- Email: [email protected]
Pirenzepine prevents form deprivation myopia in a dose dependent manner
Ophthal. Physiol. Opt. Vol. 15, No. 5, pp. 351-356, 1995 Copyright 0 1995 Elsevier Science Ltd for British College of Optometrists Printed in Great Britain. All rights reserved 0275-5408/95 $10.00 + 0.00
UTTERWORTH EINEMANN
0275-5408(95)00074-7
Pirenzepine prevents in a dose dependent E. R4. Leech,* Department Cardiff CFl
form deprivation manner
C. L. Cottriall”
of Optometry 3XF, UK
and
Vision
myopia
and N. A. McBrien” Sciences,
University
of Wales,
College
of Cardiff,
Summary Previous studies have demonstrated that muscarinic antagonists, such as atropine and pirerzepine, block form deprivation myopia in avian and mammalian models. The aim of the present investigation was to establish dose-response curves for intravitreal and subconjullctivally injected pirenzepine and to determine receptor specificity. Chicks were monccularly deprived of form vision for five days and received daily injections of either pirenzepine or saline. Keratometry, retinoscopy and A-scan ultrasonography of axial ocular dimensions were then taken. lntravitreally injected pirenzepine was effective at preventing form deprivation myopia in a dose dependent manner with an ED,, of 175pg. A 5OOpg dose totall\/ prevented induced myopia ( + 0.9 D versus - 13.7 D) and axial enlargement ( - 0.14 mm versus + 0.32 mm). Daily subconjunctival injection off pirenzepine was significantly less effec::ive in preventing form deprivation myopia. Form deprivation myopia could still be induced in animals which had undergone pirenzepine treatment. Pirenzepine was effective in preventing the axial elongation associated with experimental myopia in a dose dependent manner and via a functional not toxic mechanism.
Ophthal.
Physiol.
Opt. 1995,
15, 351-356
Introduction
recent studies have demonstrated that atropine may be acting via a non-accommodative route”,” and therefore must be having its effect at receptor sites other than those on the smooth muscle of the ciliary body. At present, five different muscarinic receptor subtypes have been identified, based on either pharmacological, morphological or functional characteristics”. The use of selective antagonists has identified M, muscarinic receptors in the retina of calf13 and chicki4, as well as post-junctional M, receptors in both ciliary muscle and iris sphincterI and pre-junctional M, receptors in the iris”j. Atropine is a broad band muscarinic antagonist which binds to all identified muscarinic receptors with equal affinity’*. Therefore, the prevention of form deprivation myopia by atropine could be occurring through either an M, , M, or M, mediated mechanism. The use of selective muscarinic antagonists make it possible to target a particular class of receptor. In an attempt to identify which receptor(s) are implicated in muscarinic control of ocular growth, selective antagonists have been used to try and prevent form deprivation myopia. It has been shown that pirenzepine prevents axial myopia in both monocularly deprived white leghorn chicks” and
Early visual experience is known to play an integral role in postnatal ocular development and disruption of a clear retinal image causes a breakdown in the process of emmetropisation’. Both environmental2.3 and pharmacologica14,5studies have implicated a pivotal role for accommodation in the regulation of eye growth. However, recent studies on animal models of refractive development question if accommodation has a primary role in the development of myopia. Investigations have demonstrated that blocking communication between the eye and higher visual centres does not prevent the development of, or recovery from, form deprivation myopia6-9, indicating that the visual signals controlling eye growth and refractive development may proceed directly from the retina to the choroid and sclera and not via higher visual centres. Consequently,
*MBCO Received: 15 May 1995 Correspondence to: N. A. McBrien
351
352
Ophthal. Physiol. Opt. 1995 15: No 5
tree shrews”. Rickers et ~2.‘~have also successfully stopped axial myopia in chicks using pirenzepine. However, the effective dose was much higher than that previously reported in chick although injections were only performed every three days rather than daily. They suggest that pirenzepine may be working through a toxic effect rather than a physiological one. The present study sought to determine whether pirenzepine effectively reduced or prevented experimentally induced myopia in a Rhode Island cross-strain of chick, and to examine the dose-response characteristics of pirenzepine when administered either intravitreally or subconjunctivally. Such information would hopefully address the issue of effective doses for pirenzepine and, more importantly, give insight as to the site of action of the drug.
using the technique described by McBrien et al.“. On each of the subsequent five days, the injection procedure was repeated; it was nearly always possible to find the same hole each time. This minimised any injection effects that may have resulted from release of growth factors or reduced intraocular pressure resulting from multiple punctures of the sclera. Subconjunctival injections were given by injecting into the tarsal subconjunctival space. Injections were performed by injecting 50 ~1 of drug or saline with a 30 gauge needle through the lid into the subconjunctival space. Following the injection, the treated eye of all animals was occluded with a hemispherical translucent plastic occluder, attached by means of a velcro ring placed around the orbit. This facilitated the daily removal and replacement of the occluders at each subsequent injection.
