August 2012
Volume 53, Issue 9
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Anatomy and Pathology/Oncology  |   August 2012
Muscarinic Antagonist Control of Myopia: Evidence for M4 and M1 Receptor-Based Pathways in the Inhibition of Experimentally-Induced Axial Myopia in the Tree Shrew
Author Notes
  • From the Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia. 
  • Corresponding author: Neville A. McBrien, Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, 3010, Australia; nmcbrien@unimelb.edu.au
Investigative Ophthalmology & Visual Science August 2012, Vol.53, 5827-5837. doi:10.1167/iovs.12-9943
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      Baskar Arumugam, Neville A. McBrien; Muscarinic Antagonist Control of Myopia: Evidence for M4 and M1 Receptor-Based Pathways in the Inhibition of Experimentally-Induced Axial Myopia in the Tree Shrew. Invest. Ophthalmol. Vis. Sci. 2012;53(9):5827-5837. doi: 10.1167/iovs.12-9943.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The broadband muscarinic antagonist atropine is effective in stopping the progression of myopia in animals and humans. The partially selective M1/M4 antagonist pirenzepine also slows progression of myopia, although not as effectively as atropine. Due to the supra maximal doses utilized in these studies, it is unclear if this antimyopia effect occurs through a receptoral-based mechanism, and if so, which receptors are involved. Studies in chicks indicate the involvement of the M4 muscarinic receptor. The current study investigated the effect of the highly selective muscarinic antagonists Muscarinic Toxin 3 (MT3) (M4 selective) and Muscarinic Toxin 7 (MT7) (M1 selective) on experimental myopia in a mammalian model.

Methods.: Tree shrews (n = 23) underwent daily intravitreal injections of MT3, MT7, or vehicle (phosphate buffered saline) for five days in the treated eye, combined with deprivation of vision with a translucent occluder (MD). The contralateral eye was unocccluded and underwent intravitreal injections of vehicle for the same period. Two additional groups (n = 10) underwent daily intravitreal injections of MT7 or vehicle for 10 days in the treated eye combined with negative lens (−9.5 diopter [D]) defocus (LIM). The control eye was injected with saline and wore a plano lens.

Results.: Both MT3 and MT7 treatment reduced the development of deprivation-induced myopia (treated-control eye [T-C]; vehicle-MD; −4.3 ± 0.6 D versus MT3-MD; −0.7 ± 0.2 D and MT7-MD; −0.7 ± 0.4 D; P < 0.001). MT7 treatment was effective at inhibiting lens-induced myopia (T-C; vehicle-LIM; −4.6 ± 0.5 D versus MT7-LIM; 0.2 ± 0.2 D; P < 0.05).

Conclusions.: The findings demonstrate that inhibition of form-deprivation myopia by muscarinic antagonists involves both M4 and M1 muscarinic receptor signaling pathways in mammals.

