused at 22 mm and driven by a Panametrics 5052pulser/receiver was coupled to a 15 mm perspex stan-doff that was perfused continuously with 0.9% saline(flow rate 0.8 ml/min). The standoff was positioned byhand so the saline column contacted the anesthetizedcornea (0.5% proxymetacaine HC1) without any ap-planation. Waveform echoes passed from the pulser/receiver into a LeCroy (Geneva, Switzerland) 9400 dig-ital storage oscilloscope (sample rate 100 megasam-ples/s). To enhance the signal-to-noise ratio, eachstored waveform was the average of 20 single incom-ing waveforms. Six stored waveforms from indepen-dent positionings of the transducer were collected foreach eye and transferred to an Opus (Reading, UK) PCV (AT) computer for subsequent measurement. Con-version of time to distance employed previously pub-lished values for the chick eye.39

In Vitro Measures and Histology

After all the in vivo optical and structural measureswere completed, the animal was deeply anesthetizedwith sodium pentobarbital and the eyes were enucle-ated. Digital caliper measurements of the medial/la-teral and superior/inferior equatorial diameters andthe axial length were taken. The eye then was weighedto the nearest 10 /*g. To determine whether the drugtreatment altered the physical integrity of the retina,eyes from atropine-injected and saline-injected chickswere placed in fixative (phosphate buffered 2.5% glu-taraldehyde) for 24 hr and dehydrated via graded alco-hols and embedded in epoxy resin for histologic evalu-ation. Semi-thin sections (2 /xm) of the posterior polewere cut and examined at the light microscopy level.

Pupil and Accommodation Measures

Evidence for the presence of extra-synaptic musca-rinic receptors recently was presented for chick iris.Although these receptors are more prominent in theembryo, they also are present after hatching.35 A simi-lar mixed cholinergic receptor population is suggestedto be present in chick ciliary muscle. In light of thesefindings, it was important to determine whether atro-pine had any measurable effect on accommodation orpupil responses; thus, two control experiments wereperformed. Age-matched chicks were given intravi-treal injections of atropine (n = 5) or saline (n = 5), asdescribed above. Horizontal and vertical pupil diame-ters were measured using a small operating micro-scope with a measuring graticule eyepiece at X10 mag-nification. Readings were taken before the intravitrealinjection at a luminance of 100 lux and approximately60 min after the injection. Further post-injection mea-sures were taken after an initial 10 min period of darkadaptation at luminances of 10, 50, 100, 500, 1000,and 2000 lux, in an ascending pattern, with 3 min dark



adaptation between readings at different luminances.The investigator who took the readings was unaware ofthe experimental treatment (atropine or saline).

To determine the effect of atropine on accommo-dative amplitude, another two groups of chicks weregiven intravitreal injections of atropine (n = 5) or sa-line (n = 5). One hour after intravitreal injection, thechicks were anesthetized with ketamine and xylazine,and baseline measures of the refractive state of in-jected and contralateral control eyes were taken usingstreak retinoscopy. A-scan ultrasonography was takenon both eyes of each chick using the procedure de-scribed above, except that only three averaged wave-forms were recorded for each eye. Carbachol 10% (nic-otinic and muscarinic agonist) then was topically ap-plied to the injected eye via corneal iontophoresis.40

The cornea was pre-treated with topical proxymeta-caine HC1 (0.5%) to enhance penetration of the drug;then a 2.5% agar button of 5 mm diameter containingcarbachol 10% was placed on the center of the cornea.The negative electrode was attached to the skin, andthe positive electrode was gently applied to the agarbutton for 20 sec at a current of 200 /xA. Because ofthe rapid and relatively short-lived response to phar-macologic stimulation of chick-striated ciliary mus-cle,36 all measures were recorded within 10 min of topi-cal application of carbachol. The pupil diameter of thecarbachol-treated eye was measured over the first 3min post-application at 30 sec intervals. Five min afterthe carbachol was applied, the refractive state wasmeasured in the horizontal and vertical meridians witha streak retinoscope. A-scan ultrasonography then wastaken on the injected eye as described above, andreadings were obtained 9 min (± 1) min after topicalcarbachol was applied. Once measures were com-pleted on the injected eye, retinoscopy and ultrasoundwere taken again on the contralateral control eye.

Statistical Analysis

For analysis of differences between groups, paramet-ric statistics were employed (analysis of variance [AN-OVA], t-test) because there was no evidence of askewed distribution.

