Muscarinic mechanisms in a mouse model of myopia 2

3.4.2 Expression of mAChR subtypes in atropine and saline treated sclera of mice that have undergone experimental myopia RT-PCR was carried out on sclera from mice receiving atropine a

3.4 Gene Expression of Muscarinic Receptor subtypes

3.4.1 Expression of mAChR subtypes; M1-M5 on naive and sclera from mice with induced myopia

RNA (1
g) sample, from each collected tissue was separated on a 1.2 % agarose gel to determine the RNA purity prior to further analysis (Figure 18) RT-PCR was performed on brain and sclera of two month old naive myopic mice After 8 weeks of lens wearing, the experimental eye was found to be myopic Myopic and contra-lateral control sclera was collected from this group of mice to establish the expression of muscarinic receptor subtypes In age matched naive mice without treatment, the sclera and brain was collected to determine the expression level of mAChR sub types 18S rRNA was used as a positive control (Figure 19) and water was used as a negative control for all samples

mAChR 1 and mAchR 4 PCR products were run on 3% agarose gels at 80 Volts for 45 minutes mAChR 2 and mAchR 5 PCR products were run on 5% agarose gels at 80 Volts for 1 hour mAChR 3 PCR products were run on 4% agarose gel at 80Volts for 45 minutes The gels were stained with ethidium bromide for 15 minutes and de stained for a subsequent 15 minutes It was determined that M1 to M5 (Figures

20, 21, 22, 23, 24 and 25 respectively) was expressed in the brain and sclera of naive, experimental and contra-lateral control All experiments were done in triplicates

3.4.2 Expression of mAChR subtypes in atropine and saline treated sclera of

mice that have undergone experimental myopia

RT-PCR was carried out on sclera from mice receiving atropine and normal saline in conjunction with myopic and contra-lateral control sclera from 2-month old mice It was determined that M1 to M5 (Figure 26, 27, 28, 29 and 30 respectively)

were also expressed in atropine and normal saline receiving myopic sclera and lateral control sclera The results obtained suggest that the gene expression of all five muscarinic receptor sub types were expressed in mouse brain and sclera of all collected samples

contra-Figure 19 Quality of RNA was determined by 1.2% agarose / formaldehyde gel electrophoresis at 100 Volts for 40 minutes Lane 1: 1 kb RNA Ladder (28S rRNA-4.7 kb, 18S rRNA- 1.9 kb as indicated on left side), Lane 2: brain, Lane 3: normal sclera, Lane 4: myopic sclera, Lane 5: control sclera, Lane 6: atropine treated myopic sclera, Lane 7: atropine treated control sclera, Lane 8: normal saline treated myopic

sclera, Lane 9: normal saline treated control sclera

Figure 20 RT-PCR photo shows 18S rRNA (positive control) gene expression of all samples collected Lane 1: 100 bp DNA ladder, lane 2: brain, lane 3: normal sclera, lane 4: myopic sclera, lane 5: contra-lateral control sclera, lane 6: atropine treated myopic sclera, lane 7: atropine treated control sclera, lane 8: saline treated myopic sclera and lane 9: saline treated control sclera

Figure 21 RT-PCR photo shows that the mAChR 1 gene expression on normal and myopic mouse sclera Lane 1: 100 bp DNA ladder, lane 2: brain, lane 3: normal sclera, lane 4: myopic sclera, lane 5: contra-lateral control sclera and lane 6: water (negative control)

Figure 22 RT-PCR photo shows that the mAChR 2 gene expression on normal and myopic mouse sclera Lane 1: 100 bp DNA ladder, lane 2: brain, lane 3: normal sclera, lane 4: myopic sclera, lane 5: contra-lateral control sclera and lane 6: water (negative control)

Figure 23 RT-PCR photo shows that the mAChR 3 gene expression on normal and myopic mouse sclera Lane 1: 100 bp DNA ladder, lane 2: brain, lane 3: normal sclera, lane 4: myopic sclera, lane 5: contra-lateral control sclera and lane 6: water (negative control)

Figure 24 RT-PCR photo shows that the mAChR 4 gene expression on normal and myopic mouse sclera Lane 1: 100 bp DNA ladder, lane 2: brain, lane 3: normal sclera, lane 4: myopic sclera, lane 5: contra-lateral control sclera and lane 6: water (negative control)