Introduction
The broadband muscarinic antagonist, atropine, has been shown to prevent the development of myopia in humans. 13 In addition, atropine and the partially selective muscarinic antagonist, pirenzepine, have been shown to reduce the development of myopia in several animal models, including rhesus monkeys, 4 chicks, 5,6 and tree shrews. 7,8 However, the disadvantage of atropine is that it is a nonselective muscarinic antagonist and has unwanted ocular and gastric adverse effects. Studies of pirenzepine, a partially selective M1/M4, 6,7,9 and himbacine, a partially selective M4/M2 antagonist, 10 have shown them to be effective at slowing the progression of myopia. However, the specific site of action of these antagonists remains unclear, partly due to the lack of specificity and high doses required to inhibit myopia. Although pirenzepine is termed an M1 selective antagonist, it also demonstrates subtype cross-reactivity to other receptors causing significant increase in pupil diameter in tree shrews, 7 significant increase in pupil size and decrease in accommodation in rhesus monkeys, 11 and blurred near vision in human children. 12 As the constriction of the iris sphincter muscle is mediated through M3 muscarinic receptors this highlights the cross-reactivity. 13,14 These findings indicate that pirenzepine, at the doses utilized to slow myopia progression, is binding at other muscarinic receptor subtypes in addition to M1. In vitro affinity constants (the concentration of drug that would occupy 50% of the receptors) of pirenzepine for the various muscarinic receptor subtypes reveals only a 4-fold lower affinity for the M4 receptor than the M1 receptor, with much lower binding affinities for M2 and M3 receptors. 15  
The fact that M1/M4 (pirenzepine) and an M4/M2 (himbacine) partially selective muscarinic antagonists have been demonstrated to show dose-dependent inhibition of axial myopia in chick and mammalian models of myopia, implicates both M1 and M4 receptor pathways in muscarinic antagonist control of myopia, as M2 antagonists have been shown to be ineffective. 6 In the tree shrew, with the exception of the muscarinic M2 receptor in the cornea, all other ocular tissues investigated have mRNA expression for all five muscarinic receptors. Immunohistochemistry confirmed translation to protein expression of all identified muscarinic receptors. 16 The sequence homology between the tree shrew M1 and M4 receptors and the human M1 and M4 receptors is 93% in both cases. M1 and M4 muscarinic receptors are found in the specific ocular tissues implicated in muscarinic antagonist inhibition of myopia, namely retina, choroid, and sclera. Similar findings are reported for mouse 17 and guinea pig models, 18 and also human. 19  
In chicks, who do not possess M1 receptor mRNA, 20 pirenzepine's inhibitory effect on myopia could be either via M2 receptors (a suggested surrogate for the M1 receptor in birds 21 ) or via M4 receptors (since there is only a 4-fold higher affinity to M1 than M4 receptor). 10,20 Although previous studies have indicated that pirenzepine has a 10-fold higher affinity for the chick M2 receptor than for the mammalian M2 receptor, 21 several studies have demonstrated that more selective M2 muscarinic antagonists were ineffective at inhibiting experimentally-induced myopia in chicks (Methoctramine, AF-DX 116 [11-(2-[diethylaminomethyl]-1-piperdinylacetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine pine-6-one] and QNB). 6,22 Furthermore, the M3 selective muscarinic antagonist, 4-DAMP, has also been demonstrated to be ineffective at reducing the development of myopia. 6  
Human clinical trials of the M1/M4 partially selective muscarinic antagonist, pirenzepine, met with only limited success. A Food and Drug Administration (FDA) phase 2 two center clinical trial in the USA and Singapore, was stopped after one year by Novartis due to only limited prevention of myopia (45%–50%) using a topical gel. The high dose needed to slow myopia- (2%) induced nonselective side effects, including partial cycloplegia and gastric problems. 12,23 The problem with clinically available selective orthosteric muscarinic antagonists, such as pirenezepine (M1/M4 selective), is that they are only partially selective. This relates to the high level of sequence homology between the five muscarinic receptors. Before further human clinical trials can be justified, a more detailed knowledge on both the specific receptor pathways involved and the site of action of muscarinic antagonist control of myopia is required to provide a more clinically acceptable drug treatment for myopia. 
The advent of highly selective muscarinic antagonists, originally extracted from the green mamba snake, 24 and which attach to an allosteric site on the muscarinic receptor, provides the opportunity to dissect out specific muscarinic receptor involvement in signaling pathways. The greater structural diversity of allosteric muscarinic receptor sites, compared with orthosteric sites, where the endogenous ligand binds, enables substantially greater selectivity of compounds that bind to these sites. 25 In recent years, synthetic muscarinic allosteric receptor agonists and antagonists are increasingly being utilized to better detail muscarinic receptor involvement in brain conditions such as Alzheimer's and Parkinson's disease. There is considerable excitement at recent results showing therapeutic treatment with M1 and M4 selective muscarinic agonists that bind to the allosteric site, improving cognition and memory function in these neurodegenerative brain diseases. 26 With respect to inhibition of myopia, the allosteric muscarinic antagonists Muscarinic Toxin 7 (MT7) (M1 antagonist with 1000-fold higher selectivity for M1 than M4) and Muscarinic Toxin 3 (MT3) (M4 antagonist with over 100-fold higher selectivity for M4 than M1) have provided new pharmacological tools to investigate the specific muscarinic receptors through which atropine is mediating its inhibitory effect on myopia progression. 
The highly selective M4 muscarinic antagonist, MT3, has been shown to reduce experimentally-induced myopia in chicks, while MT7 (M1 selective) was, as anticipated, ineffective at inhibiting myopia in chicks as the M1 gene is not present in this species. 20,27 These findings support M4 muscarinic receptor signaling involvement in the development of experimentally-induced myopia in chicks. However, the M1 receptor is known to be present in human eyes as well as other mammalian species including the tree shrew, as detailed above. The present study sought to test the efficacy of M4 and M1 muscarinic receptor mediated antagonism in the control of experimentally-induced myopic ocular growth in the tree shrew by use of the highly selective allosteric muscarinic antagonists MT3 and MT7 to further elucidate the role of muscarinic receptor signalling in myopia control. 
Materials and Methods
The tree shrew ( Tupaia belangeri ) is an established mammalian model of myopia that undergoes similar structural and pathological changes in myopia as found in humans. 2830 The tree shrew is a mammal close to the primate line, in which axial myopia can readily be induced through either deprivation of vision by translucent occluder or optical defocus by negative powered lenses. 3133 Animals were raised in a breeding colony on a 14 hour light/10 hour dark cycle. Thirteen days after eye opening, animals were removed from the maternal cage and housed in individual large steel cages, with illumination at the floor of the cage ranging between 200 and 300 lux. Food and water were available ad libitum. All animal procedures were performed in accordance with the ARVO statement for the care and use of animals in ophthalmic and vision research. 
Experimental Groups
Monocular Deprivation (MD) Study.
Fifteen days after eye opening, MD of vision was imposed with a translucent occluder fitted into one side of a spectacle goggle that was clipped to a skull mounted pedestal (see below). Tree shrews were randomly assigned to one of four treatment groups: MT3 or MT7, PBS vehicle groups (n = 6 each) or Normal MD group (n = 5). Three groups (MT3-MD, MT7-MD, and vehicle-MD) were treated with daily intravitreal injection of the respective drugs MT3, MT7, or PBS vehicle, and also monocularly deprived of vision by a translucent occluder over the same eye. The control eye of each animal received a daily intravitreal injection of PBS, but this eye was unoccluded, this was to directly control for the effect of intravitreal injection on normal growth. The vehicle-MD (PBS) group served as the control group for the MT3-MD and MT7-MD groups in that they also received intravitreal injections in one eye that was also occluded, while the fellow unoccluded eye in all three groups had vehicle injections to provide an intra-animal control for the injection procedure. The Normal-MD group animals were fitted with a translucent occluder over one eye (treated) and the other eye left unoccluded, neither eye received injections. This group acted as control for the effect of the vehicle injection alone and was compared with the vehicle-MD group. After 5 days, intravitreal injections were ceased and a full set of in vivo biometric measures were collected under anesthesia and with 1% tropicamide cycloplegia. In order to assess the duration of any inhibitory effect on myopia development after cessation of muscarinic antagonist treatment, or conversely, if the rate of change of biometric parameters in deprivation myopia altered when drug treatment ceased, animals were allowed to recover from anesthesia after biometric measures. They continued wearing the translucent occluder over the treated eye for another 7 days (total period of form deprivation was 12 days). Another full set of in vivo biometric measures and ocular refraction data (with 1% tropicamide cycloplegia) was collected. 
Lens-Induced Myopia (LIM) Study.
To assess whether a highly selective allosteric muscarinic antagonist inhibited negative LIM, the most highly selective allosteric muscarinic antagonist, MT7 (M1 antagonist) was utilized in an additional experiment. We chose to assess only MT7 in our mammalian model as a previous study in chicks had demonstrated that MT3 inhibited lens-induced myopia (Diether S, et al. IOVS 2005;46:ARVO Abstract 1986). Optical lenses were made of polymethylmethacrylate (PMMA) with a 12.5-mm total diameter. The power of the lenses used was either 0 or −9.5 diopter (D). The treatment period in the LIM study was 10 days to increase the level of induced myopia in the vehicle control group, as LIM does not induce experimental myopia as quickly as form deprivation and the repeated intravitreal injections reduce the induced myopia due to the scleral puncture. Two experimental groups (n = 5 each) received MT7 or PBS-vehicle and negative lens defocus. All tree shrews were given treatment with −9.5 D lenses fitted over one eye and treated with daily intravitreal injections of either MT7 or PBS-vehicle. The fellow eye of both groups was fitted with a plano lens and received daily PBS intravitreal injections. 
Dose Selection