Materials
In vivo ocular measures
and methods
Animals used Day old Rhode Island cross chicks (Lohmann Brown) were obtained from a local supplier and raised in a temperature controlled environment. Lighting was set for a 12 h light/ 12 h dark cycle with a mean light level of 250 lux. Food and water were made available ad libitum. Experimental protocol All animals in the study underwent monocular deprivation and either intravitreal or subconjunctival injection. Chicks were assigned to one of a number of groups depending on whether they were injected with pirenzepine (pirenzepine MD) or saline drug vehicle (saline MD). All groups were balanced for starting weights. The experimental groups in this study comprised six intravitreal injection groups (minimum of n = 7 in each group), which received one of the following doses of injected pirenzepine: 3.5 pg, 20 ,ug, 100 ,ug, 200 pg, 350 pg or 5OOpg, dissolved in 7 ~1 phosphate buffered saline (PBS). Further experimental groups comprised six subconjunctival injection groups (minimum n = 6), which received one of the following doses of pirenzepine: 3.5 pg, 5OOpg, 75Opg, 1 mg, 5 mg, or 7.5 mg made up in 50~1 PBS. Control animals received an equivalent volume of 0.9% saline vehicle, depending on the method of injection. Injection protocol Pirenzepine is a selective muscarinic M, antagonist” which has an elimination half-life of 12 hzo. Due to the reported half-life, it was considered necessary to administer the drug on a daily regimen. On the seventh day after hatching, chicks were anaesthetised with 2.0-3.0% halothane anaesthetic. Injections of drug or saline were given by intravitreal or subconjunctival route. Intravitreal injections were given
On the sixth day after occlusion, full ocular measures were taken. Chicks underwent general anaesthesia using ketamine (50 mg/kg) and xylazine (3.5 mg/kg). Body temperature and breathing were monitored. The head was supported by a dental bitebar to allow measurements along the optic axis of the eye. In vivo measurements of cornea1 radius (modified keratometry), ocular refraction (streak retinoscopy) and axial ocular dimensions (A-scan ultrasonography) were taken using the methods described previously”. Statistical analysis Data on structural component measures were entered into a spreadsheet (Excel 5.0). Measurements were expressed as the difference between the treated and the contralateral control eye and computed values transferred to a statistical package (Minitab). Analysis of variance (ANOVA) and Tukeys pairwise multiple comparison test were used to examine overall effects and assess individual group differences. Dependent or independent t-tests were used to examine specific differences within groups (P < 0.05 was taken as the minimum level for significance). Results Intravitreal
groups
Animals receiving intravitreal injections of saline vehicle (n = 11) developed a significant myopia in the deprived/ injected eye after five days of treatment compared to the contralateral control eye (- 11.1 + 1.6 D versus 2.6 f. 0.2 D; mean + SEM; t-test, P < 0.01) (Figure IA) resulting from vitreous chamber elongation (+O. 32 + 0.05 mm in the treated eye compared to the contralateral control; t-test, P < 0.001) (Figure If?). There was a highly significant, dose dependent reduction
Muscarinic
Intravitrcal
regulation
of form
deprivation
myopia:
E. M. leech
et al.
353
Subconjunctival Groups
Groups i;
5.0
g
0.0
z-; &:: nrs = g 5 I
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~.
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-5.0 FM **
-10.0.
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o r -15.0 20 4. zg :- )- -20.0 -iz
!fz
Saline
3.5
20
100
200
350
500
-25.0
IT
-30.0
Ie..pp__----..-.---
I
Sollne
-.......-. 3.5
3.5
20
100
200
350
500
Sdlne
3.5
PIRENZEPINE (pug)
samle
3.5
20
100
200
PIRENZEPINE (pg)
750
1000
5000
7500
PIRENZEPINE (,ug)
PIRENZEPINE (pg)
Saline
500
500
750
1000
5000
7500
5000
7500
PIRENZEPINE (pg)
350
500
Sdllle
3.5
500
750
1000
PIRENZEPINE (fig)
Figure 1. Differences in refractive state and ocular dimensions between deprived and contralateral open control eyes for pirenzepine treated and control groups: (A) differences in ocular refraction in intravitreally injected animals; (13) differences in vitreous chamber depth in intravitreally injected animals; (C) differences in anterior segment depth in intravitreally injected animals (the reduction in anterior chamber depth is not significant until the dose reaches 350~9); (D) differences in ocular refraction in subconjunctivally injected animals; (E) differences in vitreous chamber depth in subconjunctivally injected animals; (F) differences in anterior segment depth in subconjunctivally injected animals. In all cases, a dose dependent reduction was seen. (“P < 0.05, **P < 0.01, Tukey’s test). Error bars = 1 SEM. Minimum n = 6 for all groups.
354
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Opt.
1995
15: No 5
Intravitreal
in both induced myopia (ANOVA; F = 13.85, P < 0.001) (Figure IA), with an ED,, of 175 pg (Figure 2A), and vitreous chamber elongation (ANOVA; F = 8.44, P < 0.001) (Figure 1B) with an ED,, value of 250 pg compared to saline MD controls. An injected dose of 5OOpg was found to produce complete prevention of induced myopia (+0.9 k 1.5 D, P < 0.001) and vitreous chamber elongation (-0.14 f 0,07mm, P < 0.001). There was also a significant, dose dependent reduction in anterior segment depth (cornea1 thickness plus anterior chamber depth) compared to the saline MD controls (ANOVA; F = 5.48, P < 0.001) (Figure I C). The shallowing of the anterior segment at the higher doses of pirenzepine account for the difference between the ED,, values for ocular refraction and vitreous chamber elongation.