RESULTS

Refractive differences for all six experimental groupsare shown in Figure 1 and Table 1. In sham-injectedchicks (n = 8), monocular deprivation of pattern visionfor 8 days produced a significant myopia in the de-prived eye of-18.5±2.9D (mean ± SEM) when com-pared to the contralateral open control eye. In saline-injected MD chicks (n = 8), a relative myopia betweendeprived and control eyes of —20.9 ± 1.2 D was ob-served. In atropine-injected MD chicks (n = 8), a rela-tive myopia of —2.8 ± 1.5 D was observed. One-wayANOVA revealed a highly significant difference in ex-

Sham- ln | Saline Atropine Atropine Saline NormalMD MD MD Open Open Open

FIGURE 1. Differences in ocular refraction between opencontrol eyes and deprived eyes in monocularly deprivedchicks and between open control and injected or right andleft eyes of binocularly open chicks. Cycloplegic refraction(vercuronium bromide) as measured by streak retinoscopy.Chronic atropine administration almost completely elimi-nated the experimental myopia produced by monocular de-privation, n = 8 in each group. Error bars = J SEM.

perimentally induced myopia between MD groups (F= 24.5, P< 0.001). Duncan's multiple comparison testrevealed significant differences between the atropine-injected MD group and both the saline-injected MDgroup (P < 0.001) and sham-injected MD group (P< 0.001), whereas there was no significant differencebetween the sham-injected and saline-injected MDgroups. Thus, the muscarinic antagonist atropine, ad-ministered as described, almost completely eliminatedexperimentally induced myopia in treated chicks. Re-fractive differences between experimental and controleyes in groups that received intravitreal injections ofatropine or saline, but not MD, were not significantlydifferent from refractive differences between rightand left eyes of normal binocular chicks (F = 2.72, P= 0.09). Thus, atropine alone did not significantly af-fect the refractive state of the eye.

The major structural cause of the experimentallyinduced myopia in sham-injected and saline-injectedMD chicks was enlargement of the vitreous chamber,axially and equatorially, as found in previous stud-ies.39'41 Vitreous chamber depth (Fig. 2A; Table 1), asmeasured by A-scan ultrasonography, showed a rela-tive elongation in the deprived eye of sham-injectedand saline-injected MD chicks of 0.86 ± 0.10 mm and0.92 ± 0.06 mm, respectively. In atropine-injectedMD chicks, the deprived eye showed a relative vitreouschamber elongation of only 0.38 ± 0.05 mm, com-pared to the contralateral open control eye. There wasa highly significant difference in vitreous chamberelongation between MD groups (ANOVA, F = 16.28,P < 0.001). There was no significant difference in vit-reous chamber depth between injected and controleyes for atropine-injected (P = 0.80) or saline-injected



TABLE l. Cycloplegic Ocular Refraction and Axial Dimensions of All Deprived (Dep) andOpen Control Eyes of Monocularly Deprived (MD) Chicks and Right and Left Eyes ofBinocularly Open Chicks

Retinoscopy(D)

Cornealradius(mm)

Anteriorsegment(mm)

Lensthickness(mm)

Vitreouschamber(mm)

Axiallength(mm)

Equatorialdiameter(sup/inf; mm)

Equatorialdiameter(Med/Lat; mm)

Equatorialdimensions(average; mm)

Eyeweight(g)

DepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - ODepOpenD - O

Sham-Injected MD

-15.3 ±2.6+3.2 ±0.6

-18.5 ±2.93.15 ±0.043.18 ± 0.02

-0.03 ± 0.041.70 ±0.081.57 ± 0.040.13 ±0.062.34 ± 0.082.33 ± 0.070.01 ±0.026.36 ±0.175.50 ±0.180.86 ±0.10

10.40 ± 0.279.40 ± 0.231.00 ±0.14

12.56 ±0.1612.18 ±0.100.38 ± 0.08

12.55 ±0.1512.03 ±0.080.52 ± 0.09

12.55 ±0.1512.11 ±0.090.44 ± 0.080.77 ± 0.030.64 ± 0.010.13 ±0.02

Saline MD

-17.3 ± 1.2+3.6 ±0.7

-20.9 ± 1.53.20 ± 0.033.22 ± 0.03

-0.02 ±0.011.65 ±0.031.54 ±0.020.11 ± 0.042.28 ± 0.072.27 ±0.070.01 ±0.026.35 ±0.195.43 ± 0.130.92 ± 0.06