The injected concentrations of MT3 and MT7 were chosen based on a recent published study in chicks 27 and were 10 μM for each drug. The in vitro affinity constants for muscarinic receptors indicate that the dissociation constant (Ki) for MT3 at the M4 receptor and MT7 at the M1 receptor were 2 and 0.1 nM, respectively. These Ki values were determined by competition binding experiments using [3H] N-Methylscopolamine (NMS) on cloned and expressed human receptors. 34,35 The calculated concentration at the retino/vitreal interface for both MT3 and MT7 was 220 nM, based on the assumption the drug distributes evenly throughout the ocular volume and the tree shrew eye is a sphere with a radius of 4.2 mm. 36 The retino/vitreal concentration of 220 nM is the highest possible concentration, however, due to a number of factors (e.g., some backflow at injection site, diffusion barriers at the internal limiting membrane of the retina and other inner cellular layers) the concentration at muscarinic receptor sites in the retina would be less than 220 nM. 
Surgery and Intravitreal Injections
On day 13 after eye opening, a head mounted goggle was affixed to the skull using a technique similar to that reported previously. 37 Animals were allowed 2 days to recover from the general anesthesia, such that experimental procedures began on day 15 after eye opening. All daily intravitreal injections of MT3 or MT7 and/or PBS-vehicle (pH 7.4) were carried out using a previously reported technique. 5 In brief, on the first treatment day, tree shrews were anesthetized using 2% to 3% isoflurane (Veterinary and Medicine Supplies, VIC, Australia) in oxygen, and a drop of 0.5% proxymetacaine hydrochloride (Alcon, Fort Worth, TX) topical anesthetic was applied to the cornea of both eyes 30 seconds before the intravitreal injection. Injections were with a 30 gauge needle attached to a Hamilton micro-syringe (Hamilton, Reno, NV) via polyethylene micro tubing. On the next day of injections, it was possible to locate the puncture site in all cases and deliver the drug into the same hole, thus, minimizing the injection effects of scleral puncture on ocular growth. Each time after the injection, the tree shrew was allowed to recover completely from gaseous anesthesia before being returned to their home cage (∼ 3–5 minutes). 
Ocular Measures
In vivo refractive and structural measurements were taken at the start of the study, after 5 days of treatment (end of intravitreal injections), and after 12 days (7 days after injections ceased). For the LIM study, measurements were taken at the start and 10 days following treatment. Measurement procedures have been described in detail previously. 38 In brief, tree shrews were anaesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine intramuscularly. A dental bite bar was used to orient the head and enable accurate measurements of refraction and axial dimensions along the visual axis. In order to minimize fluctuation in measured refraction due to any active accommodation, 1% tropicamide was used as a short acting cycloplegic. Corneal curvature was measured with a modified Bausch & Lomb one position keratometer (Bausch & Lomb, Rochester, NY). Refraction was measured with a 3.5 V Keeler streak retinoscope (Keeler Instruments Inc., Windsor, Hertfordshire, UK) to the nearest 0.5 D. Refractive data were averaged for the two principal meridians and expressed as the mean spherical equivalent after correcting for the working distance (33 cm) and the vertex distance (5 mm). However, the small eye artifact 39 was not corrected for. Refractive measures were conducted separately by two experimenters. 
Ultrasonography was carried out using a 10 MHz probe driven by a pulser-receiver (Panametrics, Waltham, MA) and data acquisition controlled by a personal computer (Microsoft Windows XP; Microsoft Corporation, Redmond, WA) running a custom designed LabView program (National Instruments Corporation, Austin, TX). The probe contacted the eye through a saline interface, and was positioned with an x, y, z micro manipulator (World Precision Instruments, Sarasota, FL) to measure along the visual axis. Data consisted of six separate measurements, each the average of 20 incoming waveforms. Data were converted from time to distance using previously published velocity constants for the various ocular tissues 36 and then analyzed with the LabView software. Measures of anterior chamber depth (front of cornea to front of lens), lens thickness, vitreous chamber depth (VCD) (back of lens to retinal/vitreous interface), and retina plus choroid thickness (front of retina to back of choroid) were analyzed from the ultrasound traces. Axial length was reported as the sum of anterior chamber depth, lens thickness, and VCD. 
Tissue Processing and Histology Sectioning
After final measurements were completed, both eyes (the treated and the control eye) were enucleated and hemisected into anterior and posterior segments, followed by immediate fixation into paraformaldehyde (PFA)/glutaraldehyde (GA) (1% PFA and 2.5% GA in 0.1M phosphate buffer, pH 7.4). After 24 hours, tissues were rinsed using 0.1 M phosphate buffer and samples of the retinal/choroid/sclera complex were dissected. These tissue samples were first dehydrated using ascending concentrations of methanol (75%, 85%, 95%, and 100%), followed by 100% acetone for 5 minutes. Finally, tissues were embedded in Epon 18 resin (ProSciTech, Thuringowa, Qld, Australia). Sections were cut using an ultramicrotome (Reichert-Jung Ultracut; Reichert Technologies, Depew, NY), deplasticized, stained with toluidine blue solution, and cover slipped. Slides were visualized and photographed using a Zeiss photomicroscope (Carl Zeiss Axioplan 2; Carl Zeiss, Jena, Germany) and Kodak Megaplus camera (Axiocam; Carl Zeiss). 
Data Analyses
Data were entered into an Excel spreadsheet and analyzed using graphpad prism 5 (Graphpad Software, Inc., La Jolla, CA). Differences between right and left eyes (at the start of MD and LIM study), and treated and control eyes were assessed using paired t-tests. Comparisons within the three treatment groups were assessed using one-way ANOVA with Newman-Keuls post hoc test; differences were considered significant when P was less than 0.05. Comparison of the two groups in MT7 lens-defocus study and the vehicle-MD and Normal-MD comparisons were assessed using unpaired t-tests. 
Results
Injection Effect on Myopia Development
The largest amount of induced myopia (Fig. 1A) was found in the Normal-MD group that did not undergo scleral puncture for intravitreal injections. After 5 days of MD −7.5 ± 3.4 D (mean ± SD) of myopia was induced. Animals undergoing vehicle injection developed less myopia over the same 5 day period (−4.3 ± 1.4 D). This difference between the Normal-MD and the vehicle-MD groups was significant (P < 0.05). In keeping with the reduced myopia in the vehicle-MD group, the vitreous chamber elongation was less (Table 1 and Fig. 1B; P < 0.05) as was the change in axial length (Fig. 1C), however, axial length differences did not reach significance (P = 0.07). The combination of both the needle puncture and saline vehicle reduced myopia development due to reduced elongation of the vitreous chamber. This vehicle injection effect was of similar magnitude in mammalian eyes as that previously found in chick eyes 27 (42.9% vs. 40.9%) using the same injection technique. Although the present study did not run an additional control group that underwent scleral puncture alone, with no vehicle injection, due to the limited number of tree shrews produced by the breeding colony, such a control group was run in our recent chick experiments and demonstrated that most of the difference in induced myopia between the Normal-MD group and vehicle-MD group is accounted for by the needle puncture, with 76% of the reduction in myopia caused by needle puncture alone. 27  
Figure 1. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length in Normal-MD animals and Vehicle-MD (PBS) animals after 5 days of monocular deprivation of vision. Daily intravitreal injections produced a reduction in deprivation-induced myopia due to reduced vitreous chamber elongation. *P < 0.05. Error bars denote ±1 SD.
Figure 1. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length in Normal-MD animals and Vehicle-MD (PBS) animals after 5 days of monocular deprivation of vision. Daily intravitreal injections produced a reduction in deprivation-induced myopia due to reduced vitreous chamber elongation. *P < 0.05. Error bars denote ±1 SD.
Table 1. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT3, MT7, Vehicle and Normal MD groups for MD Study
Table 1. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT3, MT7, Vehicle and Normal MD groups for MD Study
Groups Days & Sample Sizes Refraction (D) (Mean ± SEM) Corneal Radius (mm) (Mean ± SEM) Anterior Chamber Depth (mm) (Mean ± SEM) Lens Thickness (mm) (Mean ± SEM) Vitreous Chamber Depth (mm) (Mean ± SEM) Retina + Choroid Thickness (mm) (Mean ± SEM) Axial Length (mm) (Mean ± SEM)
Eye T C T C T C T C T C T C T C
MT3 MD group Day 0
(n = 6)
9.9 ± 0.5 9.7 ± 0.5 3.32 ± 0.03 3.34 ± 0.03 0.98 ± 0.01 0.99 ± 0.02 3.19 ± 0.03 3.16 ± 0.02 2.80 ± 0.02 2.81 ± 0.02 0.22 ± 0.01 0.22 ± 0.01 6.97 ± 0.03 6.96 ± 0.02
Day 5
(n = 6)
8.2 ± 0.5 8.9 ± 0.4 3.42 ± 0.03 3.40 ± 0.02 1.01 ± 0.02 1.00 ± 0.02 3.21 ± 0.02 3.23 ± 0.03 2.83 ± 0.02 2.82 ± 0.03 0.22 ± 0.01 0.22 ± 0.01 7.05 ± 0.02 7.06 ± 0.03
Day 12
(n = 6)
1.2 ± 1.4 7.3 ± 0.3 3.47 ± 0.02 3.44 ± 0.03 1.02 ± 0.01 1.02 ± 0.02 3.30 ± 0.03 3.30 ± 0.03 2.94 ± 0.02 2.81 ± 0.03 0.22 ± 0.00 0.23 ± 0.01 7.26 ± 0.02 7.13 ± 0.03
MT7 MD group Day 0
(n = 6)
10.2 ± 0.5 10.4 ± 0.5 3.38 ± 0.02 3.41 ± 0.02 1.00 ± 0.02 0.99 ± 0.01 3.23 ± 0.03 3.22 ± 0.02 2.81 ± 0.02 2.81 ± 0.03 0.22 ± 0.00 0.22 ± 0.01 7.04 ± 0.03 7.03 ± 0.04
Day 5
(n = 6)
6.6 ± 0.7 7.4 ± 0.4 3.36 ± 0.02 3.34 ± 0.02 0.99 ± 0.03 0.97 ± 0.02 3.31 ± 0.04 3.30 ± 0.04 2.78 ± 0.03 2.77 ± 0.02 0.23 ± 0.01 0.22 ± 0.00 0.22 ± 0.01 7.04 ± 0.02
Day 12
(n = 6)
0.7 ± 0.7 8.1 ± 0.4 3.46 ± 0.04 3.47 ± 0.03 1.02 ± 0.01 1.02 ± 0.01 3.35 ± 0.03 3.36 ± 0.03 2.91 ± 0.02 2.76 ± 0.01 0.21 ± 0.00 0.22 ± 0.01 7.28 ± 0.03 7.14 ± 0.04
Vehicle MD group Day 0
(n = 6)
11.4 ± 0.3 11.3 ± 0.1 3.36 ± 0.03 3.38 ± 0.05 1.03 ± 0.02 1.02 ± 0.01 3.12 ± 0.02 3.16 ± 0.02 2.84 ± 0.03 2.84 ± 0.01 0.22 ± 0.00 0.22 ± 0.00 7.00 ± 0.05 7.03 ± 0.05
Day 5
(n = 6)
6.4 ± 0.6 10.7 ± 0.1 3.43 ± 0.03 3.43 ± 0.02 1.03 ± 0.02 1.03 ± 0.02 3.25 ± 0.02 3.23 ± 0.01 2.84 ± 0.02 2.79 ± 0.01 0.23 ± 0.00 0.24 ± 0.01 7.12 ± 0.04 7.05 ± 0.03
Day 12
(n = 6)
0.6 ± 1.6 8.6 ± 0.4 3.49 ± 0.02 3.51 ± 0.01 1.05 ± 0.02 1.03 ± 0.02 3.32 ± 0.03 3.34 ± 0.03 2.95 ± 0.03 2.79 ± 0.03 0.22 ± 0.01 0.23 ± 0.01 7.32 ± 0.04 7.16 ± 0.02
Normal MD group Day 0
(n = 5)
9.0 ± 0.5 8.6 ± 0.5 Data not collected 1.06 ± 0.02 1.07 ± 0.01 3.12 ± 0.02 3.12 ± 0.02 2.85 ± 0.02 2.85 ± 0.02 0.23 ± 0.01 0.23 ± 0.01 7.03 ± 0.05 7.04 ± 0.04
Day 5
(n = 5)
0.7 ± 1.4 8.1 ± 0.4 1.06 ± 0.01 1.06 ± 0.02 3.23 ± 0.03 3.24 ± 0.04 2.95 ± 0.02 2.81 ± 0.02 0.22 ± 0.01 0.23 ± 0.01 7.24 ± 0.04 7.11 ± 0.04
Day 12
(n = 5)
−2.4 ± 1.0 8.9 ± 0.6 1.07 ± 0.02 1.10 ± 0.02 3.29 ± 0.03 3.26 ± 0.02 3.02 ± 0.02 2.79 ± 0.03 0.21 ± 0.01 0.21 ± 0.01 7.38 ± 0.04 7.16 ± 0.02
Effect of Highly Selective Muscarinic Antagonists on Deprivation Myopia
A significant difference in induced myopia was found between the Vehicle-MD, MT3-MD, and MT7-MD groups (treated-control eye; Vehicle-MD; −4.3 ± 0.6 D; MT3-MD; −0.70 ± 0.2 D and MT7-MD; −0.70 ± 0.4 D; Mean ± SEM; ANOVA; P < 0.0001; Fig. 2A). Multiple comparison testing revealed significant differences in refractive error between the vehicle-MD group and the MT3-MD and MT7-MD groups (Newman-Keuls post hoc test; P < 0.001; P < 0.001; Fig. 2A). There was no significant refractive error difference between MT3-MD and MT7-MD (Newman-Keuls post hoc test; P > 0.05; Fig. 2A). 
Figure 2. 
 
