2011; 17:680-692 <http://www.molvis.org/molvis/v17/a78>
Received 26 January 2011 | Accepted 3 March 2011 | Published 9 March 2011
1Singapore Eye Research Institute, Singapore; 2Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Health Sciences, Singapore; 3Duke-NUS, SRP in Neurosciences and Behavioral Disorders, Singapore
Correspondence to: Dr. R.W. Beuerman, Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore, 168751; Phone: (+65) 6322 4553; FAX: (+65) 6322 4599; email: firstname.lastname@example.org
Purpose: To investigate the effect of atropine on the development of spectacle lens induced myopia in the mouse and to determine if the level of mRNAs for the muscarinic receptor subtypes (M1 - M5) is affected by atropine treatment.
Methods: Experimental myopia was developed in Balb/CJ (BJ) mice by placing −10 diopter spectacle lens on post-natal day 10 over the right eyes of 150 mice (n=10 in each group, 5 repetitions) for six weeks. After 2 weeks of lens wearing, the atropine group received a daily sub-conjunctival injection (10 µl) of 1% atropine sulfate and the saline group received daily 10 µl of 0.9% normal saline for 4 weeks. In addition, myopia was developed in C57BL/6 (B6) mice by placing −10 D spectacle lens on post-natal day 10 over the right eyes of 60 mice (n=10 in each group, 2 repetitions) for six weeks with and without atropine treatment. Refraction and axial length was measured at 2, 4, and 6 weeks after treatments. RT–PCR and northern blots were performed using specific primers for M1-M5, and products sequenced. Real-time PCR was used to quantify message levels.
Results: Axial length of myopic eyes was 111% of their controls without atropine treatment and 103% of controls after atropine (p<0.01). Refraction shifted from myopic to emmetropic after atropine was administered in both pigmented and non-pigmented eyes. Corneal thickness, anterior chamber depth, corneal curvature and retinal thickness were not significantly different with and without atropine treatment (p=0.14). The lens thickness and vitreous chamber depth were significantly reduced after receiving atropine (p<0.05). Real-time PCR showed that message levels for M1, M3, and M4 were upregulated in myopic sclera after atropine treatment, but M2 and M5 showed little change.
Conclusions: The present study shows that 1% atropine reduces myopia progression in both pigmented and non-pigmented mice eyes. Axial length and vitreous chamber depth appear to be the main morphological parameters related to myopia. The results suggest that atropine may act on one or more muscarinic receptors to differentially regulate expression levels of specific receptors.
The cardinal optical characteristic of myopia is axial elongation of the posterior segment of the eye, which is due to scleral growth and remodeling [1-4]. Axial elongation is believed to be mediated by alterations in the connective tissues of the sclera [3,4], part of the collagenous outer tunic of the eye. To date, atropine (a pan-muscarinic antagonist) has proven to be a promising pharmacological agent that significantly reduced the progression of myopia in several clinical trials [5,6] including the ATOM (Atropine Treatment Of Myopia) study that was conducted in our center . In a recent publication we demonstrated that the mouse and human scleral fibroblasts express all five types of muscarinic receptors . However, it remains to be shown that atropine also halts the progression of myopia in the mouse model .
Atropine has been extensively tested in animal models. Suppression of deprivation myopia was studied with atropine treatment in tree shrews , chicks [11,12] and rhesus monkeys . Since the mouse has recently been used as a new model [14,15] to study biologic aspects of myopia, the effects of atropine on experimental myopia are important to examine.
It is reasonable to expect that the mechanism of action of muscarinic receptor antagonists in inhibiting myopia progression to be consistent across species. Although atropine [16,17] and pirenzepine (specific muscarinic receptor 1 (M1) antagonist)  have both been shown to reduce myopia progression via slowing of axial elongation, the exact mechanism is still unknown. It was found that the M1 receptor does not exist in the chick sclera  such evidence implies that muscarinic antagonists which prevents the progression of myopia in the chick either work through another muscarinic receptor subtype or through non-specific or non-receptor mediated mechanisms. It is important to investigate the gene expression pattern during myopic development or progression may offer a productive avenue for future research.