10.27 ±0.249.23 ±0.211.04 ±0.06

12.73 ±0.1712.03 ±0.130.36 ± 0.06

12.78 ±0.1512.09 ± 0.120.69 ±0.10

12.75 ±0.1612.23 ±0.120.52 ± 0.070.79 ± 0.030.64 ± 0.020.14 ±0.01

Atropine MD

+0.7 ± 1.5+3.5 ±0.3-2.8 ± 1.7

3.29 ± 0.043.25 ± 0.030.04 ± 0.021.44 ±0.051.61 ±0.03

-0.17 ±0.052.27 ±0.072.27 ± 0.070.00 ± 0.026.04 ±0.195.66 ±0.150.38 ± 0.049.76 ± 0.259.55 ± 0.230.21 ±0.05

12.48 ± 0.1712.26 ±0.130.22 ± 0.07

12.33 ± 0.1412.04 ± 0.140.29 ± 0.08

12.40 ±0.1512.15 ±0.130.25 ± 0.060.70 ± 0.020.64 ± 0.020.06 ± 0.01

LRL - RLRL - RLRL - RLRL - RLRL - RLRL - RLRL - RLRL - RLRL - RLRL - R

Atropine Open

+4.9 ±0.5+3.6 ±0.5+ 1.3 ±0.7

3.23 ± 0.023.17 ±0.020.06 ± 0.021.51 ±0.031.60 ±0.03

-0.09 ± 0.022.37 ± 0.072.35 ± 0.070.02 ± 0.015.71 ±0.145.66 ±0.150.05 ± 0.039.60 ± 0.229.61 ±0.24

-0.01 ± 0.0412.30 ±0.0812.21 ±0.090.09 ± 0.05

12.05 ±0.0712.00 ±0.080.04 ± 0.05

12.17 ± 0.0712.10 ±0.080.07 ± 0.040.63 ± 0.010.63 ±0.010.00 ±0.01

Saline Open

+3.4 ± 0.5+3.0 ±0.3+0.4 ± 0.5

3.20 ± 0.033.20 ± 0.030.00 ± 0.021.48 ±0.021.53 ±0.03

-0.05 ± 0.022.23 ± 0.062.21 ±0.040.02 ± 0.025.31 ±0.105.32 ±0.10

-0.01 ±0.039.02 ±0.169.06 ±0.170.04 ± 0.03

12.22 ±0.1312.26 ±0.10

-0.04 ± 0.0811.98 ±0.1211.92 ± 0.140.06 ±0.11

12.10 ± 0 .1212.09 ±0.120.01 ± 0.090.64 ± 0.0.10.67 ± 0.010.00 ±0.01

Normal

+2.6 ±0.3+3.0 ±0.2-0.4 ± 0.2

3.21 ±0.033.22 ± 0.03

-0.01 ±0.011.49 ±0.021.51 ±0.02

-0.02 ±0.012.21 ±0.022.22 ± 0.02

-0.01 ±0.015.30 ± 0.045.29 ± 0.050.01 ± 0.029.01 ± 0.069.02 ± 0.07

-0.01 ±0.0212.45 ±0.0912.48 ±0.09-0.03 ±0.0312.26 ±0.0812.30 ±0.08

-0.04 ± 0.0212.36 ±0.0812.39 ±0.09

-0.03 ± 0.010.68 ± 0.020.67 ± 0.020.01 ±0.01

In all cases, the left eye received the intravitreal injection or deprivation, n = 8 in all groups. Figures are means ± SEM.

(P = 0.97) binocular groups, or between right and lefteyes of normal binocular chicks (P = 0.89).

Sham-injected (+0.13 ± 0.06 mm) and saline-in-jected (+0.11 ± 0.04 mm) MD chicks developeddeeper anterior segment depths (corneal thicknessplus anterior chamber depth) in the deprived eye com-pared to the fellow control eye (Fig. 2B; Table 1),whereas atropine-injected MD chicks developed shal-lower anterior segment depths in the deprived eye(—0.17 ± 0.05 mm). Comparison of differences foranterior segment depth between deprived and controleyes in MD groups revealed a highly significant differ-ence (F = 11.01, P < 0.001). One-way ANOVA alsorevealed significant differences in anterior segmentdepth between eyes in binocularly open groups (F= 4.07, P < 0.05) as a result of atropine-injected openeyes developing shallower anterior segment depthsthan their fellow control eyes (—0.09 ± 0.02 mm). Nosignificant differences were observed in corneal cur-vature between experimental and control eyes of MDor binocular animals (F = 2.33, P = 0.06), althoughthe atropine groups were the only groups in which theexperimental eye had a flatter corneal curve. No dif-ferences in lens thickness were found between de-

prived and control eyes of MD chicks or between in-jected and control eyes for binocularly open chicks forany experimental group (F = 0.56, P = 0.73).

