And Negative-Lens–Induced Refractive Error and Ocular Growth in Chicks

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Anatomy and Pathology The Effect of Pirenzepine on Positive- and Negative-Lens–Induced Refractive Error and Ocular Growth in Chicks

Sangeetha Metlapally and Neville A. McBrien

7–9 10 PURPOSE. The selective muscarinic antagonist pirenzepine in- rinic antagonists pirenzepine and himbacine have been hibits experimentally induced myopia in avian and mammalian shown to be effective in preventing the axial elongation and species, including nonhuman primates and adolescent hu- myopia induced by form deprivation in animal models. mans. Transient positive lens defocus has a potent inhibitory Pirenzepine has been shown to inhibit not only form-dep- effect on negative-lens–induced myopia in avian and mamma- rivation myopia in chicks, tree shrews, monkeys, and guinea lian models. The purpose of the present study was to deter- pigs,8,11–13 but also negative-lens–induced myopia in tree mine the influence of daily treatment with pirenzepine on shrews and guinea pigs.11,14 However, the effects of musca- ocular growth and refractive error in chicks wearing positive rinic antagonists on positive-lens–induced hyperopia have not lenses. been explored in detail, except by Rickers and Schaeffel.9 This METHODS. The chicks were allocated to one of eight groups (n ϭ group reported that pirenzepine did not stop positive-lens– 6 each group) on the basis of whether they wore ϩ10 or Ϫ10 induced hyperopia in chicks, even at toxic doses that were D lenses monocularly and whether they received daily intrav- effective in reducing form-deprivation myopia, while negative- itreal injections of pirenzepine (700 ␮g) or vehicle (phosphate- lens–induced myopia was suppressed partially, at the same buffered saline) in the lens-defocused eye. In vivo refractive high doses. However, in the Rickers and Schaeffel study, piren- and biometric data were collected, and glycosaminoglycan zepine was administered only once at the start of the experi- synthesis in the sclera was assessed. mental period in the lens-defocus paradigm, with an intravitreal injection on day 17 and lens defocus for 4 days. The partial RESULTS. Pirenzepine did not alter the level of positive-lens– induced hyperopia in chicks wearing ϩ10 D lenses compared inhibition of both form-deprivation myopia and negative-lens– ϩ Ϯ induced myopia was attributed to retinal toxic mechanisms, with that in the vehicle control group ( 8.1 0.6 D vs. 9 ϩ8.9 Ϯ 2.4 D, mean Ϯ SEM; P ϭ 0.76). In contrast, pirenz- rather than physiological receptor-mediated mechanisms. As epine caused significant inhibition of negative-lens–induced pirenzepine has an elimination half-life of 4 and 7 hours from Ϫ Ϯ the retina and sclera, respectively, when administered intra- myopia compared with that in the vehicle group ( 1.1 1.5 15 D vs. Ϫ8.8 Ϯ 1.1 D; P ϭ 0.001). Glycosaminoglycan synthesis vitreally, one must assume that any effect of pirenzepine in the posterior sclera was significantly increased in the nega- administration in that study was due to the effects of the single tive-lens–treated groups and showed small decreases in the dose in the first 12 hours after administration. Thus, it is not positive-lens–treated groups. surprising that only very high (toxic) doses resulted in any change in ocular growth. CONCLUSIONS. The influence of pirenzepine on ocular growth in The findings of Rickers and Schaeffel, when using the lens chicks differed by sign of lens defocus, with pirenzepine blocking defocus paradigm, were at odds with the results in later studies negative-lens effects on ocular growth, but not positive-lens ef- in which daily intravitreal injections of significantly lower fects. The most likely reason that hyperopia was not enhanced by doses of pirenzepine were administered in chicks,8,16 which pirenzepine treatment was that the rapid compensatory eye fully prevented experimentally induced myopia with no evi- growth associated with positive lenses eliminated the imposed dence of toxic damage to the retina. Pirenzepine prevented the myopic defocus, and the clear retinal image prevented any addi- excessive axial elongation accompanying form-deprivation my- tional hyperopia from developing. (Invest Ophthalmol Vis Sci. 8,16 2010;51:5438–5444) DOI:10.1167/iovs.09-4431 opia at substantially lower, nontoxic doses in chicks and tree shrews11 and inhibited negative-lens–induced myopia in tree shrews.11 It has also been found that daily injections of uscarinic antagonists are a class of drugs that have been atropine or pirenzepine induce a small degree of hyperopia in Mexamined extensively because of their inhibitory effect 5,8 on the progression of myopia in humans1–3 and in animal unoccluded eyes. models.4–7 The nonselective broad-band muscarinic antago- In the present study, we investigated the effects of daily nists atropine5 and oxyphenonium4 and the selective musca- pirenzepine treatment on positive-lens–induced hyperopia. From previous studies, we know that positive lens defocus causes more rapid changes in eye growth over a shorter treatment duration than do equal-powered negative lenses.17–19 From the Department of Optometry and Vision Sciences, The Positive-lens–induced hyperopia is more resistant to interrup- University of Melbourne, Victoria, Australia. tion with periods of normal vision than is negative-lens–in- Supported by Grant 454602 from the National Health and Medical duced myopia,20 and durations of positive- and negative-lens Research Council of Australia. defocus do not add linearly and are weighted differently, re- Submitted for publication August 4, 2009; revised December 24, sulting in induced hyperopia despite only brief episodes of 2009, February 5 and April 22, 2010; accepted May 13, 2010. positive lens wear interspersed between day-long periods of Disclosure: S. Metlapally, None; N.A. McBrien, None 17,18 Corresponding author: Neville A. McBrien, Department of Optom- negative lens wear. The marked differences in the tempo- etry and Vision Sciences, The University of Melbourne, Victoria, 3010, ral integration of the lens defocus signal and its effects on Australia; [email protected]. ocular growth between positive and negative lens defocus