The purpose of this study was to investigate the effect of atropine in a mouse model of experimental myopia and to determine if atropine prevents axial elongation after induction of spectacle lens induced myopia. The experimental myopia was developed in two different strains to determine the effect of spectacle lens induction in pigmented and non-pigmented eyes. Scleral fibroblast message levels for the muscarinic subtypes (M1 - M5) were also analyzed in conjunction with atropine treatment during myopia progression.
Pregnant BJ mice (Mus musculus) and B6 mice were obtained from the animal holding unit of the National University of Singapore. Animals gave birth in our animal holding unit. Naive control animals were housed in groups of 6 while experimental animals were housed individually in standard mouse cages after 28 days of age at 25 °C on a schedule of 12:12 h of light on and off with mouse pellets and water available ad libidum. Approval was obtained from the SingHealth Institutional Animal Care and Use of Committee (IACUC) and all procedures performed in this study complied with the Association of Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmology and Vision Research.
The effects of atropine treatment were examined using spectacle lens-induced myopia and allocated to one of three groups: One group (n=10, 5 batch) received a daily 10 μl of 1% sub-conjunctival injections of atropine sulfate (ATG), and the other group (n=10, 5 batch) received daily 10 μl of 0.9% of sterile normal saline (NSG) as a vehicle, and the third group (n=10, 5 batch) was treated with spectacle lens alone to induce myopia. The right was used as a experimental and left eye was served as contra-lateral control in all groups. The same study was conducted in B6 mice (n=10, 2 batch in each group) with −10 diopter (D) to investigate the effect of strain difference with lens treatment. In all the remaining experiments, BJ treated mice samples were used.
Sub-conjunctival injections were administered to both eyes at the same time each day (approximately 9:00 AM) commencing on the 24th day (2 weeks after initiation of spectacle lens treatment). A compatible level of atropine was determined before the in vivo use in a tissue culture study with mouse scleral fibroblasts . These concentrations (0.01% [147.929 µM], 0.1% [1479.29 µM], 0.5% [7396.45 µM], and 1% [14792.9 µM]) were then tested in vivo in a small pilot study. From our pilot study, we found that 0.5% and 1% atropine significantly reduced the elongation of axial length (data not shown). In this study, we are reporting results from the 1% atropine treatment. The injection (31.5 gauge needle) was performed under sterile conditions and microscopic control. For this procedure, mice were anaesthetized with 0.05–0.1 ml (IP) of a mixture of 0.2 ml 10% ketamine hydrochloride and 0.1 ml 2% xylazine hydrochloride, dissolved in 1.0 ml sterile saline. The eyes were examined daily and no infections were found. This treatment schedule continued for four weeks starting on post-natal day 24 continued until post-natal day 52. All measurements were taken at post-natal day 52, the equivalent of 6 weeks of spectacle lens wear.
Refractions and biometry measurements were recorded every week until the end of the study. Axial length, lens thickness, vitreous chamber depth and corneal diameter were measured with in vivo Optic Low Coherence Interferometry (OLCI-AcMaster). Refraction was measured by automated eccentric photorefractor. Details of the methods were previously described [14,15]. The corneal radius of curvature was measured in vivo by automated infrared photo-keratometry in B6 mice at all time points.
Six eyes from each of the atropine and saline treated groups were fixed in freshly prepared 4% paraformaldehyde, pH 7.4 at room temperature (RT) for 24 h. Tissues were embedded in JB-4 plastic (Electron Microscopy Sciences, Hatfield, PA) overnight. Processed sections were stained with hematoxylin and eosin before coverslipping. Tissues were observed under light a microscope (LWDPL40xFPL-6, Olympus with a Nikon CoolpiX-995 Digital Camera (Olympus, Center Valley, PA) to record images) and sclera thickness measured using an ocular micrometer at 40× magnification. Measurements were obtained from three locations: just posterior to the limbus, at the equator and immediately lateral to the optic nerve head. For each location, three adjacent fields were measured and then averaged. Six sections from each eye were placed on a slide and the measurements were made from five individual mouse eye sections. The values used for each calculations represents the mean of ninety values for each locations.