DOSE
groups
Animals subconjunctivally injected with the saline vehicle developed a significant degree of myopia (- 15.4 & 1.4 D versus 1.9 + 0.3 D; mean k SEM; P < 0.001) (Figure 1D) and vitreous chamber elongation (0.56 f 0.07 mm; P < 0.001) (Figure IE) after five days of treatment compared to the contralateral control eye. Subconjunctivally injected pirenzepine again had a dose dependent effect on the reduction of induced myopia (ANOVA; F = 7.91, P < 0.001) (Figure 1D) and vitreous chamber elongation (ANOVA; F = 4.22, P < 0.05) (Figure 1E). However, much higher doses were required to produce a significant reduction with an ED,, value for induced myopia of 5.0 mg (28-fold higher than intravitreal injection) (Figure 2B). No ED,, value was achieved for reduction in vitreous chamber elongation where the maximum reduction was found to be only 38.0%. The difference in ED,, values was again accounted for by changes in anterior segment depth. Increasing doses of pirenzepine were found to reduce anterior segment enlargement in a dose dependent manner which reached significance at a dose of 5.0 mg (5.0 mg, -0.04 * O.O3mm, P < 0.01; 7.5mg, -0.03 *O.O3mm, P < 0.05) (Figure 1F). Evidence of normal retinal function in pirenzepine treated animals was given by the finding that age-matched chicks (n = 2) which underwent daily intravitreal pirenzepine injections (500 pg), were still susceptible to form deprivation myopia post-treatment. Significant levels of myopia were induced in the treated eye, compared to the contralateral control eye, after one week of occlusion following pirenzepine treatment (-20.8D versus +1.4D, P < 0.001). This was comparable to that found in untreated animals. The induced myopia resulted from vitreous chamber elongation (6.82mm versus 5.64mm, P < 0.001) (Figure 3). Furthermore, histological examination of retinal tissue of treated animals revealed no evidence of toxic damage to the retina”.
OF PIRENZEPINE
(fig)
Subconjunctival Groups E; -2 z
Subconjunctival
Groups
P 2
B
T
I
i
1'0
100 DOSE
1000
OF PIRENZEPINE
(pg)
Figure 2. Dose-response curves for intravitreally and subconjunctivally injected pirenzepine on induced myopia (log scale). The ordinate indicates the fraction of the myopia expressed as a percentage of the control group. Values represent mean for each group. (A) for intravitreally injected pirenzepine, the ED,, for the reduction of myopia was calculated as 175 pg. (8) for subconjunctivally injected of myopia pirenzepine, the ED,, value for the reduction was calculated as 5.0mg. Error bar = 1 SEM. Minimum n = 6 for all groups
L
I
L
I
OCULAR REFRACTION
VITREOUS CHAMBER
Figure 3. Form deprivation myopia and vitreous chamber elongation can still be induced following pirenzepine treatment, demonstrating intact retinal function. Error bar = 1 SD.
Muscarinic
regulation
Discussion The results demonstrate that form deprivation myopia can be blocked by administration of pirenzepine, an M, antagonist. Furthermore, the dose-response characteristics of both intravitreally and subconjunctivally injected pirenzepi:le on form deprivation myopia were investigated in order to elucidate the possible mode and site of action of the drug. in particular as to whether its effects are toxic or not. 1n:ravitreal administration of pirenzepine was effective at reducing vitreous chamber elongation at doses above 100 ,ug. In comparison, pirenzepine administered by a tarsal subconjllnctival route had to be given in substantially higher doses to achieve a similar structural effect (2OOpg intravitreally and 5.0 mg subconjunctivally-a 25-fold difference). Ever at the highest subconjunctival doses, an ED,, value was not obtained and the drug effect on vitreous chamber elongation plateaued at a 38% reduction of the full saline MD control value. Thus, the vitreous chamber elongation associated with form deprivation could not be completely eliminated; this presumably reflects the inability of the drug to reach its site of action in a functional dose by this deli, ery route. It could be argued that, due to the greater effectiveness of the intravitreal route of administration, pirenzepine may be acting at muscarinic receptor sites in the retina or choroid as a->posed to the sclera. It is well known that there are chol:nergic amacrine cells in the retina and M, receptors have allso been identified in the retina. It is possible that pirenzepine is altering retinal neurotransmission and this influ’srices ocular growth regulation. However, it is also possible that an intravitreal route may deliver more pirenzepine to the sclera than a subconjunctival route. The results demonstrate that while subconjunctival administration is effective at reducing anterior segment growth, it is much less effective at reducing vitreous chamber elongation. This could be due to the drug being eliminated from the eye prior to reaching the posterior segment in sufficient dose, whereas intravitreal administration would maintain the drug in the eye longer allowing diffusion to the sclera. A direct scleral mechanism has recently been proposed as an alternative explanation for muscarinic antagonist effects in fcrm deprivation myopia, by demonstrating that muscarinic antagonists can act directly on in vitro scleral proteoglycan synthesis and so could affect ocular growth and developmentz2. The doses found to be effective in this study differ from those previously reported. Stone et al.” found almost comy:llete cessation of axial length increase with a daily dose of 3.5 ,ug of pirenzepine, when given via subconjunctival injection, but neither our own results reported here, nor those of Rickers et a1.18 have been able to repeat these effects at such low doses. This may be due to the wide species variations reported in experimental models23. Furthermore,
of form deprivation
myopia:
