Molecular mechanisms of muscarinic receptors in mouse scleral fibroblasts: Prior to and after induction of experimental myopia with atropine treatment

Animals

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.

Treatment protocols

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 [8]. 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.

Refraction and axial length

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.

Histological studies

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.

RNA Isolation and RT–PCR

Total RNA was isolated using TRIzol reagent (Invitrogen life technologies, Carlsbad, CA) in accordance with the manufacturer’s instructions [20]. 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 [8].

Northern blotting

Northern blot hybridizations were performed as previously described [21]. 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

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 [14] and data was analyzed by comparative C_T (ΔΔC_T) method as previously described [22].

Data Analysis

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.

Atropine treatment reduced axial elongation, lens thickness and vitreous chamber depth

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).

Refractive error changes with atropine treatment

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).

Comparative analysis by real time PCR

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.

Gene expression of myopic and atropine treated myopic sclera

Initially, C_T values for M₁-M₅ in MS and AMS were compared against NSMS of muscarinic receptors gene C_T 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.

Gene expression of myopic, atropine treated, saline treated compared with contra-lateral control sclera

Message levels for the muscarinic receptors from MS was compared against the CS from the same animal using the gene C_T 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.

Gene expression of experimental and control sclera against naive sclera

Experimental, control scleral, and mouse cerebellum muscarinic receptor subtypes C_T values were compared against naive sclera of M₁-M₅ C_T 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.

Northern blot analysis

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.

Expression of muscarinic receptor subtypes

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 [8].

Atropine treatment reversed scleral thinning

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).