Total RNA was isolated using TRIzol reagent (Invitrogen life technologies, Carlsbad, CA) in accordance with the manufacturer’s instructions . Pairs of eyes were enucleated. The retina and choroid were stripped away from the sclera and immediately frozen in liquid nitrogen. Six different tissue samples were analyzed: 1. Atropine treated myopic sclera (AMS), 2. Control sclera from atropine treated mice (AMCS), 3. Normal saline treated myopic sclera (NSMS), 4. Normal saline treated control sclera (NSMCS), 5. Naive sclera (S) and 6. mouse cerebellum (C). Ten eyes were separately pooled and packed into aluminum foil and frozen as one sample. This experiment was repeated five times with different batches for all five tissues (n=10 in each group from 3 batch). A single pool of RNA from the cerebellum of three naive mice was used throughout the experiments as a control. In addition to the above mentioned tissues, myopic sclera (MS) and contra-lateral control sclera (CS) samples were also used for real-time PCR experiments (described below). PCR was performed as previously described .
Northern blot hybridizations were performed as previously described . Briefly, 25 µg of total RNA was loaded in each lane, run on a 1% agarose gel, transferred to a positively charged nylon membrane, and hybridized to a fluorescein-labeled mouse M1 EcoRI enzyme digested insert cDNA clone.
Real-Time comparative PCR was performed in a 96-well microtiter plate format on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) equipped with a Sequence Detection System (SDS) software version 1.6.3. PCR was performed using 250 ng of cDNA of each sample. The primers/probes for muscarinic receptor subtypes; M1-M5 were obtained from Taqman, Assays On Demand, PE Applied Biosystems. Primer sequences and conditions are presented in Table 1 Quantum RNA classic II 18S Internal Standard (Ambion, Austin, TX) was used as an endogenous control. The detailed method was previously described  and data was analyzed by comparative CT (ΔΔCT) method as previously described .
Independent t-tests were used to analyze the means of the axial length, scleral thickness, and the refraction of the eyes. A difference at p value <0.05 was considered statistically significant. Data are reported as the mean±SD. The statistical significance of changes in M1 - M5 mRNA levels was analyzed using Student’s t-test. The Mann–Whitney U-test was used to determine differences between groups.
Spectacle lens induction for a period of 42 days resulted in statistically significant in axial elongation, increased vitreous chamber depth of the treated eyes in BJ (p<0.01, n=50) and B6 mice (p<0.05, n=20) when compared to the contra-lateral control eyes. However the axial elongation, lens thickness, and vitreous chamber depth was appeared to be most significant in the BJ treated eyes as compared to the B6 treated eyes. There was no significant difference in corneal thickness and anterior camber depth between lens treated and contra-lateral eyes in both strains. (Figure 1A-E). Daily injections of the normal saline vehicle, into spectacle lens induced eyes did not alter the change in axial elongation, lens thickness and vitreous chamber depth in BJ (p<0.01, n=50) and B6 mice (p<0.05, n=20). Daily injections of atropine, at a concentration of 1%, blocked most of the elongation of axial length, lens thickness and the vitreous chamber depth associated with spectacle-lens induced myopia, but did not significantly affect the rate of axial elongation in contra-lateral control eyes. The other eye biometry measurements were not significantly different in treated or control eyes in non-pigmented and pigmented mice (Figure 2A-E and Figure 3A-E, respectively).
Eyes wearing −10 D lens in both non-pigmented and pigmented mice, the refraction was shifted from hyperopic to myopic (Figure 1F, p<0.01, n=50 and n=20, respectively) after 6 weeks of induction. Eyes wearing −10 D lens, treated with atropine sulfate for 4 weeks was shifted from myopic to hyperopic in both strains (Figure 2F, p<0.01, n=50 and 3F, p<0.01, n=20, respectively) were significant when compared to normal saline treatment. Whereas the minus lens wearing eye, treated with saline for 4 weeks was still showed a myopic shift in both strains (Figure 2F and Figure 3F, respectively). This result indicates that the atropine treatment reduces the progression of myopia and not the saline vehicle treatment. There was no significant difference seen in the contra-lateral control eyes in both strains (Figure 2F and Figure 3F, respectively). In B6 mice, the minus lens wearing eye’s corneal curvature was slightly flatter than the control and atropine treated eyes (Figure 3G). This was significant at 4 and 6 weeks after induction of myopia (p<0.05, n=20).