E. M. Leech et al.
355
Rickers et al.” found an injected dose of 2000 pg pirenzepine was required to produce significant reductions in either induced myopia or axial elongation which proved toxic to the retina. However, their results also showed a reduction in myopia with doses of 2OOp.g or more; these differences may be explained by methodological differences between the two studies (daily injection versus injection every three days). The present study demonstrated that the ocular growth mechanism in myopia development was still functional after pirenzepine (500 pg) treatment. This study has shown that deprivation myopia in chicks can be prevented with daily intravitreal injections of the M, antagonist pirenzepine. Furthermore, the use of M, and M, antagonists has shown that pirenzepine is mediating its effect via M, receptors2’. By comparing the doses required to achieve a partial effect with subconjunctival injections, it has proved that intravitreal administration of the drug is significantly more efficient at blocking myopia. These results show that selectively blocking M, receptors, possibly in the retina or the choroid, prevents myopia. Acknowledgements We would like to acknowledge grant support from the BBSRC (GR/J33265), the British College of Optometrists and the Wellcome Trust. References 1 Robb, R. M. Refractive errors associated with haemangiomas of the eye-lids and orbit in infancy. Am. J Ophthalmol. 83, 52-58 (1977) 2 Young, F. A. The effect of restricted visual suace on the refractive error of the young monkey eye. Int: Ophthalmol. 2, 571-577 (1963) 3 Miles, F. A. and Wallman, J. Local ocular compensation for imposed local refractive error. v&on Res. 30, 339-349 (1990) 4 Bedrossian, R. H. The effect of atropine on myopia. Am. .I. Ophthalmol. 86, 713-717 (1979) 5 McKanna, J. A. and Casagrande, V. A. Atropine affects lid suture myopia development. Dot. Ophthalmol. Proc. Series 28, 187-192 (1981) 6 Raviola, E. and Wiesel, T. N. An animal model of myopia. New Engl. J. Med. 312, 1609-1615 (1985) 7 Schaeffel, F., Troilo, D., Wallman, J. and Howland, H. C. Developing eyes that lack accommodation grow to compensate for imposed defocus. Visual Neurosci. 4, 177-183 (1990) 8 Norton, T. T., Essinger, J. A. and McBrien, N. A. Lidsuture myopia in tree shrews with retinal ganglion cell blockade. Visual Neurosci. 11, 143-153 (1994) 9 McBrien, N. A., Moghaddam, H. 0.) Cottriall, C. L., Leech, E. M. and Cornell, L. M. The effects of blockade of retinal cell action potentials on ocular growth, emmetropization and form deprivation myopia in chicks. Vision Res. 35, 1141-1152 (1995) 10 Stone, R. A.,.- Lin, T. and Laties, A. M. Muscarinic antagonist effects on experimental chick myopia. Exp. Eye Rex 52, 755-758 (1991)
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11 McBrien, N. A., Moghaddam, H. 0. and Reeder, A. P. Atropine reduces experimental myopia and eye enlargement via a non-accommodative mechanism. Invest. Ophthalmol. Visual Sci. 34, 205-215 (1993) 12 Goyal, R. K. Muscarinic receptor subtypes: physiology and clinical implications. New. Engl. J. Med. 321, 1022-1029 (1989) 13 Vanderheyden, P., Ebinger, G. and Vauquelin, G. Characterization of M, and M, muscarinic receptors in calf retina membranes. Vision Res. 28, 247-250 (1988) 14 Gremo, F., Marchisio, A. M. and Vernadakis, A. Muscarinic receptor subclasses in the chick embryo retina: influence of corticosteroid treatment. J. Neurochem. 45, 345-3.5 1 (1985) 15 Pang, I. H., Matsumoto, S., Tamm, E. and DeSantis, L. Characterization of muscarinic receptor involvement in human ciliary muscle cell function. J. Ocular Pharmacol. 10, 125-136 (1994) 16 Jumblatt, J. E. and Ha&miller, R. C. M,-type muscarinic receptors mediate prejunctional inhibition of norepinephrine release in human iris-ciliary body. Exp. Eye Rex 58, 175-180 (1994) 17 McBrien, N. A. and Cottriall, C. L. Pirenzepine reduces axial elongation and myopia in monocularly deprived tree
shrews. Invest. Ophthalmol. Visual Sci. 34(4), 1210 (1993) 18 Rickers, R., Schaeffel, F., Hagel, G. and Zrenner, E. Dose dependent effects of intravitreal pirenzepine on deprivation myopia and lens-induced refractive errors in chickens. Invest. Ophthalmol. Visual Sci. 35(4), 1801 (1994) 19 Hammer, R., Berrie, C. P., Birdsall, N. J. M., Burgen, A. S. V. and Hulme, E. C. Pirenzepine distinguishes between different sub-classes of muscarinic receptor. Nature 283, 90-92 (1980) 20 Carmine, A. A. and Brogden, R. N. Pirenzepine, a review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy in peptic ulcer disease and other allied diseases. Drugs 30, 85-126 (1985) 21 McBrien, N. A., Cottriall, C. L. and Leech, E. M. The role of selective muscarinic antagonists in form deprivation myopia. Curr. Eye Res. (submitted) (1995) 22 Marzani, D., Lind, G. J., Chew, S. J. and Wallman, J. The reduction of myopia by muscarinic antagonist may involve a direct effect on scleral cells. Invest. Ophthalmol. Visual Sci. 35(4), 1801 (1994) 23 Troilo, D., Glasser, A., Li, T. and Howland, H. Different strains of chick have different eye growth responses to visual deprivation. Invest. Ophthalmol. Visual Sci. 33(4), 711 (1992)
UTTERWORTH EINEMANN
0275-5408(95)00074-7
Pirenzepine prevents in a dose dependent E. R4. Leech,* Department Cardiff CFl