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warrant further investigation. In particular, the purpose of the powers of ϩ10 and Ϫ10 D (Australian Contact Lenses, Carlton, Victo- present study was to elucidate further the underlying mecha- ria, Australia), placed in front of the treated eye by means of a goggle nisms of positive-lens defocus by assessing the inﬂuence of fashioned from Velcro. The calculated effective powers at the corneal pharmacologic intervention with pirenzepine on the develop- plane were ϩ10.5 and Ϫ9.5 D, since the posterior lens surfaces were ϭ Ϫ ⅐ ment of hyperopia in positive-lens–treated eyes. 5 mm in front of the corneal plane of the eye: Feff Fopt/1 d Fopt, Speciﬁcally the purpose was to assess whether pirenzepine where Feff is the effective lens power at the cornea, d refers to the would work additively with positive-lens treatment to induce distance in meters between the corneal plane and the posterior lens

hyperopia, in excess of the power of the positive lens defocus. surface, and Fopt is the optical power of the PMMA lens used. However, Both treatments have been shown to inhibit myopic eye they will be referred to as the ϩ10 or Ϫ10 D groups on the basis of the growth and also to induce hyperopia when applied to normal optical rather than the effective lens powers. The lenses were cleaned open eyes. The combined treatments may provide an en- daily. hanced signal to slow ocular elongation and induce hyperopia more than when they are used individually. If they are found to Sulfate Labeling of Scleral Glycosaminoglycans work through separate ocular growth pathways that are addi- tive in effect, it may provide a new treatment approach for the To obtain a metabolic correlate of any ocular growth changes, we prevention of progression of myopia in humans. measured glycosaminoglycan (GAG) synthesis in the posterior sclera (8 mm punch). On the final experimental day (fourth day), 1 hour after the final intravitreal injection or sham injection, each chick was in- MATERIALS AND METHODS ␮ 35 jected intraperitoneally with 100 Ci of radiolabeled sulfate ( SO4)in Animals a volume of 200 ␮L. The animals were then returned to the brooder for a further 2 hours of visual experience before the eyes were measured Newly hatched chicks (Gallus gallus domesticus) of the White Leg- by retinoscopy and A-scan ultrasound, and tissue was taken for analy- horn and Black Australorp cross variety were obtained from a local sis. supplier and kept in a temperature-controlled brooder maintained at 28°C, on a 12 hour light–dark cycle. The illumination at the floor of the brooder was 300 lux. The chicks were acclimatized to the conditions Ocular Measurements of the brooder for 7 days before experimental procedures. Food and On the final (fourth) experimental day, 3 hours after the last intravitreal water were available ad libitum. All procedures were performed in injection, the chicks were anesthetized with 75 mg/kg ketamine (100 accordance with the ARVO Statement for the Use of Animals in Oph- mg/mL) and 5 mg/kg xylazine (20 mg/mL), and body temperature was thalmic and Vision Research. maintained with a heating pad. A dental bite bar was taped to the beak Experimental Groups and In Vivo Procedures of the chick fixed in a universal joint, to enable accurate orientation of the head to allow optical and biometric data to be collected on the ϭ The chicks were allocated to one of eight groups (n 6 each group) central axis of the eye. Ocular measurements, namely refraction and on the basis of whether they wore a lens and whether they received an axial dimensions were collected by using retinoscopy and A-scan ␮ intravitreal injection (7 L) of pirenzepine (pz) or the vehicle PBS (sal) ultrasound with a 10-MHz transducer, as described elsewhere.5 Axial ϩ ϩ Ϫ in the treated eye. The groups were 10 D-pz, 10 D-sal, 10 D-pz, length refers to the internal axial length, defined as the sum of the Ϫ ϩ Ϫ 10 D-sal, 10 D no-injection, 10 D no-injection, open-pz, and anterior chamber depth (front of cornea to front of crystalline lens), ϩ Ϫ open-sal. The 10 D-pz and 10 D-pz were the two main treatment lens thickness, and the vitreous chamber depth (back of crystalline ␮ groups, which were given a daily intravitreal injection of 700 g lens to front of vitreoretinal interface). Each recorded value of axial ϩ Ϫ pirenzepine in the lens-wearing eye. The 10 D-sal and 10 D-sal biometric measurements was the mean of six averaged waveforms groups received an intravitreal injection of PBS alone in the lens- (each in itself an average of 20 incoming waveforms).The results were wearing eye and acted as the control for the effects of the