(A) Ocular refraction, (B) axial length, and (C) combined retina plus choroid thickness following 5 days of Vehicle, MT3, or MT7 treatment. MT3-MD and MT7-MD groups had significantly less deprivation-induced myopia compared with the vehicle-MD group. ***P < 0.001. Error bars denote ±1 standard error of the mean.
Figure 2. 
 
(A) Ocular refraction, (B) axial length, and (C) combined retina plus choroid thickness following 5 days of Vehicle, MT3, or MT7 treatment. MT3-MD and MT7-MD groups had significantly less deprivation-induced myopia compared with the vehicle-MD group. ***P < 0.001. Error bars denote ±1 standard error of the mean.
Overall differences in VCD across the three treatment groups were found not to be statistically significant (treated-control eye; vehicle-MD; 0.06 ± 0.02 mm; MT3-MD; 0.01 ± 0.02 mm MT7-MD; 0.01 ± 0.02 mm; ANOVA; P > 0.05; Table 1). However, schematic modeling of VCD changes showed that they accounted for at least 70% of the refractive change. Differences in axial length between the three treatment groups were found to reach significance at the P equals 0.05 level (treated-control eye; vehicle-MD; 0.07 ± 0.03 mm; MT3-MD; −0.01 ± 0.03 mm and MT7-MD; 0.03 ± 0.02 mm; ANOVA; P = 0.05; Fig. 2B). Multiple comparison testing (Newman-Keuls post hoc test) revealed a significant difference between Vehicle-MD group and the MT3-MD group (P = 0.05), but not between the Vehicle-MD group and the MT7-MD group (P > 0.05), or the MT3-MD and MT7-MD groups (P > 0.05).There were no significant differences in any other ocular biometric parameter measured (retina + choroid thickness [Fig. 2C], corneal curvature, anterior chamber depth, lens thickness; data not shown). 
Refractive and Biometric Measures in MT3, MT7, and Vehicle MD Groups after 12 Days of Deprivation Myopia (Drug and Vehicle Injections Ceased after 5 Days)
After injections were ceased at 5 days, monocular deprivation of vision was continued for Normal-MD, vehicle-MD, MT3-MD, and MT7-MD groups in the treated eye for another 7 days (in total 12 days). This was to assess whether muscarinic antagonist effects were sustained after treatment ceased, or conversely, whether the rate of eye growth was accelerated in previously treated MT3-MD and MT7-MD animals compared with the vehicle-MD animals, as found after cessation of atropine treatment in humans. 40 There were no significant differences in refractive error between the three injection treatment groups (treated-control eye; vehicle-MD; −8.1 ± 1.6 D; MT3-MD; −6.2 ± 1.3 D and MT7-MD; −7.4D ± 0.8 D; Mean ± SEM; ANOVA; P > 0.05; Fig. 3A). However, when the Normal-MD group was included in analysis there was a significant difference found between Normal-MD (−11.3 ± 1.2 D) and the MT3-MD group (Newman-Keuls post hoc test; P < 0.05; Fig. 3A). Similarly, VCD elongation was not significantly different between the three injected MD groups after 12 days (treated-control eye; vehicle-MD; 0.16 ± 0.03 mm; MT3-MD; 0.13 ± 0.03 mm and MT7-MD; 0.15 ± 0.02 mm; ANOVA; P > 0.05; Fig. 3B). Again when the Normal-MD was included in analysis a significant difference between Normal-MD (0.23 ± 0.02 mm) and the MT3-MD group was found (Newman-Keuls post hoc test; P < 0.05; Fig. 3B). There was no significant difference observed in axial length difference between groups (ANOVA; P > 0.05; Fig. 3C) or in corneal radius, anterior chamber depth, or lens thickness at any of the measurement times across any of the groups (P > 0.05; data not shown). 
Figure 3. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length following 12 days of MD (another 7 days continued with deprivation after the termination of injections). Refractive error and VCD changes in Normal-MD group were significantly different to MT3-MD following 12 days. *P < 0.05. Error bars denote ±1 standard error of the mean.
Figure 3. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length following 12 days of MD (another 7 days continued with deprivation after the termination of injections). Refractive error and VCD changes in Normal-MD group were significantly different to MT3-MD following 12 days. *P < 0.05. Error bars denote ±1 standard error of the mean.
Effect of M1 Muscarinic Antagonist MT7 on Negative Lens-Induced Myopia following 10 Days of Treatment
Following 10 days of MT7 treatment there was a significant reduction in the development of lens-induced myopia compared with vehicle treatment (treated-control eye; vehicle-LIM; −4.6 ± 0.5 D and MT7-LIM; −1.5 ± 0.9 D; Mean ± SEM; unpaired t-test; P < 0.05; Fig. 4A). The VCD showed a significant elongation in the vehicle-LIM group compared with the MT7-LIM group (treated-control eye; vehicle-LIM; 0.07 ± 0.01 mm and MT7-LIM; 0.01 ± 0.02 mm; unpaired t-test; P < 0.05; Fig. 4B). The combined retina plus choroid thickness, axial length, corneal curvature, anterior chamber depth, and lens thickness (data not shown) were not significantly different between the vehicle-LIM and MT7-LIM groups (P > 0.05; Table 2). 
Figure 4. 
 