form deprivation manner
C. L. Cottriall”
of Optometry 3XF, UK
and
Vision
myopia
and N. A. McBrien” Sciences,
University
of Wales,
College
of Cardiff,
Summary Previous studies have demonstrated that muscarinic antagonists, such as atropine and pirerzepine, block form deprivation myopia in avian and mammalian models. The aim of the present investigation was to establish dose-response curves for intravitreal and subconjullctivally injected pirenzepine and to determine receptor specificity. Chicks were monccularly deprived of form vision for five days and received daily injections of either pirenzepine or saline. Keratometry, retinoscopy and A-scan ultrasonography of axial ocular dimensions were then taken. lntravitreally injected pirenzepine was effective at preventing form deprivation myopia in a dose dependent manner with an ED,, of 175pg. A 5OOpg dose totall\/ prevented induced myopia ( + 0.9 D versus - 13.7 D) and axial enlargement ( - 0.14 mm versus + 0.32 mm). Daily subconjunctival injection off pirenzepine was significantly less effec::ive in preventing form deprivation myopia. Form deprivation myopia could still be induced in animals which had undergone pirenzepine treatment. Pirenzepine was effective in preventing the axial elongation associated with experimental myopia in a dose dependent manner and via a functional not toxic mechanism.
Ophthal.
Physiol.
Opt. 1995,
15, 351-356
Introduction
recent studies have demonstrated that atropine may be acting via a non-accommodative route”,” and therefore must be having its effect at receptor sites other than those on the smooth muscle of the ciliary body. At present, five different muscarinic receptor subtypes have been identified, based on either pharmacological, morphological or functional characteristics”. The use of selective antagonists has identified M, muscarinic receptors in the retina of calf13 and chicki4, as well as post-junctional M, receptors in both ciliary muscle and iris sphincterI and pre-junctional M, receptors in the iris”j. Atropine is a broad band muscarinic antagonist which binds to all identified muscarinic receptors with equal affinity’*. Therefore, the prevention of form deprivation myopia by atropine could be occurring through either an M, , M, or M, mediated mechanism. The use of selective muscarinic antagonists make it possible to target a particular class of receptor. In an attempt to identify which receptor(s) are implicated in muscarinic control of ocular growth, selective antagonists have been used to try and prevent form deprivation myopia. It has been shown that pirenzepine prevents axial myopia in both monocularly deprived white leghorn chicks” and
Early visual experience is known to play an integral role in postnatal ocular development and disruption of a clear retinal image causes a breakdown in the process of emmetropisation’. Both environmental2.3 and pharmacologica14,5studies have implicated a pivotal role for accommodation in the regulation of eye growth. However, recent studies on animal models of refractive development question if accommodation has a primary role in the development of myopia. Investigations have demonstrated that blocking communication between the eye and higher visual centres does not prevent the development of, or recovery from, form deprivation myopia6-9, indicating that the visual signals controlling eye growth and refractive development may proceed directly from the retina to the choroid and sclera and not via higher visual centres. Consequently,
*MBCO Received: 15 May 1995 Correspondence to: N. A. McBrien
351
352
Ophthal. Physiol. Opt. 1995 15: No 5
tree shrews”. Rickers et ~2.‘~have also successfully stopped axial myopia in chicks using pirenzepine. However, the effective dose was much higher than that previously reported in chick although injections were only performed every three days rather than daily. They suggest that pirenzepine may be working through a toxic effect rather than a physiological one. The present study sought to determine whether pirenzepine effectively reduced or prevented experimentally induced myopia in a Rhode Island cross-strain of chick, and to examine the dose-response characteristics of pirenzepine when administered either intravitreally or subconjunctivally. Such information would hopefully address the issue of effective doses for pirenzepine and, more importantly, give insight as to the site of action of the drug.
using the technique described by McBrien et al.“. On each of the subsequent five days, the injection procedure was repeated; it was nearly always possible to find the same hole each time. This minimised any injection effects that may have resulted from release of growth factors or reduced intraocular pressure resulting from multiple punctures of the sclera. Subconjunctival injections were given by injecting into the tarsal subconjunctival space. Injections were performed by injecting 50 ~1 of drug or saline with a 30 gauge needle through the lid into the subconjunctival space. Following the injection, the treated eye of all animals was occluded with a hemispherical translucent plastic occluder, attached by means of a velcro ring placed around the orbit. This facilitated the daily removal and replacement of the occluders at each subsequent injection.