Data from three different comparisons was analyzed. Seven different tissues were used for quantification of muscarinic receptor gene expression: 1. MS (minus lens wearing), 2. CS (no lens), 3. AMS (minus lens with atropine), 4. AMCS (no lens with atropine) 5. NSMS (minus lens with saline), 6. NSMCS (no lens with saline), 7. S (Naive sclera), 8. Mouse Brain cerebellum was used as positive control and 18s rRNA was used as endogenous internal control for analysis.
Initially, CT values for M1-M5 in MS and AMS were compared against NSMS of muscarinic receptors gene CT values after normalizing with 18s rRNA. Similar analysis method was applied to the control group. M1, M3, and M4 mRNA levels were upregulated in the AMS and AMCS. In contrast, M2 and M5 mRNA levels were down regulated in the AMS and AMCS (Figure 4A). There were no changes in the MS and CS M1-M5 mRNA levels.
Normalized fold change expression of M1-M5 in AMS as compared with NSMS were 11, −8, 9, 8.5, −9.2 fold change (p<0.01, n=5 experimental repeats), respectively. The effects of atropine on message levels for M1, M3, and M4 were greater in experimental myopic eyes than the contra-lateral control (non-myopic) eyes. Similarly atropine effect on M2 and M5 was much greater in experimental myopic eyes than control eyes. It was reported that atropine reduces the axial length even in the naive eyes [23,24] however the effect was much higher in the spectacle lens wearing eyes.
Message levels for the muscarinic receptors from MS was compared against the CS from the same animal using the gene CT value after normalizing with 18s rRNA. Normalized fold change expression of M1 (−2.12 fold), M3 (−2.27 fold), and M4 (−2.02) mRNA levels were down-regulated in the MS and NSMS (−1.93 fold, −2.09 fold and −1.83 fold, respectively; p<0.01) whereas M1, M3, and M4 mRNA levels were upregulated after receiving atropine (10.58 fold, 5.01 fold and 6.78 fold, respectively; p<0.01). In contrast, M2 (2.98 fold) and M5 (3.47 fold) mRNA levels were upregulated in the MS and NSMS (1.96 fold, 2.3 fold respectively; p<0.01). This was opposite in the AMS (M2: −2.28 and M5: −5.39 fold; Figure 4B).
Analysis showed that atropine effect on M1 was much greater in MS as compared to their control after being normalized with 18s rRNA than on M4 and M3 (125%, 115%, and 112%, respectively; p<0.01). Similarly atropine effect on M5 was much greater in MS than it was on M2 (116%. and 101%, respectively; p<0.01). Our results showed that M1, M3, and M4 levels of scleral mRNA reduction, which led to the suppression of excessive axial elongation while M2 and M5 showed little change.
Experimental, control scleral, and mouse cerebellum muscarinic receptor subtypes CT values were compared against naive sclera of M1-M5 CT values after being normalized with 18s rRNA. M1 mRNA levels were down regulated (−9.52 fold) after the induction of myopia and treated with normal saline (−9.22 fold) and this expression pattern was reversed with atropine treatment (4.34 fold). The mRNA level of M3 and M4 showed a similar expression trend to that of M1. In contrast, M2 and M5 mRNA levels were upregulated in MS and NSMS whereas it was highly down regulated in the AMS (Figure 4C). The atropine effect on M1, M2, and M4 was much greater in experimental myopic eyes when compared to naive sclera.
In all set of data, MS and NSMS gene expression patterns for M1-M5 was similar but not the exact values as expected. The morphological and structural changes of sclera in both treatments were not different. Moreover, the mRNA levels were slightly different in these 2 groups of sclera; this may be due to some external stress received during saline injection or other factors involved.