(A) Ocular refraction and (B) VCD differences between MT7 and vehicle groups following 10 days of negative lens treatment. MT7 treatment significantly inhibited lens-induced myopia compared with the vehicle group. *P < 0.05. Error bars denote ±1 standard error of the mean.
Figure 4. 
 
(A) Ocular refraction and (B) VCD differences between MT7 and vehicle groups following 10 days of negative lens treatment. MT7 treatment significantly inhibited lens-induced myopia compared with the vehicle group. *P < 0.05. Error bars denote ±1 standard error of the mean.
Table 2. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT7 and Vehicle Groups for 10 Days LIM Study
Table 2. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT7 and Vehicle Groups for 10 Days LIM Study
Treat- ment Groups Time Points and Sample Sizes Refraction (D) (Mean ± SEM) Corneal Radius (mm) (Mean ± SEM) Anterior Chamber Depth (mm) (Mean ± SEM) Lens Thickness (mm) (Mean ± SEM) Vitreous Chamber Depth (mm) (Mean ± SEM) Retina + Choroid Thickness (mm) (Mean ± SEM) Axial Length (mm) (Mean ± SEM)
Eye T C T C T C T C T C T C T C
MT7 lens group Day 0
(n = 5)
12.8 ± 0.2 12.5 ± 0.2 Not done 1.03 ± 0.04 1.04 ± 0.04 3.17 ± 0.03 3.14 ± 0.04 2.77 ± 0.03 2.76 ± 0.04 0.24 ± 0.02 0.24 ± 0.04 6.98 ± 0.03 6.95 ± 0.04
(T-C) 0.2 ± 0.2 –0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01 0.00 ± 0.00 0.03 ± 0.01
Day 10
(n = 5)
7.7 ± 0.2 9.2 ± 0.2 3.33 ± 0.05 3.32 ± 0.04 1.01 ± 0.05 1.02 ± 0.04 3.31 ± 0.03 3.29 ± 0.04 2.73 ± 0.04 2.73 ± 0.04 0.23 ± 0.02 0.23 ± 0.02 7.05 ± 0.05 7.04 ± 0.04
(T-C) –1.5 ± 0.9 0.01 ± 0.02 –0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.02 0.01 ± 0.01 0.01 ± 0.03
Vehicle lens group Day 0
(n = 5)
13.6 ± 0.2 13.5 ± 0.1 Not done 1.01 ± 0.04 1.01 ± 0.04 3.16 ± 0.06 3.17 ± 0.06 2.80 ± 0.05 2.77 ± 0.06 0.23 ± 0.02 0.23 ± 0.02 6.97 ± 0.09 6.94 ± 0.08
(T-C) 0.1 ± 0.3 0.00 ± 0.02 –0.01 ± 0.02 0.03 ± 0.01 0.00 ± 0.01 0.02 ± 0.02
Day 10
(n = 5)
6.7 ± 0.3 11.3 ± 0.3 3.37 ± 0.05 3.38 ± 0.05 1.02 ± 0.04 1.01 ± 0.03 3.29 ± 0.04 3.30 ± 0.03 2.79 ± 0.08 2.71 ± 0.09 0.23 ± 0.02 0.23 ± 0.02 7.09 ± 0.09 7.02 ± 0.10
(T-C) –4.6 ± 0.5 –0.01 ± 0.01 0.01 ± 0.01 –0.01 ± 0.01 0.07 ± 0.01 0.00 ± 0.01 0.07 ± 0.02
Daily Rate of Refractive Change during Drug Treatment
Animals treated with MT3 (MT3-MD) had a rate of change of 0.19 D/day, MT7-MD treated animals changed 0.12 D/day, while the vehicle-MD animals had a rate of change of 0.87 D/day, the daily rates of change were significantly different (ANOVA, P < 0.001; Fig. 5A). Multiple comparison testing revealed that the rate of refractive change was significantly faster in vehicle-MD animals than both MT3-MD and MT7-MD animals (P < 0.01). The rate of refractive change in the 5 day treatment period was significantly slower in vehicle-MD treated animals than Normal-MD (1.58 D/day) animals (P < 0.01), highlighting the effect of the daily injection procedure on induced myopia. There was no significant difference in the rate of refractive change between MT3-MD and MT7-MD during the 5 days of drug treatment. In the negative LIM study, animals treated with MT7 over 10 days had a rate of change of 0.17 D/day compared with a rate of change of 0.47 D/day in vehicle-LIM animals over the same 10 day period (unpaired t-test; P < 0.05). 
Figure 5. 
 
Rate of change of (A) refraction, (B) VCD, and (C) axial length during the treatment period from 0 to 5 days and also from 6 to 12 days after drug treatments and intravitreal injections were ceased at day 5. Error bars denote ±1 standard error of the mean.
Figure 5. 
 