Figure 25 RT-PCR photo shows that the mAChR 5 gene expression on normal and myopic mouse sclera Lane 1: 100 bp DNA ladder, lane 2: brain, lane 3: normal sclera, lane 4: myopic sclera, lane 5: contra-lateral control sclera and lane 6: water (negative control)

Figure 26 RT-PCR photo shows that the mAChR 1 gene expression on atropine treated and saline treated sclera Lane 1: 100 bp DNA ladder, lane 2: atropine treated myopic sclera, lane 3: atropine treated control sclera, lane 4: saline treated myopic sclera, lane 5: saline treated control sclera

Figure 27 RT-PCR photo shows that the mAChR 2 gene expression on atropine treated and saline treated sclera Lane 1: atropine treated myopic sclera, lane 2: atropine treated control sclera, lane 3: saline treated myopic sclera, lane 4: saline treated control sclera and lane 5: water (negative control), lane 6: 50 bp DNA ladder

Figure 28 RT-PCR photo shows that the mAChR 3 gene expression on atropine treated and saline treated sclera Lane 1: 100 bp DNA ladder, lane 2: atropine treated myopic sclera, lane 3: atropine treated control sclera, lane 4: saline treated myopic sclera, lane 5: saline treated control sclera and lane 6: water (negative control)

Figure 29 RT-PCR photo shows that the mAChR 4 gene expression on atropine treated and saline treated sclera Lane 1: atropine treated myopic sclera, lane 2: atropine treated control sclera, lane 3: saline treated myopic sclera, lane 4: saline treated control sclera

Figure 30 RT-PCR photo shows that the mAChR 5 gene expression on atropine treated and saline treated sclera Lane 1: 100 bp DNA ladder, lane 2: atropine treated myopic sclera, lane 3: atropine treated control sclera, lane 4: saline treated myopic sclera, lane 5: saline treated control sclera and lane 6: water (negative control)

3.5 Sequencing of Plasmid DNA with insert RT-PCR products

3.5.1 mAChR insert DNA for the five receptor subtypes were sequenced

Mouse primer sequences and NCBI Blast mAChR sub-types of mouse sequences match are shown in Table 8 and chromatography of M1-M5 are shown in Figure 31, 32, 33, 34 and 35 respectively

Table 8 Mouse mAChR 1- 5 insert DNA sequencing results

Figure 31 Electrophorogram of M1 primer sequence was analysed by ABI Prism DNA sequencer Colour on image: A - Green, C – Blue, G – Black and T – Red

Figure 32 Electrophorogram of M2 primer sequence was analysed by ABI Prism DNA sequencer Colour on image: A - Green, C – Blue, G – Black and T – Red

Figure 33 Electrophorogram of M3 primer sequence was analysed by ABI Prism DNA sequencer Colour on image: A - Green, C – Blue, G – Black and T – Red

Figure 34 Electrophorogram of M4 primer sequence was analysed by ABI Prism DNA sequencer Colour on image: A - Green, C – Blue, G – Black and T – Red

Figure 35 Electrophorogram of M5 primer sequence was analysed by ABI Prism DNA sequencer Colour on image: A - Green, C – Blue, G – Black and T – Red

3.6 Northern Blot Analysis

To establish the positive results obtained from the PCR, Northern blot was carried out which is a standard method for detection of mRNA levels and at the same time it also provides a direct relative comparison of message abundance between samples on a single membrane Northern blot analysis confirmed the presence of all five mAChR sub types of M1 (Figure 36 and Figure 41), M2 (Figure 37 and Figure 42), M3 (Figure 38 and Figure 43), M4 (Figure 39 and Figure 44) and M5 (Figure 40 and Figure 45) transcripts in the mouse brain, naive sclera, experimental myopic sclera, contra lateral-control sclera and atropine treated experimental myopic sclera, atropine treated contra lateral control sclera, normal saline treated experimental myopic sclera and normal saline treated contra lateral control sclera respectively

Figure 36 Northern blot of mAChR1 mRNA expression in 2-month-old mouse brain, naive sclera, experimental myopic sclera and control sclera Total RNA (25
g) 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 In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m1 (2.6 kb) are indicated to the left

Figure 37 Northern blot of mAChR2 mRNA expression in 2-month-old mouse brain, naive sclera, experimental myopic sclera and control sclera Total RNA (25
g) 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 M2 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m2 (1.8 kb) are indicated to the left

Figure 38 Northern blot of mAChR3 mRNA expression in 2-month-old mouse brain, naive sclera, experimental myopic sclera and control sclera Total RNA (25
g) 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 M3 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m3 (3.1 kb) are indicated to the left