Materials
In vivo ocular measures
and methods
Animals used Day old Rhode Island cross chicks (Lohmann Brown) were obtained from a local supplier and raised in a temperature controlled environment. Lighting was set for a 12 h light/ 12 h dark cycle with a mean light level of 250 lux. Food and water were made available ad libitum. Experimental protocol All animals in the study underwent monocular deprivation and either intravitreal or subconjunctival injection. Chicks were assigned to one of a number of groups depending on whether they were injected with pirenzepine (pirenzepine MD) or saline drug vehicle (saline MD). All groups were balanced for starting weights. The experimental groups in this study comprised six intravitreal injection groups (minimum of n = 7 in each group), which received one of the following doses of injected pirenzepine: 3.5 pg, 20 ,ug, 100 ,ug, 200 pg, 350 pg or 5OOpg, dissolved in 7 ~1 phosphate buffered saline (PBS). Further experimental groups comprised six subconjunctival injection groups (minimum n = 6), which received one of the following doses of pirenzepine: 3.5 pg, 5OOpg, 75Opg, 1 mg, 5 mg, or 7.5 mg made up in 50~1 PBS. Control animals received an equivalent volume of 0.9% saline vehicle, depending on the method of injection. Injection protocol Pirenzepine is a selective muscarinic M, antagonist” which has an elimination half-life of 12 hzo. Due to the reported half-life, it was considered necessary to administer the drug on a daily regimen. On the seventh day after hatching, chicks were anaesthetised with 2.0-3.0% halothane anaesthetic. Injections of drug or saline were given by intravitreal or subconjunctival route. Intravitreal injections were given
On the sixth day after occlusion, full ocular measures were taken. Chicks underwent general anaesthesia using ketamine (50 mg/kg) and xylazine (3.5 mg/kg). Body temperature and breathing were monitored. The head was supported by a dental bitebar to allow measurements along the optic axis of the eye. In vivo measurements of cornea1 radius (modified keratometry), ocular refraction (streak retinoscopy) and axial ocular dimensions (A-scan ultrasonography) were taken using the methods described previously”. Statistical analysis Data on structural component measures were entered into a spreadsheet (Excel 5.0). Measurements were expressed as the difference between the treated and the contralateral control eye and computed values transferred to a statistical package (Minitab). Analysis of variance (ANOVA) and Tukeys pairwise multiple comparison test were used to examine overall effects and assess individual group differences. Dependent or independent t-tests were used to examine specific differences within groups (P < 0.05 was taken as the minimum level for significance). Results Intravitreal
groups
Animals receiving intravitreal injections of saline vehicle (n = 11) developed a significant myopia in the deprived/ injected eye after five days of treatment compared to the contralateral control eye (- 11.1 + 1.6 D versus 2.6 f. 0.2 D; mean + SEM; t-test, P < 0.01) (Figure IA) resulting from vitreous chamber elongation (+O. 32 + 0.05 mm in the treated eye compared to the contralateral control; t-test, P < 0.001) (Figure If?). There was a highly significant, dose dependent reduction
Muscarinic
Intravitrcal
regulation
of form
deprivation
myopia:
E. M. leech
et al.
353
Subconjunctival Groups
Groups i;
5.0
g
0.0
z-; &:: nrs = g 5 I
__----.-..
-.-
~.
D -
-5.0 FM **
-10.0.
c
o r -15.0 20 4. zg :- )- -20.0 -iz
!fz
Saline
3.5
20
100
200
350
500
-25.0
IT
-30.0
Ie..pp__----..-.---
I
Sollne
-.......-. 3.5
3.5
20
100
200
350
500
Sdlne
3.5
PIRENZEPINE (pug)
samle
3.5
20
100
200
PIRENZEPINE (pg)
750
1000
5000
7500
PIRENZEPINE (,ug)
PIRENZEPINE (pg)
Saline
500
500
750
1000
5000
7500
5000
7500
PIRENZEPINE (pg)
350
500
Sdllle
3.5
500
750
1000
PIRENZEPINE (fig)
Figure 1. Differences in refractive state and ocular dimensions between deprived and contralateral open control eyes for pirenzepine treated and control groups: (A) differences in ocular refraction in intravitreally injected animals; (13) differences in vitreous chamber depth in intravitreally injected animals; (C) differences in anterior segment depth in intravitreally injected animals (the reduction in anterior chamber depth is not significant until the dose reaches 350~9); (D) differences in ocular refraction in subconjunctivally injected animals; (E) differences in vitreous chamber depth in subconjunctivally injected animals; (F) differences in anterior segment depth in subconjunctivally injected animals. In all cases, a dose dependent reduction was seen. (“P < 0.05, **P < 0.01, Tukey’s test). Error bars = 1 SEM. Minimum n = 6 for all groups.
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Intravitreal
in both induced myopia (ANOVA; F = 13.85, P < 0.001) (Figure IA), with an ED,, of 175 pg (Figure 2A), and vitreous chamber elongation (ANOVA; F = 8.44, P < 0.001) (Figure 1B) with an ED,, value of 250 pg compared to saline MD controls. An injected dose of 5OOpg was found to produce complete prevention of induced myopia (+0.9 k 1.5 D, P < 0.001) and vitreous chamber elongation (-0.14 f 0,07mm, P < 0.001). There was also a significant, dose dependent reduction in anterior segment depth (cornea1 thickness plus anterior chamber depth) compared to the saline MD controls (ANOVA; F = 5.48, P < 0.001) (Figure I C). The shallowing of the anterior segment at the higher doses of pirenzepine account for the difference between the ED,, values for ocular refraction and vitreous chamber elongation.