Rate of change of (A) refraction, (B) VCD, and (C) axial length during the treatment period from 0 to 5 days and also from 6 to 12 days after drug treatments and intravitreal injections were ceased at day 5. Error bars denote ±1 standard error of the mean.
Histology Examination following MT3, MT7, and Vehicle Injections in the Tree Shrew
To rule out the possibility that nanomolar doses of MT3 and MT7 affected structural integrity, histologic sections of the retina, choroids, and sclera were collected. No signs of structural abnormality (cellular edema or damage) were observed (data not shown) indicating MT3 and MT7 inhibition of myopia was not caused by any toxic effects on the retina or other ocular structures. 
Discussion
Based on a recent study in chicks, we anticipated that the M4 muscarinic antagonist (MT3) would be effective at inhibiting deprivation-induced myopia in tree shrews, while it was unknown if the M1 muscarinic antagonist (MT7) would inhibit myopia in a mammal. The current study demonstrates that the highly selective muscarinic antagonists of M4 (MT3) and M1 (MT7) receptors are effective at inhibiting deprivation-induced myopia in tree shrews. The present study carried out intravitreal injections in both eyes to control for the surgical intervention, thus, enabling evaluation of the actual drug treatment effect. The present study also demonstrated that eyes that received MT3 and MT7 to inhibit myopia were still susceptible to deprivation-induced myopia (after the termination of intravitreal injections) indicating no functional damage to the signaling pathways of deprivation myopia. In addition, the present study demonstrated that MT7 was also effective at inhibiting negative lens-induced myopia in tree shrew. 
The present results demonstrated approximately 80% inhibition of deprivation-induced myopia with MT3-MD (−73% ± 9%) and MT7-MD (−88% ± 13%) treatment, when compared with vehicle-MD animals over a 5 day treatment period. MT7 inhibited −66 ± 20% of negative lens-induced myopia in tree shrew over a 10 day treatment period. The major structural parameter contributing to the inhibition of myopia was reduced vitreous chamber elongation, which was 67% less in both MT3-MD and MT7-MD animals when compared with vehicle-MD animals. The vitreous chamber elongation was 86% less in MT7-LIM animals compared with vehicle-LIM controls. Unlike inhibition of myopia in chicks where choroidal thickening plays a significant role, 27,41 choroidal thickness changes in mammalian models of myopia are substantially smaller in magnitude, with previous studies in tree shrews reporting changes of approximately 20 μm. 38,42 In the present study, although the A-scan ultrasonography resolution was not sufficient to consistently differentiate just choroidal thickness due to occasional poor definition of the retina-choroid interface, retina plus choroid thickness was measurable. Retina plus choroid measures (Table 1 and Fig. 2C) show that in both MT3-MD and MT7-MD treated animals there was a slight thickening, while in Normal-MD and vehicle-MD animals a slight thinning occurred. Although these changes were in the expected directions for a retinal or choroidal based effect, they were not significantly different from the fellow eye values (< 10 μm) and made little contribution to the axial or refractive differences between groups. 
The present findings further our understanding of which muscarinic receptors are involved in atropine's control of myopia in human and mammalian models of refractive development. A previous study in chicks by our laboratory 27 demonstrated that the M4 antagonist, MT3, was effective in inhibiting experimentally-induced myopia, while the M1 antagonist, MT7, was ineffective. As it has previously been demonstrated that the chick does not possess an M1 receptor gene, 20 this finding was confirmatory of this fact and demonstrates that the partially selective M1/M4 muscarinic antagonist, pirenzepine, must inhibit myopia in the chick via an M4 receptor pathway. The present study adds to this work by demonstrating in a mammalian model of refractive development, which possesses all five muscarinic receptors, similar to humans, that the M1 receptor pathway is also involved in muscarinic antagonist inhibition of myopia. The findings from these two recent studies are complimentary, and add clarity to interpretation of previous studies using partially selective muscarinic antagonists to control myopia. 
Changes in Rate of Myopia Development Post Drug Treatment
It has been demonstrated in a study on Singaporean children given atropine treatment to slow myopia progression, that when treatment ceased after 24 months, the previously treated eyes then elongated at a faster rate, and, consequently, myopia progression was greater than the fellow eye over the following 12 months. 40 It can be seen from Figure 5A that the rate of change in myopia development was significantly less in the MT3-MD and MT7-MD groups compared with vehicle-MD and Normal-MD groups during the treatment period (analysis of regression slopes F = 17.2; P < 0.0001). However, once drug treatment was ceased, the rate of myopia development in MT3-MD and MT7-MD animals was now greater than in vehicle-MD or Normal-MD animals (Fig. 5A). Although there was no significant difference in the regression slopes from day 6 to 12 (F = 0.96; P > 0.05), the intercepts were significantly different (F = 12.6; P < 0.0001). Thus, similar to atropine use in humans, the previously treated eyes of MT3-MD and MT7-MD animals were more sensitive to the myopiagenic signal, in this case deprivation of pattern vision. The rates of change for axial dimensions (VCD and axial length) were confirmatory of refractive changes with similar statistical significance (Figs. 5B, 5C). Why this super sensitivity occurs in previously treated animals is uncertain, but may relate to a hypersensitivity of previously blocked muscarinic receptors to the endogenous ligand and, thus, accelerate ocular elongation and myopia development. 
Evidence for a Receptoral-Based Mechanism of Muscarinic Antagonist Control of Myopia
Previous studies on both humans and animals have employed high doses of either the broadband antagonist atropine or the partially selective M1/M4 muscarinic antagonist, pirenzepine, in order to inhibit the development of myopia. 35,7,9,12 Previous studies, including our own laboratory, have employed doses of atropine as much as 10,000 times or higher than theoretically required for binding at the M4 receptors or 17,500 times or higher than required for binding at the M1 receptor 5,43,44 based on published receptor affinity values. 45 Consequently, a number of studies have questioned whether muscarinic antagonist drug effects on myopia inhibition are working via retinal receptors and proposed either a nonreceptoral mechanism of action or at muscarinic receptors in the sclera. 16,46,47 The present study utilized highly selective muscarinic antagonists (MT3 and MT7) in an animal model that has both M4 and M1 receptors, and used doses to provide physiologically relevant concentrations at the receptor binding site for ocular-based receptors, specifically retinal-based receptors. The finding that these highly selective antagonists inhibited experimentally-induced axial myopia gives much greater confidence in the hypothesis that muscarinic antagonist inhibition of myopia is initiated via a receptoral-based mechanism. It is well established that MT3 and MT7 are highly selective allosteric antagonists at the M4 and M1 muscarinic receptors, respectively. 26,48,49 These antagonists bind to the allosteric binding site and bring a change in the affinity of the receptor for the endogenous orthosteric ligand (in this case acetylcholine). The use of physiological concentrations is critical for appropriate conformation changes at the respective receptor. For example, alcuronium has a biphasic effect on binding of 3H-NMS to M2 receptors, where low concentrations (10 μM) have agonist effects and the high concentrations (1 mM) are antagonistic. 50 In addition, low concentrations (1–10 μM) of gallamine are shown to increase the dissociation of [3H] quinuclidinyl benzilate at M1 and M2 receptors, while 1 mM gallamine slows it. 51 Although to our knowledge there is no study to date that has reported the above biphasic effect for MT3 or MT7 at their receptor subtypes, as noted the use of high concentrations can lead to different pharmacological drug receptor effects than when using physiological concentrations. The in vitro affinity constants indicate that MT7 has its effects at the M1 receptor at doses of approximately 0.1 nM and for the M4 receptors at doses of approximately 2 μM. The present study employed a calculated maximum concentration of 220 nM of MT3 and MT7 at the retinal/vitreous interface. However, the concentration at the retinal, choroidal, or scleral muscarinic receptors would be less than 220 nM, due to passage through ocular tissue barriers. Thus, it is highly likely that the inhibition of myopia, due to reduced vitreous chamber elongation, by MT3 and MT7 was caused by pharmacological actions at their respective receptor targets with little or no cross reactivity with other muscarinic receptors. 
Evidence for a Retinal Site of Action for Muscarinic Antagonism of Myopia
Although there is strong evidence that the signaling cascade initiated by either deprivation myopia or negative lens-induced myopia begins at the retinal level with, for example, sign of defocus changes in ZENK (zif268, EGR-1, NGFI-A, and krox24) levels of amacrine cells, 52,53 this does not automatically mean that muscarinic inhibition of myopia occurs at a retinal site of action. As stated above, in mammalian species, the retina, choroid, and sclera have all five muscarinic receptors present, and as such topically or intravitreally applied muscarinic antagonists could be binding to receptors in any of the posterior segment tissues. A previous study 57 of ocular drug distribution found that levels from a single intravitreal injection of 700 ug 3HPirenzepine (the minimum dose needed to completely inhibit myopia in a chick) delivered supra threshold concentrations of the drug in the retina, choroid, and scleral tissues. It is likely that doses of 1% atropine and 2% pirenzepine, both of which have been used in human studies, would also provide supra threshold doses in all posterior segment tissues. In a recently published study of reduced doses of atropine on myopia inhibition in Singaporean children, it was found that 0.01% was nearly as effective as 1% atropine in slowing myopia progression. 54 The use of such high doses to inhibit myopia had led to the suggestion that muscarinic antagonists work at a nonretinal site, most likely the sclera, in preventing the axial elongation of the eye. This has been supported by studies that have directly applied muscarinic antagonists on scleral fibroblasts either in cell culture (mouse) or eyecup preparations (chick) and reported reductions in either collagen synthesis or glycosaminoglycan synthesis, respectively, 17,55 although use of a highly selective muscarinic antagonist (MT3) at nanomolar doses did not influence glycosaminoglycan synthesis in chick scleral buttons. 56 The current study employed much lower doses of MT3 and MT7, in the nanomolar range at the vitreal/retinal interface, which are close to M4 and M1 receptor affinity constants, and successfully inhibited deprivation and lens-induced myopia (MT7). This supports a retinal site of action as the concentration at choroidal or scleral muscarinic receptors would likely be in the picomolar range, based on previous drug delivery studies. 57 More direct evidence for a retinal (or choroidal) site(s) of action by MT3 comes from recent findings that MT3 inhibition of myopia in chicks not only prevents axial elongation of the eye in myopia, but also prevents the choroidal thinning normally associated with deprivation-induced myopia in chicks, thus, inhibiting a myopic response of the tissue prior to reaching the sclera. 27 In addition, Bitzer and colleagues 53 proposed a retinal site of action for MT3, as it caused a significant increase in the synthesis of retinal ZENK in glucagon amacrine cells of the chick retina and it is known that increased ZENK expression suppresses axial eye growth. 
The M1 and M4 muscarinic receptors have different secondary signaling pathways, with M1 linked to mobilization of intracellular calcium through activation of the phospholipase Cβ, and M4 linked to the inhibition of adenylyl cyclase. Some insight on how these signaling pathways might interact, or converge, in downstream signaling is provided by recent findings that report that the inhibitory effect of both MT3 and MT7 on myopia development can be prevented by co-application of the dopamine D2 antagonist spiperone (Arumugam B and McBrien N, IOVS 2010;51:ARVO E-Abstract 1195; Arumugam B and McBrien N, IOVS 2012;53:ARVO E-Abstract 3431). Research on the interaction of M1 and M4 receptor pathways has been very active in the last few years due to their involvement in cognition and particularly in neurodegenerative conditions such as Alzheimer's and Parkinson's disease and schizophrenia. 58,59 More specifically, cholinergic activity has been implicated in the etiology of schizophrenia, and M1 and M4 muscarinic receptors have been strongly implicated as potential targets for ameliorating the cognitive and behavioral deficits of the disease. 58 In Alzheimer's disease there is major disturbance of cholinergic signaling in the central nervous system where M1 and M4 receptor expression predominate and the M1 receptor is considered the primary mediator of cognitive efficacy, while the M4 subtype seems to underlie antipsychotic efficacy. 59 In regard to these neurodegenerative conditions, the aim has been to develop highly selective M1 and M4 agonists and this has led to recent breakthroughs in allosteric M1 and M4 agonists, and positive allosteric modulators that have shown significant improvement in cognition and memory in recent clinical trials. The relevance to the present results is that highly selective muscarinic allosteric drugs are proving to be effective clinical treatment approaches, where previous partially selective muscarinic orthosteric drugs have proved problematic. Of particular interest, with respect to the present study, was that the most effective approaches were found when M1 and M4 therapy was combined, 26,60 such as with the M1/M4 muscarinic agonist xanomeline. 6163 Important next steps in deciphering the mechanism of muscarinic antagonist control of myopia will be (1) to test if combining MT3 and MT7 treatment, at even lower nanomolar concentrations than the present study, provide an additive inhibitory effect preventing axial myopia, without atropine's ocular or systemic side effects, and (2) the interaction of MT3 and MT7 with dopamine receptors, which are also implicated in the control of ocular growth and myopia. 
In summary, both the highly selective muscarinic antagonists MT3 and MT7 are effective at inhibiting deprivation-induced myopia in tree shrews. The highly selective M1 muscarinic antagonist, MT7, is also effective at inhibiting negative lens-induced myopia in this mammalian model. Based on the above findings the inhibition of experimentally-induced myopia by the highly selective allosteric muscarinic antagonists implicates both M4 and M1 receptor signaling pathways in mammals. 
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Footnotes
 Supported by National Health & Medical Research Council of Australia Grant 454602.
Footnotes
 Disclosure: B. Arumugam, None; N.A. McBrien, None
Figure 1. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length in Normal-MD animals and Vehicle-MD (PBS) animals after 5 days of monocular deprivation of vision. Daily intravitreal injections produced a reduction in deprivation-induced myopia due to reduced vitreous chamber elongation. *P < 0.05. Error bars denote ±1 SD.
Figure 1. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length in Normal-MD animals and Vehicle-MD (PBS) animals after 5 days of monocular deprivation of vision. Daily intravitreal injections produced a reduction in deprivation-induced myopia due to reduced vitreous chamber elongation. *P < 0.05. Error bars denote ±1 SD.
Figure 2. 
 