injection on expressed as the mean of final values for the six animals in each group ocular growth. Two groups of animals were injected intravitreally with or the average difference between the values for the treated and pirenzepine (open-pz) or PBS (open-sal) in one eye, and neither eye control eyes within a group. wore lenses (binocularly open groups). These groups served as the control for the effect of the injected substance on normal ocular growth. Two groups wore ϩ10 or Ϫ10 D lenses over one eye and Enucleation and Equatorial acted as controls for the effect of lenses alone (ϩ10 D no-injection and Diameter Measurements Ϫ10 D no-injection). The lens and/or injection treatments were mon- After all in vivo measurements, the chicks were given a lethal dose ocular, and the left eye was the treated eye in all cases. The contralat- (325 mg/kg) of pentobarbitone sodium (325 mg/mL) intraperitoneally. eral eye was left untreated for comparison. Treatment was either The eyeball was enucleated after the 12 o’clock position was marked. started on the seventh day after hatching or was staggered by a day to Once the eye was enucleated, the mean equatorial diameter (H ϩ V)/2 enable appropriate timing of measurements on the final experimental was measured with digital vernier calipers, and the results were ex- 5 day. Intravitreal injections were performed as described elsewhere. pressed as the differences between the treated and control eyes. The chicks were anesthetized with 2% halothane; the upper lid re- tracted; the superior temporal anterior sclera viewed under an oper- Tissue Dissection and Measurement ating microscope (Carl Zeiss Meditec, Dublin, CA); a single injection of GAG Synthesis hole made with the 30g needle, which was inserted 3 mm into the vitreous cavity; and pirenzepine or PBS injected. The same injection After equatorial diameter measurements, the eye was separated into hole was used for the four daily injections in each injected eye, to avoid anterior and posterior halves with a razor blade. An 8-mm diameter the effect of multiple puncture holes in the sclera on ocular growth.5 surgical trephine was used to punch a button out of the posterior Animals that did not undergo intravitreal injection were anesthetized segment of the eyes, which included the posterior pole but avoided every morning with 2% halothane for 4 minutes (average time for the pecten and optic nerve head. The retina and choroid were carefully intravitreal injection procedure) to control for the effects of daily removed from the sclera. The posterior scleral tissue punch was then anesthesia. snap frozen in liquid nitrogen. The tissues were stored at Ϫ80°C until assayed. Induction of Refractive Error The tissue samples were freeze-dried overnight (MicroModulyo; Induction of refractive error was achieved by using polymethylmethac- Edwards, Crawley, UK). Dry weights were measured on a microbal- rylate (PMMA) lenses (base curve, 7 mm; total diameter, 12.5 mm) with ance (R200D; Satorius, AG, Go¨ttingen, Germany) to the nearest 0.001

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mg. Scleral tissue was then digested as previously described,16 and the digested scleral tissue samples were stored at Ϫ80°C until assayed. GAG incorporation of the radiolabel was assessed by selective precipitation, with a modified version of the Alcian blue dye–binding method. Details of the assay, as used on chick sclera, have been published.16 Sulfate incorporation was measured as counts per minute in 10 mL of scintillation solution (Cytoscint; Fisher Scientific, Scoresby, VIC, Australia) over a 10-minute period with a liquid scintillation counter (Wallac 1409; Perkin Elmer, Boston, MA). All tissue samples were analyzed in triplicate and the results expressed as decays per minute after correction for quenching effects by using an external standard. The final results were expressed as the percentage difference in the incorporation of sulfate into GAGs in the treated and control eyes and normalized to both dry weight and DNA content. DNA content was measured with the Hoechst 33258 assay (Sigma-Aldrich, Castle Hill, NSW, Australia), as described elsewhere.21 Statistical Analyses Paired Student’s t-tests were used to compare treated and control eyes of the same animal (all analysis by GraphPad Software, Inc., San Diego, CA). Unpaired t-tests were used to compare either the absolute treated control eye differences or the percentage differences between different groups. A one-way ANOVA with Tukey’s multiple comparison test was used for comparisons involving more than two groups of animals. All results are presented as the mean Ϯ SEM, unless otherwise stated.