DOSE
groups
Animals subconjunctivally injected with the saline vehicle developed a significant degree of myopia (- 15.4 & 1.4 D versus 1.9 + 0.3 D; mean k SEM; P < 0.001) (Figure 1D) and vitreous chamber elongation (0.56 f 0.07 mm; P < 0.001) (Figure IE) after five days of treatment compared to the contralateral control eye. Subconjunctivally injected pirenzepine again had a dose dependent effect on the reduction of induced myopia (ANOVA; F = 7.91, P < 0.001) (Figure 1D) and vitreous chamber elongation (ANOVA; F = 4.22, P < 0.05) (Figure 1E). However, much higher doses were required to produce a significant reduction with an ED,, value for induced myopia of 5.0 mg (28-fold higher than intravitreal injection) (Figure 2B). No ED,, value was achieved for reduction in vitreous chamber elongation where the maximum reduction was found to be only 38.0%. The difference in ED,, values was again accounted for by changes in anterior segment depth. Increasing doses of pirenzepine were found to reduce anterior segment enlargement in a dose dependent manner which reached significance at a dose of 5.0 mg (5.0 mg, -0.04 * O.O3mm, P < 0.01; 7.5mg, -0.03 *O.O3mm, P < 0.05) (Figure 1F). Evidence of normal retinal function in pirenzepine treated animals was given by the finding that age-matched chicks (n = 2) which underwent daily intravitreal pirenzepine injections (500 pg), were still susceptible to form deprivation myopia post-treatment. Significant levels of myopia were induced in the treated eye, compared to the contralateral control eye, after one week of occlusion following pirenzepine treatment (-20.8D versus +1.4D, P < 0.001). This was comparable to that found in untreated animals. The induced myopia resulted from vitreous chamber elongation (6.82mm versus 5.64mm, P < 0.001) (Figure 3). Furthermore, histological examination of retinal tissue of treated animals revealed no evidence of toxic damage to the retina”.
OF PIRENZEPINE
(fig)
Subconjunctival Groups E; -2 z
Subconjunctival
Groups
P 2
B
T
I
i
1'0
100 DOSE
1000
OF PIRENZEPINE
(pg)
Figure 2. Dose-response curves for intravitreally and subconjunctivally injected pirenzepine on induced myopia (log scale). The ordinate indicates the fraction of the myopia expressed as a percentage of the control group. Values represent mean for each group. (A) for intravitreally injected pirenzepine, the ED,, for the reduction of myopia was calculated as 175 pg. (8) for subconjunctivally injected of myopia pirenzepine, the ED,, value for the reduction was calculated as 5.0mg. Error bar = 1 SEM. Minimum n = 6 for all groups
L
I
L
I
OCULAR REFRACTION
VITREOUS CHAMBER
Figure 3. Form deprivation myopia and vitreous chamber elongation can still be induced following pirenzepine treatment, demonstrating intact retinal function. Error bar = 1 SD.
Muscarinic
regulation
Discussion The results demonstrate that form deprivation myopia can be blocked by administration of pirenzepine, an M, antagonist. Furthermore, the dose-response characteristics of both intravitreally and subconjunctivally injected pirenzepi:le on form deprivation myopia were investigated in order to elucidate the possible mode and site of action of the drug. in particular as to whether its effects are toxic or not. 1n:ravitreal administration of pirenzepine was effective at reducing vitreous chamber elongation at doses above 100 ,ug. In comparison, pirenzepine administered by a tarsal subconjllnctival route had to be given in substantially higher doses to achieve a similar structural effect (2OOpg intravitreally and 5.0 mg subconjunctivally-a 25-fold difference). Ever at the highest subconjunctival doses, an ED,, value was not obtained and the drug effect on vitreous chamber elongation plateaued at a 38% reduction of the full saline MD control value. Thus, the vitreous chamber elongation associated with form deprivation could not be completely eliminated; this presumably reflects the inability of the drug to reach its site of action in a functional dose by this deli, ery route. It could be argued that, due to the greater effectiveness of the intravitreal route of administration, pirenzepine may be acting at muscarinic receptor sites in the retina or choroid as a->posed to the sclera. It is well known that there are chol:nergic amacrine cells in the retina and M, receptors have allso been identified in the retina. It is possible that pirenzepine is altering retinal neurotransmission and this influ’srices ocular growth regulation. However, it is also possible that an intravitreal route may deliver more pirenzepine to the sclera than a subconjunctival route. The results demonstrate that while subconjunctival administration is effective at reducing anterior segment growth, it is much less effective at reducing vitreous chamber elongation. This could be due to the drug being eliminated from the eye prior to reaching the posterior segment in sufficient dose, whereas intravitreal administration would maintain the drug in the eye longer allowing diffusion to the sclera. A direct scleral mechanism has recently been proposed as an alternative explanation for muscarinic antagonist effects in fcrm deprivation myopia, by demonstrating that muscarinic antagonists can act directly on in vitro scleral proteoglycan synthesis and so could affect ocular growth and developmentz2. The doses found to be effective in this study differ from those previously reported. Stone et al.” found almost comy:llete cessation of axial length increase with a daily dose of 3.5 ,ug of pirenzepine, when given via subconjunctival injection, but neither our own results reported here, nor those of Rickers et a1.18 have been able to repeat these effects at such low doses. This may be due to the wide species variations reported in experimental models23. Furthermore,