(A) Ocular refraction, (B) axial length, and (C) combined retina plus choroid thickness following 5 days of Vehicle, MT3, or MT7 treatment. MT3-MD and MT7-MD groups had significantly less deprivation-induced myopia compared with the vehicle-MD group. ***P < 0.001. Error bars denote ±1 standard error of the mean.
Figure 2. 
 
(A) Ocular refraction, (B) axial length, and (C) combined retina plus choroid thickness following 5 days of Vehicle, MT3, or MT7 treatment. MT3-MD and MT7-MD groups had significantly less deprivation-induced myopia compared with the vehicle-MD group. ***P < 0.001. Error bars denote ±1 standard error of the mean.
Figure 3. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length following 12 days of MD (another 7 days continued with deprivation after the termination of injections). Refractive error and VCD changes in Normal-MD group were significantly different to MT3-MD following 12 days. *P < 0.05. Error bars denote ±1 standard error of the mean.
Figure 3. 
 
(A) Ocular refraction, (B) VCD, and (C) axial length following 12 days of MD (another 7 days continued with deprivation after the termination of injections). Refractive error and VCD changes in Normal-MD group were significantly different to MT3-MD following 12 days. *P < 0.05. Error bars denote ±1 standard error of the mean.
Figure 4. 
 
(A) Ocular refraction and (B) VCD differences between MT7 and vehicle groups following 10 days of negative lens treatment. MT7 treatment significantly inhibited lens-induced myopia compared with the vehicle group. *P < 0.05. Error bars denote ±1 standard error of the mean.
Figure 4. 
 
(A) Ocular refraction and (B) VCD differences between MT7 and vehicle groups following 10 days of negative lens treatment. MT7 treatment significantly inhibited lens-induced myopia compared with the vehicle group. *P < 0.05. Error bars denote ±1 standard error of the mean.
Figure 5. 
 
Rate of change of (A) refraction, (B) VCD, and (C) axial length during the treatment period from 0 to 5 days and also from 6 to 12 days after drug treatments and intravitreal injections were ceased at day 5. Error bars denote ±1 standard error of the mean.
Figure 5. 
 