RESULTS Refractive and Ocular Biometry To confirm that differences in measurements of treated versus control eyes were due only to treatment-induced changes, we compared the control eye data sets of the eight groups, across the nine experimental measurements. Since statistical analysis (ANOVA) revealed no significant differences in any parameter among the fellow control eyes in any of the eight experimental groups, the treated–control eye differences indicate only changes induced in the treated eye. Therefore, in the sections that follow, the changes in all parameters in the treated eye are expressed as differences (treated minus control). Treated eyes in the ϩ10 D group were significantly hyperopic relative to the contralateral control eyes (ϩ13.9 Ϯ 0.8 D vs. ϩ4.0 Ϯ 0.3 D, P Ͻ 0.001), giving a treated–control eye difference of ϩ9.9 Ϯ 0.5 D (Fig. 1A). This equated to 94% compensation for the effective lens power (ϩ10.5 D). The treated eyes in the Ϫ10 D group were significantly myopic compared with the control eyes (Ϫ5.8 Ϯ 1.3 D vs. ϩ2.9 Ϯ 0.4 D, P Ͻ 0.001), with a difference between treated and control eyes of Ϫ8.7 Ϯ 1.3 D (Fig. 1A). This difference equated to 92% compensation for the effective lens power (Ϫ9.5 D). Treated eyes in the ϩ10 D group had shallower vitreous chambers (Ϫ0.39 Ϯ 0.04 mm, P Ͻ 0.001, Fig 1B) than did the contralateral control eyes, whereas treated eyes in the Ϫ10 D group had deeper vitreous chambers than did the control eyes (ϩ0.20 Ϯ 0.05 mm, P Ͻ 0.01, Fig 1B). The axial length changes were in FIGURE 1. (A) Differences in ocular refraction between treated and contralateral control eyes in the experimental groups: Positive and negative lenses induced relative hyperopia and myopia, respectively. Daily intravitreal injections of pirenzepine (700 ␮g) did not alter the amount of hyperopia induced by the positive lenses, but prevented the The vitreous chamber depth changes observed in binocularly open myopia induced by the negative lenses. Pirenzepine induced a signif- animals were not significantly different. (C) Differences in ocular axial icant relative hyperopia in binocularly open animals. (B) Differences in length between treated and contralateral control eyes in the experi- vitreous chamber depth between treated and contralateral control eyes mental groups: The vitreous chamber was the major contributor to the in the experimental groups: The relative hyperopia and myopia in- changes observed in the ocular axial length. (A–C) Asterisks not duced by the lenses and/or pirenzepine were due to shorter or en- associated with brackets denote P-values from comparisons between larged vitreous chambers, respectively. Pirenzepine treatment did not treated and control eyes in a single group, and those associated with inhibit the changes in vitreous chamber depth induced by positive brackets between bars are from comparisons of treated minus control lenses, but prevented the vitreous chamber elongation associated with differences between the two groups. *P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ the use of negative lenses, relative to the vehicle (sal)-injected groups. 0.001, n.s. represents P Ͼ 0.05. Error bars, 1 SEM; n ϭ 6, all groups.

Downloaded from jov.arvojournals.org on 09/25/2021 IOVS, November 2010, Vol. 51, No. 11 Pirenzepine and Positive-Lens–Induced Refractive Error 5441 5.8 (3.0) 2.6 (1.1) 7.9 (4.0) 3.1 (0.9) 4.6 (2.3) 8.92 (0.41) 8.48 (0.25) 8.17 (0.40) 8.56 (0.16) 8.38 (0.20) Ϫ

IGURE Differences in mean equatorial diameters between treated ) 1.42 (0.07) 1.31 (0.09)) 1.29 (0.12) 5.39 (0.35) 1.34 (0.04) 5.02 (0.18) 1.37 (0.08) 4.78 (0.37) 5.09 (0.17) 4.92 (0.19) F 2. 10 D-sal Open-pz Open-sal 06) 2.11 (0.08) 2.15 (0.02) 2.10 (0.03) 2.13 (0.06) 2.09 (0.07) and contralateral control eyes in the experimental groups. The treated ؊ eyes in the Ϫ10 D group and Ϫ10 D-pz group had signiﬁcantly enlarged diameters compared with the respective control eyes. Inter- group comparisons did not reveal statistically signiﬁcant differences. *P Ͻ 0.05, **P Ͻ 0.01. Error bars, 1 SEM; n ϭ 6, all groups. 7) 11.87 (0.39) 11.77 (0.65) 11.54 (0.30) 11.70 (0.38) 11.78 (0.20) 11.85 (0.27)

the same direction as the vitreous chamber depth changes (Fig.