Combining interocular axial dimensions, obtainedby A-scan ultrasonography, to give axial length mea-sures revealed marked differences between atropine-injected MD chicks and the other MD groups (Fig. 2C;Table 1). Atropine-injected MD chick eyes developed arelative axial elongation of only 0.21 ± 0.15 mm, com-pared to their fellow control eye, whereas saline-in-jected MD chick eyes underwent a relative axial elonga-tion of 1.04 ± 0.06 mm and sham-injected MD chickeyes underwent an axial elongation of 1.0 ± 0.14 mm.

Equatorial dimensions, as measured with digitalcalipers, also showed differences for atropine-treatedMD chicks compared to saline-injected or sham-in-jected MD chicks. Atropine-injected MD chicks un-derwent less enlargement of the superior/inferiorequatorial dimension (0.22 ± 0.07 mm) than did sa-line-injected (0.36 ± 0.06 mm) or sham-injected (0.38± 0.08 mm) MD chicks, although this enlargement wasnot statistically significant (F = 1.46, P = 0.25). How-ever, measurement of the medial/lateral equatorial di-mension revealed a significant reduction in the


210 Investigative Ophthalmology 8c Visual Science, January 1993, Vol. 34, No. 1

5 -0.2Sham-ln| Sallna Atroplna Atroplna Sallna Normal

MD MD MD Opan Opan Opan

5 -0.4Sham-ln| Sallna Atroplna Atroplna Sallna Normal

MD MD MD Opan Opan Opan

Sham-ln| Sallna Atroplna Atroplna Sallna NormalMD MD MD Opan Opan Opan

-0.2

D

N=8 (aach group)

Sham-ln| Sallna Atroplna Atroplna Sallna NormalMD MD MD Opan Opan Opan

FIGURE 2. Differences in ocular dimensions between deprived and contralateral open controleyes in monocularly deprived (MD) chicks and between injected and contralateral controleyes or left and right eyes of binocularly open chicks. (A) Differences in vitreous chamberdepth. Chronic atropine administration to the deprived eye caused a significant reduction invitreous chamber elongation in MD chicks compared to control MD chicks (P < 0.001). Nodifferences were observed in binocularly open chicks. (B) Differences in anterior segmentdepth (corneal thickness plus anterior chamber depth). Chronic atropine administrationcaused a reduction in anterior segment depth in deprived chick eyes in contrast to the usualdeepening of anterior segment depth in control MD chicks (P < 0.001). (C) Differences inaxial length. Chronic intravitreal atropine administration almost completely eliminated theaxial elongation produced by monocular deprivation, but had no effect on axial length inopen eyes. (D) Differences in equatorial diameters (superior-inferior plus medial-lateral/2).Chronic intravitreal atropine administration caused a significant reduction in mean equato-rial eye enlargement in deprived chick eyes compared to control MD chicks (P < 0.05). N = 8in each group. Error bars = 1 SEM.

amount of enlargement in the deprived eye of atro-pine-injected MD chicks (0.29 ± 0.09 mm) comparedto saline-injected (0.69 ±0.10 mm) or sham-injected(0.52 ± 0.09 mm) MD chicks (F = 4.92, P < 0.02).Averaging the equatorial dimensions (Fig. 2D; Table1) also resulted in significant differences betweenatropine MD chicks (0.25 ± 0.06 mm) and saline (0.52± 0.07 mm) or sham (0.44 ± 0.08 mm) MD chicks (F= 3.72, P < 0.05). Digital caliper measures of axiallength confirmed in vivo ultrasound measures byshowing a significant reduction in axial elongation inatropine MD chicks.

Histologic evaluation of the retina (posterior pole)at the light microscope (magnification XI000) levelrevealed no apparent toxic effects on the retina ofatropine-treated eyes when compared to saline-treated eyes (Fig. 3).