To corroborate the positive results obtained from PCR, northern blots, a standard method for detection of mRNA levels, were performed. At the same time northern blots also provide a direct comparison of message abundance between samples on a single membrane. Northern blot analysis confirmed the same pattern of gene expression for all five M1-M5 in the minus lens treated sclera (Figure 5A), lens with atropine or normal saline treated experimental sclera and control sclera (Figure 5B) as like Real-Time PCR results.
RT–PCR was performed on sclera from mice treated with minus lens, receiving atropine and normal saline in conjunction with development of myopia, contra-lateral controls, and naive sclera at the end of each experiment. It was determined that M1-M5 was differentially expressed in mice treated with minus lens with and without drug treatment and, contra-lateral control sclera (Figure 6A,B, respectively). In preliminary studies it was found that the pigment epithelium was often closely adherent to the sclera. Therefore, a procedure was developed to quickly remove the pigment epithelium, which was checked by histological evaluation of sections from different regions of the sclera. Therefore the PCR results were considered to represent only the sclera fibroblasts .
As known from previous observations in the normal mammalian eye, it was found in the mouse that scleral thickness increased from anterior to posterior in naive and control eyes. Scleral thickness was measured from photomicrographs using the calibrated stage micrometer (accuracy ±7.0 µm) for as well as by magnified video imaging using trans-illuminated globes (accuracy ±4.0 µm). As shown in Table 2 (all at a level of p<0.05, n=6 in each group from 3 batch), spectacle lens induced myopia (Figure 7A) combined with daily injections of normal saline resulted in reductions in scleral thickness at equatorial and posterior locations (Figure 7C) when compared to aged-matched untreated eyes (Figure 7B) and with only normal saline without lens treatment (Figure 7D). Spectacle lens induced myopia with daily injection of atropine sulfate, at a concentration of 1%, blocked the posterior scleral thinning (Figure 7E), this was closely similar to their fellow eyes without lens treatment (Figure 7F).
In this study, BJ mouse was used to induce experimental myopia as per prior established method . Experimental myopia was achieved for our study even though we had used albino strain which suggests that visual cortex is not important to control the excessive expansion of the posterior segment of the eye or refractive error changes in mouse. Our results attained were similar to past reported studies of different species which are elaborated below [25-28]. Deprivation of various parts of the visual field produces myopia and elongation even in animals with optic nerve severed or those affected by degraded retinal image (even if it is in one sector of the eye) impling that accommodation is not involved. The mechanism by which the nervous system influences eye growth appears to be different even in closely related primate species which exhibit different responses to form deprivation conditions, suggesting differing mechanisms of eye growth control. Paralysis of the ciliary muscle or optic nerve section does not prevent the development of myopia in the rhesus macaque, indicating that in this species the axial growth is controlled by the retina. These results suggest that some factor in part of the retina can influence the growth of the sclera or the level of the scleral ocular elongation results from local growth, because we found increases in scleral fibroblasts cell proliferation in the induced eye (data not shown). It implies that the control of eye growth by vision seems to take place in local regions of the eye.
B6 mice were also used in our study to determine the effect of atropine in pigmented eyes with myopia. The results confirm that both pigmented and non-pigmented eyes were influenced by the minus lens treatment. Elongation of axial length and vitreous chamber depth appeared to be the main morphological parameters related to myopia in both strains. Moreover the axial length elongation was most significant in BJ mice when compared to B6 mice which indicates that the eye growth was higher in the BJ mice with age or with minus lens treatment.