1C). The axial change in the vitreous chamber depths ac- 10 D-pz counted for the main structural correlate to the induced refrac- ؊ tive error. Animals in the Ϫ10 D group were found to have a signiﬁcantly enlarged mean equatorial diameter in the treated eyes (11.74 Ϯ 0.40 mm vs. 11.57 Ϯ 0.43 mm, P Ͻ 0.01; Fig 2), whereas there were no signiﬁcant differences in mean equatorial diameter between the treated and control eyes in the ϩ10

D group (11.71 Ϯ 0.27 mm vs. 11.68 Ϯ 0.16 mm, P Ͼ 0.05, Fig SD of results in six eyes in each group. 10 D-sal Ϯ These results are comparable to data from an earlier study ؉ .(2 on lens-induced refractive errors in chicks.22 Daily pirenzepine injections (700 ␮g) were effective in inhibiting nearly all the myopia induced by negative lenses in the Ϫ10 D-pz group when compared with the Ϫ10 D-sal group (Ϫ1.1 Ϯ 1.5 D vs. Ϫ8.8 Ϯ 1.1 D, P Ͻ 0.01, Fig. 1A). The interocular vitreous chamber depth difference in the Ϫ10 D-pz

group was signiﬁcantly less than in the Ϫ10 D-sal group 10 D-pz Ϫ0.03 Ϯ 0.04 mm vs. ϩ0.26 Ϯ 0.06 mm, P Ͻ 0.01, Fig. 1B). ؉) Pirenzepine treatment also caused a signiﬁcant reduction in the anterior chamber depth in the treated eyes of the Ϫ10 D-pz group compared with that in the control eyes (1.26 Ϯ 0.03 mm vs. 1.40 Ϯ 0.04 mm, P ϭ 0.01, Table 1). This relative decrease 5.8 (3.3) 2.5 (0.5) 10.5 (1.6) 4.0 (1.8) 12.9 (4.5) 2.8 (0.8) 1.7 (3.3) 3.0 (0.5)

in anterior chamber depth was signiﬁcant when compared Ϫ

with that in the Ϫ10 D-saline group (Ϫ0.14 Ϯ 0.04 mm vs. 10 D Ϯ 0.04 mm, P Ͻ 0.01; Table 1). The equatorial diameter ؊ 0.02 changes in the Ϫ10 D-pz group were not significantly different from those in the Ϫ10 D-sal and the Ϫ10 D groups (ANOVA, P Ͼ 0.05). Daily pirenzepine injections (700 ␮g) did not significantly alter the refractive changes induced by positive lenses. The interocular difference in refractive error in the ϩ10 D-pz group 10 D was not significantly different from that in the ϩ10 D-sal group ؉

(ϩ8.1 Ϯ 0.6 D vs. ϩ8.9 Ϯ 2.4 D, P ϭ 0.76; Fig. 1A). The CTCTCTCTCTCTCTC T changes in interocular vitreous chamber depth were not significantly different between the ϩ10 D-pz and the ϩ10 D-sal groups (Ϫ0.44 Ϯ 0.05 mm vs. Ϫ0.49 Ϯ 0.10 mm, P ϭ 0.66, Fig. 1B), in keeping with the refractive findings. The relative changes in equatorial diameter did not differ significantly between the ϩ10 D-pz, ϩ10 D-sal, and ϩ10 D groups (ANOVA,

P Ͼ 0.05). Summary of Ocular Refractive and Structural Data in Experimental Groups Collected on the Fourth Treatment Day