Intravitreally injected atropine did not cause a re-duction in the direct pupil response to light (Fig. 4).Pupil area was not significantly different betweenatropine-injected chicks (n = 5) and saline injected (n= 5) controls for illuminance levels ranging from 10-2000 lux (P> 0.17 at all levels). Corneal iontophoresisof 10% carbachol induced the same degree of accom-


Atropine Reduces Experimental Myopia

(a) \ - - ~ - -

211

(b)

FIGURE 3. Histologic appearance of the retina, choroid, and sclera at the posterior pole from(a) an atropine MD chick eye and (b) a saline MD chick eye. No signs of toxic damage to theretina of the atropine-treated retina could be observed when compared to the saline MDretina. The saline MD retina was thinner than the atropine MD retina because of the signifi-cantly greater axial elongation and myopia in this eye. Stained with loluidine blue. (Scale bar= 100 /itn.)

2.01

0.00.0

A A ATROPINE

A A SALINE/—s

EE

LJ

ElQ_

8.0-

6.0-

4.0-

T

A

\I

1.0 2.0 3.0 4.0

ILLUMINANCE (LOG LUX)

FIGURE 4. Constriction of the pupil in response to increasinglight levels in chick eyes after intravitreal injection of atro-pine or saline. No significant difference in pupil diametersat any light level was observed between atropine and salineinjected chick eyes (P < 0.1 7). N = 5 in each group. Errorbars = 1 SEM.

modation (9.9 +1 .6D versus 9.1 ± 1.4 D; P = 0.70)and increase in lens thickness (0.10 ± 0.01 mm versus0.10 ± 0.02 mm; P = 0.77) in atropine-injected (n = 5)and saline-injected (n = 5) chicks (Fig. 5).

DISCUSSION

The finding that the muscarinic antagonist atropinealmost completely prevents the development of experi-mentally induced myopia is not a new finding. Pre-vious studies, however, usually concluded that atro-pine was preventing myopia development by blocking-accommodation.l718'22 However, because no signifi-cant differences in carbachol-induced accommoda-tion or pupillary constriction to light were observedbetween atropine- and saline-injected chicks, the pres-ent study demonstrates that chronic atropine adminis-tration prevents experimentally induced myopia inchick via a nonaccommodative mechanism.

The reduction in axial myopia was accounted formainly by a reduction in vitreous chamber elongationin the deprived eye—the major structural alteration inexperimentally induced myopia in chick. However, it is



12 >

INDUCEDACCOMMODATION

FIGURE 5. Change in lens thickness and amount of accommo-dation induced by corneal iontophoresis of 10% carbacholin chick eyes after intravitreal injection of atropine or saline.No significant difference in lens thickness changes (P= 0.77) or induced accommodation (P = 0.70) were ob-served between atropine- and saline-injected chick eyes. N= 5 in each group. Error bars = 1 SEM.

interesting that there also was a reduction in anteriorsegment depth in atropine-injected MD eyes whencompared to their contralateral control eye. This re-duction, which also contributed to reducing the ob-served myopia, indicates that atropine effectively pre-vents or attenuates MD-induced axial enlargement ofthe anterior and posterior segments of the chick eye.

The finding that ocular enlargement was atten-uated in the axial and—albeit to a lesser extent—inthe equatorial dimension is contrary to a previous re-port of pharmacologic control of experimentally in-duced myopia using atropine.42 Stone et al reportedthat muscarinic antagonists attenuated ocular enlarge-ment exclusively in the axial dimension, with no effectin the equatorial dimension. This is similar to theirearlier findings with dopamine agonists43. These ap-parently conflicting results may be the result of meth-odologic differences between the two studies. Thepresent study used a Rhode Island cross chick; depri-vation was achieved by translucent occluder, lasted for8 days, and induced 0.89 mm axial elongation and0.69 mm equatorial elongation in MD vehicle controlchicks. In contrast, Stone et al,42 using White Leghornchicks and 2 wk deprivation via lid-suture, inducedonly 0.35 mm axial elongation and 0.84 mm equatorialelongation (all measures from digital caliper readingsfor comparability). In addition to differences in in-duced ocular enlargement in control MD animals forthe two studies, Stone et al42 used a subconjunctivalroute for drug delivery that would result in a lowerfinal concentration of atropine at the retina thanwould result from the intravitreal route used in thepresent study. It is possible that higher doses of atro-pine are needed to attenuate experimentally inducedequatorial enlargement in chick than are required toattenuate axial enlargement.