Figure 39 Northern blot of mAChR4 mRNA expression in 2-month-old mouse brain, naive sclera, experimental myopic sclera and control sclera Total RNA (25
g) 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 M4 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m4 (1.5kb) are indicated to the left

Figure 40 Northern blot of mAChR5 mRNA expression in 2-month-old mouse brain, naive sclera, experimental myopic sclera and control sclera Total RNA (25
g) 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 M5 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m5 (1.6 kb) are indicated to the left

Figure 41 Northern blot of muscarinic M1 receptor mRNA expression in old mouse atropine received experimental myopic sclera, atropine received control sclera, and normal saline received experimental myopic sclera and normal saline received control sclera Total RNA (25
g) 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 In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m1 (2.6 kb) are indicated to the left

Figure 42 Northern blot of muscarinic M2 receptor mRNA expression in old mouse atropine received experimental myopic sclera, atropine received control sclera, and normal saline received experimental myopic sclera and normal saline received control sclera Total RNA (25
g) 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 M2 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m2 (1.8 kb) are indicated to the left

Figure 43 Northern blot of muscarinic M3 receptor mRNA expression in old mouse atropine received experimental myopic sclera, atropine received control sclera, and normal saline received experimental myopic sclera and normal saline received control sclera Total RNA (25
g) 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 M3 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m3 (3.1 kb) are indicated to the left

Figure 44 Northern blot of muscarinic M4 receptor mRNA expression in old mouse atropine received experimental myopic sclera, atropine received control sclera, and normal saline received experimental myopic sclera and normal saline control sclera Total RNA (25
g) 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 M4 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m4 (1.5 kb) are indicated to the left

Figure 45 Northern blot of muscarinic M5 receptor mRNA expression in old mouse atropine received experimental myopic sclera, atropine received control sclera, and normal saline received experimental myopic sclera and normal saline received control sclera Total RNA (25
g) 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 M5 EcoRI enzyme digested insert cDNA clone In the upper panel the sizes of 28S (4.7 kb), 18S (1.9 kb) rRNA and m5 (1.6 kb) are indicated to the left

2-month-3.7 Quantification of Real-Time PCR

3.7.1 Amplification Plot

PCR amplification patterns for of all five mAchR sub types and 18S rRNA internal standards are shown in Figures 46 (M1), 47 (M 2), 48 (M 3), 49 (M 4), 50 (M 5) and 51 (18S) Each gene of all samples collected was amplified in triplicates with internal standards in the same microtiter plate

Figure 46 Amplification plot of mAChR 1 The triplicates each cDNA of all eight tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed

Figure 47 Amplification plot of mAChR 2 The triplicates each cDNA of all eight tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed

Figure 48 Amplification plot of mAChR 3 The triplicates each cDNA of all eight tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed

Figure 49 Amplification plot of mAChR 4 The triplicates each cDNA of all eight tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed

Figure 50 Amplification plot of mAChR 5 The triplicates each cDNA of all eight tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed

Figure 51 Amplification plot of 18S rRNA internal standards for all five mAChRs The triplicates each cDNA of all eight tissues of 18S were amplified with that the same gene The CT value of each amplification reaction was exported and analysed

3.7.2 Comparative analysis by real time PCR

Data was analysed in four different comparisons

3.7.2.1 Gene Expression of myopic and atropine treated myopic sclera compared with normal saline treated sclera

In the first set, experimental myopic and atropine treated experimental myopic

scleral gene of interest CT values were compared against normal saline treated

experimental myopic sclera of that gene CT values after normalizing with 18s rRNA,

this was applicable to the control group [Table 9A-E (M1-M5 respectively)] M1 (Figure 52), M3 (Figure 54) and M4 (Figure 55) mRNA levels decreased (down regulated) after induction of myopia and increased (up regulated) in the atropine treated myopic and contra-lateral control M2 (Figure 53) and M5 (Figure 56) mRNA levels increased (up regulated) after induction of myopia and decreased (down regulated) in the atropine treated myopic and contra-lateral control

In this set of data collated, atropine had an effect on M1 expression only in experimental myopic eyes as compared with normal saline treated after normalizing with 18s rRNA, endogenous internal control Atropine effects on M3 and M4 were larger 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

3.7.2.2 Gene Expression of Experimental Sclera compared with their contra-lateral control sclera

In the second set, experimental scleral gene of interest CT values were compared against their own contra-lateral control sclera of that gene CT values after

normalizing with 18s rRNA [Table 10A - E (M1 – M5 respectively)] M1, M3 and

M4 (Figure 57) mRNA levels decreased (down-regulated) in the experimental myopic eye whereas increased (up-regulated) after receiving atropine and also in contra-lateral control eye M2 (Figure 58) mRNA levels increased (up-regulated) after induction of myopia and after receiving normal saline In contrast, M2 mRNA levels decreased (down-regulated) in the atropine treated myopic and contra-lateral control eyes The mRNA level of M5 (Figure 58) showed a similar expression trend to that of M2.