There were no differences in the induced refractive error or 1. ocular growth changes between the ϩ10 D-sal and the ϩ10 D C denotes the control and T denotes the treated eye of the chicks. The data are the mean chamber, mm Mean (SD)thickness, mm 1.38 Mean (0.02) (SD)chamber, mm 1.37 (0.04) 2.12 (0.05) Mean (SD) 1.39length, (0.06) mm 2.06 (0.05) 5.10 (0.18) 1.45 (0.11) 2.10 (0.07) Mean 4.71 (SD) (0.16) 1.35 (0.08) 2.08 (0.06) 8.60 5.09 (0.17) (0.19) 1.32 (0.13) 2.18 (0.10) 8.15 5.29 (0.17) (0.20) 1.37 (0.09) 2.10 (0.08) 8.57 5.07 (0.27) (0.20) 1.39 (0.11) 2.08 (0.04) 8.83 4.64 (0.31) (0.12) 1.40 (0.09) 2.05 (0.03) 8.60 5.06 (0.26) (0.13) 1.26 (0.08) 2.08 (0.06) 8.05 4.57 (0.21) (0.16) 1.40 (0.08 2.11 (0.07) 8.51 5.03 (0.18) (0.23) 2.11 (0. 8.01 5.00 (0.25) (0.17) 8.51 5.13 (0.33) (0.22 8.36 (0.27) 8.64 (0.34) diameter, mm Mean (SD) 11.68 (0.16) 11.71 (0.27) 11.57 (0.43) 11.74 (0.40) 11.84 (0.33) 11.82 (0.37) 11.39 (0.30) 11.56 (0.30) 11.68 (0.27) 11.77 (0.2 refraction, D Mean (SD) 4.0 (0.8) 13.9 (2.0) 2.9 (0.9) ABLE Anterior Lens Vitreous Axial Equatorial Effective groups or between the Ϫ10 D-sal and the Ϫ10 D groups (P Ͼ T

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0.05), suggesting that the daily injection procedure did not all showed an increase in sulfate incorporation when com- induce significant structural changes per se. pared with the contralateral untreated control eyes (Fig. 3). Chicks with no lens defocus that were treated with pirenz- There was no significant difference between the three Ϫ10 D epine (open-pz) developed significant hyperopia in the treated negative-lens–treated groups in the increase in sulfate incorpo- eyes compared with that in the contralateral control eyes (ϩ5.3 Ϯ ration (one-way ANOVA, P Ͼ 0.05), and these increases were 1.5 D, P Ͻ 0.01, Fig. 1A), which was due to significant shortening significantly different from 0 (t-test, P Ͻ 0.05) in all Ϫ10 D of the vitreous chamber depth in the treated eyes (Ϫ0.24 Ϯ 0.08 negative-lens–treated groups (Fig 3). mm, P Ͻ 0.05, Fig. 1B). The open-pz group was significantly No significant difference in sulfate incorporation was ob- hyperopic compared with the open-sal group (ϩ5.3 Ϯ 1.5 D served between the open-pz and open-sal groups (16.3% Ϯ vs. ϩ1.6 Ϯ 1.2 D, P Ͻ 0.05, Fig. 1A), indicating that pirenz- 15.2% vs. Ϫ26.1% Ϯ 9.7%, P Ͼ 0.05; Fig 3) and the mean epine alone caused relative hyperopia. The vitreous chamber difference in sulfate incorporation was not significantly differ- depth differences between the open groups were not statisti- ent from 0 for both groups (t-test, P Ͼ 0.05). cally significant, although the changes were in the direction No significant differences were observed in the DNA con- that explained the refractive effects (Ϫ0.24 Ϯ 0.08 mm vs. tent of the posterior scleral tissue button between treated and Ϫ0.17 Ϯ 0.04 mm, P ϭ 0.22; Fig. 1B). A nonsignificant differ- control eyes in the eight groups or in the dry tissue weight ence in the axial length was observed between the open-pz differences of each button (one-way ANOVA, P Ͼ 0.05, data group and the open-sal group (Ϫ0.31 Ϯ 0.07 mm vs. Ϫ0.18 Ϯ not shown). Consequently, when GAG synthesis was normal- 0.04 mm, P ϭ 0.07). Equatorial dimensions in the treated eyes ized to milligrams dry tissue weight (decays per minute per of binocularly open groups (open-pz and open-sal groups) milligram dry weight; data not shown) instead of DNA content, were not significantly different from the contralateral control no differences were found from results expressed as sulfate eyes (P Ͼ 0.05), and there were no differences between these incorporation per microgram of DNA (decays per minute per groups (P Ͼ 0.05). milligram DNA).