of form deprivation
myopia:
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Rickers et al.” found an injected dose of 2000 pg pirenzepine was required to produce significant reductions in either induced myopia or axial elongation which proved toxic to the retina. However, their results also showed a reduction in myopia with doses of 2OOp.g or more; these differences may be explained by methodological differences between the two studies (daily injection versus injection every three days). The present study demonstrated that the ocular growth mechanism in myopia development was still functional after pirenzepine (500 pg) treatment. This study has shown that deprivation myopia in chicks can be prevented with daily intravitreal injections of the M, antagonist pirenzepine. Furthermore, the use of M, and M, antagonists has shown that pirenzepine is mediating its effect via M, receptors2’. By comparing the doses required to achieve a partial effect with subconjunctival injections, it has proved that intravitreal administration of the drug is significantly more efficient at blocking myopia. These results show that selectively blocking M, receptors, possibly in the retina or the choroid, prevents myopia. Acknowledgements We would like to acknowledge grant support from the BBSRC (GR/J33265), the British College of Optometrists and the Wellcome Trust. References 1 Robb, R. M. Refractive errors associated with haemangiomas of the eye-lids and orbit in infancy. Am. J Ophthalmol. 83, 52-58 (1977) 2 Young, F. A. The effect of restricted visual suace on the refractive error of the young monkey eye. Int: Ophthalmol. 2, 571-577 (1963) 3 Miles, F. A. and Wallman, J. Local ocular compensation for imposed local refractive error. v&on Res. 30, 339-349 (1990) 4 Bedrossian, R. H. The effect of atropine on myopia. Am. .I. Ophthalmol. 86, 713-717 (1979) 5 McKanna, J. A. and Casagrande, V. A. Atropine affects lid suture myopia development. Dot. Ophthalmol. Proc. Series 28, 187-192 (1981) 6 Raviola, E. and Wiesel, T. N. An animal model of myopia. New Engl. J. Med. 312, 1609-1615 (1985) 7 Schaeffel, F., Troilo, D., Wallman, J. and Howland, H. C. Developing eyes that lack accommodation grow to compensate for imposed defocus. Visual Neurosci. 4, 177-183 (1990) 8 Norton, T. T., Essinger, J. A. and McBrien, N. A. Lidsuture myopia in tree shrews with retinal ganglion cell blockade. Visual Neurosci. 11, 143-153 (1994) 9 McBrien, N. A., Moghaddam, H. 0.) Cottriall, C. L., Leech, E. M. and Cornell, L. M. The effects of blockade of retinal cell action potentials on ocular growth, emmetropization and form deprivation myopia in chicks. Vision Res. 35, 1141-1152 (1995) 10 Stone, R. A.,.- Lin, T. and Laties, A. M. Muscarinic antagonist effects on experimental chick myopia. Exp. Eye Rex 52, 755-758 (1991)
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11 McBrien, N. A., Moghaddam, H. 0. and Reeder, A. P. Atropine reduces experimental myopia and eye enlargement via a non-accommodative mechanism. Invest. Ophthalmol. Visual Sci. 34, 205-215 (1993) 12 Goyal, R. K. Muscarinic receptor subtypes: physiology and clinical implications. New. Engl. J. Med. 321, 1022-1029 (1989) 13 Vanderheyden, P., Ebinger, G. and Vauquelin, G. Characterization of M, and M, muscarinic receptors in calf retina membranes. Vision Res. 28, 247-250 (1988) 14 Gremo, F., Marchisio, A. M. and Vernadakis, A. Muscarinic receptor subclasses in the chick embryo retina: influence of corticosteroid treatment. J. Neurochem. 45, 345-3.5 1 (1985) 15 Pang, I. H., Matsumoto, S., Tamm, E. and DeSantis, L. Characterization of muscarinic receptor involvement in human ciliary muscle cell function. J. Ocular Pharmacol. 10, 125-136 (1994) 16 Jumblatt, J. E. and Ha&miller, R. C. M,-type muscarinic receptors mediate prejunctional inhibition of norepinephrine release in human iris-ciliary body. Exp. Eye Rex 58, 175-180 (1994) 17 McBrien, N. A. and Cottriall, C. L. Pirenzepine reduces axial elongation and myopia in monocularly deprived tree
shrews. Invest. Ophthalmol. Visual Sci. 34(4), 1210 (1993) 18 Rickers, R., Schaeffel, F., Hagel, G. and Zrenner, E. Dose dependent effects of intravitreal pirenzepine on deprivation myopia and lens-induced refractive errors in chickens. Invest. Ophthalmol. Visual Sci. 35(4), 1801 (1994) 19 Hammer, R., Berrie, C. P., Birdsall, N. J. M., Burgen, A. S. V. and Hulme, E. C. Pirenzepine distinguishes between different sub-classes of muscarinic receptor. Nature 283, 90-92 (1980) 20 Carmine, A. A. and Brogden, R. N. Pirenzepine, a review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy in peptic ulcer disease and other allied diseases. Drugs 30, 85-126 (1985) 21 McBrien, N. A., Cottriall, C. L. and Leech, E. M. The role of selective muscarinic antagonists in form deprivation myopia. Curr. Eye Res. (submitted) (1995) 22 Marzani, D., Lind, G. J., Chew, S. J. and Wallman, J. The reduction of myopia by muscarinic antagonist may involve a direct effect on scleral cells. Invest. Ophthalmol. Visual Sci. 35(4), 1801 (1994) 23 Troilo, D., Glasser, A., Li, T. and Howland, H. Different strains of chick have different eye growth responses to visual deprivation. Invest. Ophthalmol. Visual Sci. 33(4), 711 (1992)