Atropine was injected sub-conjunctively through the upper bulbar conjunctiva, a route that optimizes scleral exposure to the drug to determine the muscarinic receptor gene expression pattern during myopia development. It has been proposed that muscarinic antagonists might produce their effect by acting directly on scleral muscarinic receptors; however, that hypothesis is still being investigated . We have confirmed that atropine effectively reduced axial elongation in mouse eyes undergoing spectacle lens treatment to induce myopia. Atropine did not impose significant changes in the axial length of control eyes, which received clear visual input. This finding is in agreement with previous studies that investigated the effects of atropine on FDM in chicks [11,12], in tree shrews  and in monkeys [13,31,32]. Atropine has been shown to effectively prevent the progression of human juvenile myopia [33,34]. In mouse, the atropine reduces the eye growth at both low (data not shown) and high doses (this study) but the role of muscarinic receptors remains unclear. The application of anti-muscarinic drugs were able to change collagen structure and production of scleral extracellular proteins during myopia development in animal models [35,36]. This tend to suggest that investigation of scleral remodeling by atropine in the mouse model may be a good strategy to discover other anti-myopiagenic drugs for humans.
The mouse sclera, a connective tissue consisting of fibroblasts embedded in an extracellular matrix (ECM) of largely collagen Type I and proteoglycans, defines the shape and axial length of the eye. Because both biochemical analysis and histological analysis showed, significant changes only occur within the posterior region of sclera in chick model [37,38] but without evidence in the mouse model, the histological study was performed on the whole sclera. From our findings, in anterior and equator sclera of experimental eye, there was no obvious change when compared to that of control eyes. As such, the posterior sclera was thinner than that of control eye. These results are consistent with those from the chick . The fibrous sclera responds in the same manner across species (birds, mammals, and human) in response to hyperopic defocus or form deprivation stimuli, thinning in experimental eyes. The change in axial length could potentially be attributed to changes in posteriorly located ocular tissues, such as vitreous, retina or sclera. These ambiguous findings suggest that in the chick, atropine may work via other mechanism to inhibit myopia development because it was reported that the chick does not possess a functional M1 receptor . It would be important to determine if muscarinic antagonists interact with other pathways implicated in the regulation of ocular growth.
M1, M3, and M4 gene expression levels were upregulated in sclera of the experimental eyes following atropine treatment. After the treatment, myopic sclera produced less message for M2 and M5 and more in saline treated myopic sclera. This is the first study that reports differential regulation and expression of mRNA levels following chronic administration of atropine in experimentally induced myopic mice. Comparison of these results with those of previous studies on rat brain , chick cerebral neurons , and rat cortex [42,43], indicates that differential regulation of M1-M5; M1, M3, and M4 were upregulated whereas M2 and M5 were down regulated in response to chronic administration of atropine. Thus, at both the protein and message level, these two receptor systems seem to be about equal in abundance in cortex.
In rat cerebellar cells, M2 and M3 mRNAs were down regulated with carbachol treatment  however, antagonist induced upregulation of M2 and M3 mRNA level . In rabbit tracheal smooth muscle, M3 was upregulated following atropine treatment . In guinea pig posterior sclera, M1 and M4 mRNA levels were increased after induction of form-deprived myopia . It is clear that the regulation of M1-M5 subtypes varies between species and different tissues or cell types. Differential expression of muscarinic receptors suggests that different routes of administration and concentrations of atropine would have influence on the mRNA levels.
In conclusion, we have confirmed that atropine effectively reduces progression of myopia in both pigmented and non-pigmented mice. Muscarinic receptor antagonists most certainly seem to be promising drugs to inhibit human myopia. Additional knowledge on the actions of the drug, its toxicity and the mechanism are required. We have determined the mechanism of atropine at the gene expression level however there are avenues to be explored on the molecular and protein level. In this regard, the study of changing patterns of gene expression within and among species during emmetropization and myopic progression may offer a productive avenue for future research. Elucidating deficient steps in the regulatory pathway would mark significant advance given myopia's tremendous impact. No data are available on the systemic toxic effect in the long term usage of atropine. By studying the atropine treatment on muscarinic knock out mouse model would help us in understanding the effect of long-term usage of atropine or specific muscarinic blockers in human subjects and further investigations that need to be conducted with new therapeutic agents to treat myopia with this new model.
This work was supported by the National Medical Research Council Grants, NMRC/SERI MG/97–01/0002, NMRC/SERI IBG and NMRC/NIG/003/2007.