GAG Synthesis Radiolabeled sulfate incorporation (DPM/␮g DNA) in the pos- DISCUSSION terior sclera was not significantly different between eyes The principal finding of this study is that, at the minimum dose treated with positive lenses (ϩ10 D) when compared with the (700 ␮g) demonstrated to be effective in preventing form- contralateral untreated control eyes (Ϫ2.4% Ϯ 9.0%, P Ͼ 0.05, deprivation–induced axial myopia in the chick, daily pirenz- Fig 3), whereas eyes treated with negative lenses (Ϫ10 D) epine did not alter the magnitude of positive-lens (ϩ10 D) showed significant increases compared with the contralateral induced hyperopia. Specifically daily pirenzepine treatment control eyes (61.2% Ϯ 23.5%, P Ͻ 0.05, Fig. 3). There was no neither enhanced the amount of induced hyperopia, nor inhib- significant difference between the three ϩ10 D positive-lens– ited it, contrary to proposed hypotheses. In addition, daily treated groups in the reduction in sulfate incorporation (one- pirenzepine treatment (700 ␮g) prevented nearly all negative- way ANOVA, P Ͼ 0.05), and the reduction was only signifi- lens (Ϫ10 D)–induced axial myopia, which is the first demon- cantly different from 0 in the ϩ10 D-pz group (Ϫ31.6% Ϯ 8.6%, stration of inhibition of negative-lens–induced myopia in the P Ͻ 0.01, Fig 3). Chick eyes treated with Ϫ10 D negative lenses chick model using nontoxic doses of pirenzepine. Pirenzepine has been reported to prevent negative-lens– induced myopia and axial ocular elongation in mammalian animal models, including tree shrew11 and guinea pig.14 How- ever, pirenzepine did not affect the relative equatorial enlargement during negative-lens–induced myopia (Fig 2), contrary to the findings of the effect of the broad-band muscarinic antagonist atropine on form-deprivation myopia,5 which may relate to the stronger relative affinity of atropine for all muscarinic receptors. All three groups of chicks treated with Ϫ10 D lenses had significant increases in GAG synthesis in the posterior sclera of the treated eye, relative to the contralateral control eyes, but there was no significant difference between the three groups, even though the Ϫ10 D pirenzepine–treated chicks developed significantly less myopia and elongation of the vitreous chamber. A previous study reported that pirenzepine transiently prevented the increased GAG synthesis after 2 hours in form- deprived chicks.16 This same study also demonstrated that a similar transient decrease in GAG synthesis was observed when form-deprivation was interrupted with 3 hours of unoccluded vision, which also prevents myopia development. These findings demonstrate that a transient decrease in GAG synthesis can produce a sustained decrease in the rate of FIGURE 3. Differences in sulfate incorporation into GAGs per micro- myopia development, whether produced by the physiological gram DNA in the posterior sclera between treated and contralateral regulation of ocular growth of unoccluded vision or a pharma- control eyes in the experimental groups. Eyes treated with negative cologic regulation of ocular growth with pirenzepine.16 The lenses showed a significant increase in sulfate incorporation but no significant difference between pirenzepine- or saline-treated groups. lack of a transient decrease in GAG synthesis in the present Eyes treated with positive lenses were not significantly different be- study was most likely due to the known differences in the tween groups, and only the ϩ10 D-pz eyes showed a significant time-course of ocular growth responses of chicks to negative- reduction from the contralateral eyes. *P Ͻ 0.05, **P Ͻ 0.01. Error bars, lens defocus and to diffusers.23 Negative-lens defocus induces 1 SEM; n ϭ 6, all groups. a more rapid elongation than diffusers and thus assessment at