It is interesting that although atropine effectivelyprevented the excessive enlargement of the eye in MDchicks, it had little effect on ocular growth in binocu-larly open animals. There was no effect on growth ofthe vitreous chamber in open animals, although therewas a small but significant reduction in anterior seg-ment depth. This finding would suggest that growth ofan eye deprived of pattern vision and normal oculargrowth may be mediated by different mechanisms thatdo not depend on the same retinal signals, as previ-ously suggested.44

Atropine has been found to effectively reduce theexcessive elongation of the eye and consequent myo-pia in other animal models of experimental myopia (ie,monkey,18-22 tree shrew17) and has been shown to effec-tively prevent the progression of human juvenile myo-pia.1920 It seems reasonable to assume that the modeof action by which atropine prevents excessive elonga-tion of the eye and myopia is similar in the variousspecies investigated. The present investigation hasdemonstrated that, contrary to previous hypothe-ses,1718'22 the mechanism by which atropine reduces orprevents myopia is nonaccommodative. If accommoda-tion does not play a major role in myopia develop-ment, the question arises regarding how atropine ef-fectively prevents the excessive enlargement of the eyein myopia. Acetylcholine is a retinal neurotransmit-ter,45"47 and the retina contains a cholinergic amacrinecell population48"50 that includes muscarinic acetyl-choline receptors.51"53 It is possible that atropine af-fects ocular growth by altering retinal neurotransmis-sion. Recent neurochemical studies of experimentallymyopic eyes have found altered concentrations ofother retinal neuropeptides, such as vasoactive intes-tinal polypeptide54 and dopamine.55'56 Interestingly, ithas been previously reported that, similar to musca-rinic antagonists, dopamine agonists (eg, apomor-phine)43'55 also inhibit deprivation-induced axial elon-gation of the eye. Evidence suggests that dopaminecan regulate cholinergic neurotransmission in the ret-ina57 and that dopaminergic cells are driven excitator-ily by cholinergic synapses in the retina.58 It seems fea-sible that inhibition of experimentally induced axialelongation of the eye by muscarinic antagonists anddopamine agonists is controlled by similar neuromo-dulatory mechanisms within the retina.

Particularly pertinent to the present study is a re-cent report42 that found that the nonselective musca-rinic antagonist atropine blocked deprivation-inducedaxial elongation and that the Ml selective antagonistpirenzepine had similar effects. In addition, M2 andM3 selective antagonists did not prevent axial elonga-tion. This supports a retinal locus, because Ml musca-rinic receptors are located in neural tissue59 as op-posed to intraocular muscle, where M3 receptors pre-dominate.60 Information on retinal acetylcholine



content in experimental myopia is needed to help sub-stantiate a role for retinal muscarinic receptors in thecontrol of ocular growth in myopia. If substantiated,Ml selective antagonists such as pirenzepine couldprove to be a more acceptable treatment modalitythan atropine for preventing human myopia progres-sion. Pirenzipine—compared to atropine—has onlylimited effect on the amount and particularly the dura-tion of pupil dilation, with minimal reduction in ac-commodative amplitude in mammalian intraocularmusculature (tree shrew; McBrien NA, Cottriall CL,and Reeder AP, unpublished observations). Further-more, pirenzepine is a safe and well-tolerated drug-that has been used widely to treat peptic ulcer diseasein humans.

Interestingly, it recently has been shown that atro-pine and pirenzepine can inhibit physiologic growthhormone release in normal human subjects.6162 Al-though this is thought to involve the inhibitory effectof acetylcholine on somatostatin release from the hy-pothalamus, the precise mechanism by which cholin-ergic blockade influences growth hormone secretion isunclear. Because recent investigations have demon-strated increased synthesis of scleral tissue in experi-mentally induced myopia in chick,31"33 it is feasiblethat atropine may work by inhibiting release of someyet unidentified growth factor in eyes deprived ofform vision, thus preventing myopia progression. Nowthat it is known that the effective locus of action ofcholinergic antagonists in reducing myopia progres-sion is not the accommodative system, subsequent in-vestigations should help clarify the site and role ofcholinergic control of ocular growth.

Key Words

accommodation, atropine, chick, experimental myopia, mus-carinic antagonists

Acknowledgments

The authors thank Drs, Margaret Woodhouse, Chris McCor-mack, and William Hodos for critically reading the manu-script; an anonymous reviewer for providing helpful com-ments; and Lisa Thomas and Christine Dunn for secretarialassistance.

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