Muscarinic mechanisms in a mouse model of myopia 2

103 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). RTPCR was performed on brain and sclera of two month old naive myopic mice. After 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 and mAchR PCR products were run on 3% agarose gels at 80 Volts for 45 minutes. mAChR and mAchR PCR products were run on 5% agarose gels at 80 Volts for hour. mAChR 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) 104 were also expressed in atropine and normal saline receiving myopic sclera and contralateral 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. Figure 19. Quality of RNA was determined by 1.2% agarose / formaldehyde gel electrophoresis at 100 Volts for 40 minutes. Lane 1: kb RNA Ladder (28S rRNA4.7 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. 105 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. 106 Figure 21. RT-PCR photo shows that the mAChR 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). 107 Figure 22. RT-PCR photo shows that the mAChR 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). 108 Figure 23. RT-PCR photo shows that the mAChR 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). 109 Figure 24. RT-PCR photo shows that the mAChR 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). 110 Figure 25. RT-PCR photo shows that the mAChR 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). 111 Figure 26. RT-PCR photo shows that the mAChR 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. 112 Figure 27. RT-PCR photo shows that the mAChR 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 218 219. Rada, JA, Thoft JA, Hassel JR. Extracellular matrix changes in the sclera of chickens with experimental myopia. 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The influence of study habits on myopia in Jewish teenagers J Pediatr Ophthalmol Strabismus. 1993;30,319-322. 233 Appendices Figure 2.6 Plasmid map [...]... 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 115 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... mRNA levels decreased (down-regulated) in the experimental myopic eye whereas increased (up-regulated) after receiving atropine and also in contralateral 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... were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed 135 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 136 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,... tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed 131 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 1 32 Figure 48 Amplification plot of mAChR 3 The triplicates each cDNA of all eight... 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... upper panel the sizes of 28 S (4.7 kb), 18S (1.9 kb) rRNA and m1 (2. 6 kb) are indicated to the left  126 Figure 42 Northern blot of muscarinic M2 receptor mRNA expression in 2- monthold 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,... 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 1 0A - 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... (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... tissues were amplified in the same microtiter plate The CT value of each amplification reaction was exported and analysed 133 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 134 Figure 50 Amplification plot of mAChR 5 The triplicates each cDNA of all eight . 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. 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,. 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