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the same time period in both studies (2 hours after labeling) and the sclera. Thus, the access of pirenzepine to scleral or was probably too late to assess the transient inhibition of GAG choroidal sites may be important.24 synthesis in the sclera. Another possible reason that intravitreally injected pirenz- Although the exact mechanism by which pirenzepine epine was ineffective in preventing positive-lens–induced hy- causes this reduction in myopia and ocular elongation has not peropia could be that the sustained action of positive defocus yet been elucidated, pirenzepine’s effects are unlikely to be (all day) exceeded the duration of pirenzepine’s bioactivity in mediated by toxicity. No retinal or scleral toxic effects have ocular tissues. Although the positive lens signal has been been reported with the doses of pirenzepine (500–700 ␮g) shown to be resistant to 2 hours of nonlens visual exposure used during form-deprivation studies in chicks and tree daily,20 this visual exposure may be a more powerful signal shrews.11,16 In these studies, pirenzepine did not alter retinal than pirenzepine (half-life of 4 hours in the retina) (Yin GC, et and scleral tissue morphology, DNA content, or the ability of al. IOVS 2003;44:ARVO E-Abstract 4339).15 Thus, pirenz- pirenzepine-treated eyes to become myopic after pirenzepine epine’s effect on ocular growth is overridden by the sustained treatment was discontinued. effect of positive-lens defocus. Pirenzepine did not enhance or attenuate the hyperopia and Although pirenzepine is classed as an M1-selective musca- axial ocular growth changes induced by positive lenses alone rinic antagonist, the chick model does not possess an M1- (Fig. 1A). We had hypothesized that combining positive-lens receptor (Yin GC, et al. IOVS 2003;44:ARVO E-Abstract 4339), defocus and pirenzepine would enhance the amount of hyper- which raises questions about the specific receptor-based mech- opia induced, as pirenzepine not only antagonizes myopia anism by which pirenzepine mediates ocular growth. How- development, but also induces a small amount hyperopia in ever, the binding affinity of pirenzepine to the mammalian eyes with no induced defocus. The most likely explanation of M4-receptor is only four times lower than for the mammalian why this did not occur is that the functional emmetropization M1-receptor, and the affinity of pirenzepine for the chick M2- mechanism inherent in the eye prevented further shortening of receptor (M1 surrogate in the chick) is nearly the same as its 25 the vitreous chamber once full compensation for the induced affinity for the M4-receptor. Thus, pirenzepine may mediate optical defocus was reached (94% compensation for the ϩ10 D its effects through the M4 or M2 receptor (Yin GC, et al. IOVS defocus used in this study) producing a clear image on the 2003;44:ARVO E-Abstract 4339).25 More recently, the highly retina. Previous studies have demonstrated that animal models selective M4 antagonist MT3 has been shown to be effective in of refractive error functionally emmetropize to the degree of reducing experimentally induced myopia and axial elongation induced defocus and no more.21 Specifically how a clear retinal in chicks (Morgan IG, et al. IOVS 2005;46:ARVO E-Abstract image stabilizes ocular growth and refractive state, whether it 3342; Diether S, et al. IOVS 2005;46:ARVO E-Abstract 1986; is overriding a pharmacologic intervention such as pirenzepine McBrien NA, et al. IOVS 2009;50:ARVO E-Abstract 3850). Al- or is a recovery response with optical correction of the in- though the M4-receptor pathway seems most likely, recently it duced myopia is not known, other than that it is likely to be has been reported (Diether S, et al. IOVS 2005;46:ARVO E-Ab- related to peak firing of specific retinal neurons that sense stract 1986; McBrien NA, et al. IOVS 2009;50:ARVO E-Abstract image clarity and feed into regulation of ocular growth (e.g., 3850) that the highly selective M1 muscarinic antagonist MT7 is amacrine cells). partially effective in reducing experimental myopia in the In addition, there was no evidence to indicate that the chick model of refractive development. This finding could be speed of compensation in response to the induced defocus was interpreted to indicate that multiple muscarinic receptors are faster in the eyes treated with a combination of positive-lens involved in the regulation of ocular growth and myopia, al- defocus and pirenzepine. While full refractive and ocular bi- though the highly selective nature of MT3 and -7 have yet to be ometry measurements were recorded only on the final day of confirmed with binding assays; thus, nonspecific effects can- the experiment, retinoscopy was performed before every in- not be ruled out. travitreal injection, first to confirm that the ocular media were In summary, combining pirenzepine treatment with posi- clear and no interocular injury or infection had occurred due to tive lens defocus did not alter the amount of induced hyper- injections and second to perform an approximate assessment opia nor the reduction in the rate of GAG synthesis caused by of the refractive error of the treated eye by noting the direction positive-lens–induced hyperopia. We propose that compensa- and speed of movement of the retinoscopic reflex. No marked tory eye growth under imposed myopic defocus rapidly pro- differences were noted in the rate of compensation with duces a clear retinal image, which prevents pirenzepine from ϩ10 D lenses between the three groups of chicks treated with causing a further hyperopic shift. In contrast, pirenzepine ϩ10 D lenses. This conclusion is further supported by finding prevented the development of induced myopia due to negative that all three groups of ϩ10 D-treated chicks had similar re- lens treatment (hyperopic defocus). We propose that because ductions in GAG synthesis in the posterior sclera, indicating changes in ocular growth are slower to achieve compensation similar metabolic rates of scleral remodeling. during imposed hyperopic defocus than during myopic defo- The possibility that pirenzepine may simply attenuate any cus, more time is available for pirenzepine to exert its inhibi- deviation from normal ocular growth, whether positive- or tory effect on the development of myopia. negative-lens induced was also considered. It has been reported that the hyperopic shift induced by positive-lens defo- References cus is due to the anterior displacement of the retina, in which a major contributor is choroidal thickening, whereas, the my- 1. Bedrossian RH. The effect of atropine on myopia. Ophthalmology. opic shift induced by negative lens defocus is predominantly 1979;86:713–719. due to elongation of the vitreous chamber depth, to which 2. Chou AC, Shih YF, Ho TC, Lin LL. 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