The analysis showed that atropine effect on M1 was much greater in experimental myopic eyes as compared to their control after being normalized with 18s rRNA than on M3 and M4 Similarly atropine effect on M5 was much greater in experimental myopic eyes than it was on M2

3.7.2.3 Gene Expression of all Scleral samples compared with mouse brain

In the third set, all scleral gene of interest CT values was compared against brain of that gene CT values after being normalized with 18s rRNA [Table 10A - E (M1 – M5 respectively)] M1 (Figure 59) mRNA levels decreased (down regulated) after induction of myopia and increased (up regulated) in the atropine treated myopic eye The mRNA level of M3 (Figure 61) and M4 (Figure 62) showed a similar expression trend to that of M1

The atropine effect on M4 was much greater in experimental myopic eyes as compared to brain after being normalized with 18s rRNA than it was on M1 and M3

M2 (Figure 60) mRNA levels decreased (down-regulated) after induction of myopia and normal saline treated myopic eye whereas it was highly decreased in the atropine receiving myopic eye In contrast, M5 (Figure 63) mRNA levels increased

(up-regulated) after induction of myopia and normal saline treated myopic eye, mRNA levels decreased in the atropine receiving myopic eye

Similarly atropine effect on M2 was much greater in experimental myopic eyes as compared to brain being normalized with 18s rRNA than it was on M5

3.7.2.4 Gene Expression of experimental and control Sclera compared with naive sclera

In the fourth set, brain, all experimental and control scleral gene of interest CTvalues was compared against naive sclera of that gene CT values after being

normalized with 18s rRNA [Table 12A - E (M1 – M5 respectively)] M1 (Figure 64) mRNA levels decreased (down regulated) after induction of myopia and increased (up regulated) in the atropine treated myopic eye The mRNA level of M3 (Figure 66) and M4 (Figure 67) showed a similar expression trend to that of M1 M2 mRNA (Figure 65) levels increased (up-regulated) after induction of myopia and normal saline treated myopic sclera whereas it was highly decreased in the atropine receiving myopic eye In contrast, M5 (Figure 68) mRNA levels increased (up-regulated) after induction of myopia and normal saline treated myopic sclera, mRNA levels was slightly different in the atropine receiving myopic eye

Gene expression of M1, M2 and M5 were up regulated in brain as compared with naive sclera after being normalized with 18s rRNA whereas it was down-regulated in M3 and M4 The atropine effect on M1, M2 and M4 was much greater in experimental myopic eyes in comparison to naive sclera than on M3 However the atropine effect on M5 was slightly different in the atropine treated myopic eye

Table 9 Comparative quantification of mAChRs using comparative CT method

9A Comparative quantification of MS, AMS M1 to normal saline treated (NSMS) CT

value and CS, AMCS to NSMCS

CT

SD Delta

CT

Delta Delta

CT

SD 2-

CT

Gene Expression Relative to normal saline treated

9B Comparative quantification of MS, AMS M2 to normal saline treated (NSMS) CT

value and CS, AMCS to NSMCS

CT

SD Delta

CT

Delta Delta

CT

SD 2-

CT

Gene Expression Relative to normal saline treated

All experiments were repeated in triplicates from five different batches of sclera, n = 10 sclera per experiment

9C Comparative quantification of MS, AMS M3 to normal saline treated (NSMS) CT

value and CS, AMCS to NSMCS

CT

SD Delta

CT

Delta Delta

CT

SD 2-

CT

Gene Expression Relative to normal saline treated

All experiments were repeated in triplicates from five different batches of sclera, n = 10 sclera per experiment

9D Comparative quantification of MS, AMS M4 to normal saline treated (NSMS) CT

value and CS, AMCS to NSMCS

CT

SD Delta

CT

Delta Delta

CT

SD 2-

CT

Gene Expression Relative

to normal saline treated

All experiments were repeated in triplicates from five different batches of sclera, n = 10 sclera per experiment