Rate of change of (A) refraction, (B) VCD, and (C) axial length during the treatment period from 0 to 5 days and also from 6 to 12 days after drug treatments and intravitreal injections were ceased at day 5. Error bars denote ±1 standard error of the mean.
Table 1. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT3, MT7, Vehicle and Normal MD groups for MD Study
Table 1. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT3, MT7, Vehicle and Normal MD groups for MD Study
Groups Days & Sample Sizes Refraction (D) (Mean ± SEM) Corneal Radius (mm) (Mean ± SEM) Anterior Chamber Depth (mm) (Mean ± SEM) Lens Thickness (mm) (Mean ± SEM) Vitreous Chamber Depth (mm) (Mean ± SEM) Retina + Choroid Thickness (mm) (Mean ± SEM) Axial Length (mm) (Mean ± SEM)
Eye T C T C T C T C T C T C T C
MT3 MD group Day 0
(n = 6)
9.9 ± 0.5 9.7 ± 0.5 3.32 ± 0.03 3.34 ± 0.03 0.98 ± 0.01 0.99 ± 0.02 3.19 ± 0.03 3.16 ± 0.02 2.80 ± 0.02 2.81 ± 0.02 0.22 ± 0.01 0.22 ± 0.01 6.97 ± 0.03 6.96 ± 0.02
Day 5
(n = 6)
8.2 ± 0.5 8.9 ± 0.4 3.42 ± 0.03 3.40 ± 0.02 1.01 ± 0.02 1.00 ± 0.02 3.21 ± 0.02 3.23 ± 0.03 2.83 ± 0.02 2.82 ± 0.03 0.22 ± 0.01 0.22 ± 0.01 7.05 ± 0.02 7.06 ± 0.03
Day 12
(n = 6)
1.2 ± 1.4 7.3 ± 0.3 3.47 ± 0.02 3.44 ± 0.03 1.02 ± 0.01 1.02 ± 0.02 3.30 ± 0.03 3.30 ± 0.03 2.94 ± 0.02 2.81 ± 0.03 0.22 ± 0.00 0.23 ± 0.01 7.26 ± 0.02 7.13 ± 0.03
MT7 MD group Day 0
(n = 6)
10.2 ± 0.5 10.4 ± 0.5 3.38 ± 0.02 3.41 ± 0.02 1.00 ± 0.02 0.99 ± 0.01 3.23 ± 0.03 3.22 ± 0.02 2.81 ± 0.02 2.81 ± 0.03 0.22 ± 0.00 0.22 ± 0.01 7.04 ± 0.03 7.03 ± 0.04
Day 5
(n = 6)
6.6 ± 0.7 7.4 ± 0.4 3.36 ± 0.02 3.34 ± 0.02 0.99 ± 0.03 0.97 ± 0.02 3.31 ± 0.04 3.30 ± 0.04 2.78 ± 0.03 2.77 ± 0.02 0.23 ± 0.01 0.22 ± 0.00 0.22 ± 0.01 7.04 ± 0.02
Day 12
(n = 6)
0.7 ± 0.7 8.1 ± 0.4 3.46 ± 0.04 3.47 ± 0.03 1.02 ± 0.01 1.02 ± 0.01 3.35 ± 0.03 3.36 ± 0.03 2.91 ± 0.02 2.76 ± 0.01 0.21 ± 0.00 0.22 ± 0.01 7.28 ± 0.03 7.14 ± 0.04
Vehicle MD group Day 0
(n = 6)
11.4 ± 0.3 11.3 ± 0.1 3.36 ± 0.03 3.38 ± 0.05 1.03 ± 0.02 1.02 ± 0.01 3.12 ± 0.02 3.16 ± 0.02 2.84 ± 0.03 2.84 ± 0.01 0.22 ± 0.00 0.22 ± 0.00 7.00 ± 0.05 7.03 ± 0.05
Day 5
(n = 6)
6.4 ± 0.6 10.7 ± 0.1 3.43 ± 0.03 3.43 ± 0.02 1.03 ± 0.02 1.03 ± 0.02 3.25 ± 0.02 3.23 ± 0.01 2.84 ± 0.02 2.79 ± 0.01 0.23 ± 0.00 0.24 ± 0.01 7.12 ± 0.04 7.05 ± 0.03
Day 12
(n = 6)
0.6 ± 1.6 8.6 ± 0.4 3.49 ± 0.02 3.51 ± 0.01 1.05 ± 0.02 1.03 ± 0.02 3.32 ± 0.03 3.34 ± 0.03 2.95 ± 0.03 2.79 ± 0.03 0.22 ± 0.01 0.23 ± 0.01 7.32 ± 0.04 7.16 ± 0.02
Normal MD group Day 0
(n = 5)
9.0 ± 0.5 8.6 ± 0.5 Data not collected 1.06 ± 0.02 1.07 ± 0.01 3.12 ± 0.02 3.12 ± 0.02 2.85 ± 0.02 2.85 ± 0.02 0.23 ± 0.01 0.23 ± 0.01 7.03 ± 0.05 7.04 ± 0.04
Day 5
(n = 5)
0.7 ± 1.4 8.1 ± 0.4 1.06 ± 0.01 1.06 ± 0.02 3.23 ± 0.03 3.24 ± 0.04 2.95 ± 0.02 2.81 ± 0.02 0.22 ± 0.01 0.23 ± 0.01 7.24 ± 0.04 7.11 ± 0.04
Day 12
(n = 5)
−2.4 ± 1.0 8.9 ± 0.6 1.07 ± 0.02 1.10 ± 0.02 3.29 ± 0.03 3.26 ± 0.02 3.02 ± 0.02 2.79 ± 0.03 0.21 ± 0.01 0.21 ± 0.01 7.38 ± 0.04 7.16 ± 0.02
Table 2. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT7 and Vehicle Groups for 10 Days LIM Study
Table 2. 
 
Ocular Refraction and Structural Dimensions of Treated and Control Eyes of Tree Shrews in the MT7 and Vehicle Groups for 10 Days LIM Study
Treat- ment Groups Time Points and Sample Sizes Refraction (D) (Mean ± SEM) Corneal Radius (mm) (Mean ± SEM) Anterior Chamber Depth (mm) (Mean ± SEM) Lens Thickness (mm) (Mean ± SEM) Vitreous Chamber Depth (mm) (Mean ± SEM) Retina + Choroid Thickness (mm) (Mean ± SEM) Axial Length (mm) (Mean ± SEM)
Eye T C T C T C T C T C T C T C
MT7 lens group Day 0
(n = 5)
12.8 ± 0.2 12.5 ± 0.2 Not done 1.03 ± 0.04 1.04 ± 0.04 3.17 ± 0.03 3.14 ± 0.04 2.77 ± 0.03 2.76 ± 0.04 0.24 ± 0.02 0.24 ± 0.04 6.98 ± 0.03 6.95 ± 0.04
(T-C) 0.2 ± 0.2 –0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01 0.00 ± 0.00 0.03 ± 0.01
Day 10
(n = 5)
7.7 ± 0.2 9.2 ± 0.2 3.33 ± 0.05 3.32 ± 0.04 1.01 ± 0.05 1.02 ± 0.04 3.31 ± 0.03 3.29 ± 0.04 2.73 ± 0.04 2.73 ± 0.04 0.23 ± 0.02 0.23 ± 0.02 7.05 ± 0.05 7.04 ± 0.04
(T-C) –1.5 ± 0.9 0.01 ± 0.02 –0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.02 0.01 ± 0.01 0.01 ± 0.03
Vehicle lens group Day 0
(n = 5)
13.6 ± 0.2 13.5 ± 0.1 Not done 1.01 ± 0.04 1.01 ± 0.04 3.16 ± 0.06 3.17 ± 0.06 2.80 ± 0.05 2.77 ± 0.06 0.23 ± 0.02 0.23 ± 0.02 6.97 ± 0.09 6.94 ± 0.08
(T-C) 0.1 ± 0.3 0.00 ± 0.02 –0.01 ± 0.02 0.03 ± 0.01 0.00 ± 0.01 0.02 ± 0.02
Day 10
(n = 5)
6.7 ± 0.3 11.3 ± 0.3 3.37 ± 0.05 3.38 ± 0.05 1.02 ± 0.04 1.01 ± 0.03 3.29 ± 0.04 3.30 ± 0.03 2.79 ± 0.08 2.71 ± 0.09 0.23 ± 0.02 0.23 ± 0.02 7.09 ± 0.09 7.02 ± 0.10
(T-C) –4.6 ± 0.5 –0.01 ± 0.01 0.01 ± 0.01 –0.01 ± 0.01 0.07 ± 0.01 0.00 ± 0.01 0.07 ± 0.02
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