| Objective To determine the existence of the muscarinic acetylcholine 1(M1) receptor in posterior sclera , choroids , and retina in guinea pigs, and to evaluate changes in the expression of muscarinic acetylcholine 1 receptor in form-deprived myopia.To determine the existence of the muscarinic acetylcholine 1(M1) receptor in cultured guinea pig scleral fibroblasts. To investigate effects of pirenzepine on expression of muscarinic acetylcholine 1 receptor, cell proliferation, and type I collagen syntheses in cultured guinea pig scleral fibroblasts, and to study the mechanism of pirenzepine on preventing myopia.Methods Four-week-old pigmented guinea pigs (n=30) with the same refraction in both eyes were used in this study. One eye of each animal was selected randomly to wear a occlusive goggle for 2 weeks continuously as treated group. The other eye as control group. Before and after experiment, refraction was measured using retinoscopy and the ocular axial were determined by A-scan ultrasonography. At the end of experiment, animals were given over dose of pentobarbital and dead. The eyes were enucleated and residual orbital tissue carefully removed. Ocular weight was recorded by electric scale. Then anterior ocular tissues and vitreous body were removed. Posterior retina, choroids and sclera were carefully dissected. The total tissue RNA was extracted from the posterior retina, choroids and sclera respectively.The expression of mRNA of Ml receptor in posterior retina, choroids and sclera was determined by quantitative analysis of products of reverse-transcription polymerase chain reaction.Four-week-old pigmented guinea pigs (n=5) were used in this study and primery cultures were established using the sclera. The cultured cells was determined to be fibroblasts using immunocytolchemistory methods. The primary and the 7rd passage cells were collected when they became conflunt. The total cellular RNA was extracted from the cells, and the expression of mRNA of M receptor was analyzed by RT-PCR. The 7rd passage cells were cultured in medium containing 10'3M pirenzepine for 48 h, and the expression of mRNA of Ml receptor was analyzed by RT-PCR. Cultured guinea pig scleral fibroblasts were incubated with various concentration 10~2M, 10~3 M, 10"4 M> 10~5 M> 10~6M)of pirenzepine for 48 h .Morphologic alterations were observed using a phase contrast microscope and scanning electric microscope. Effects of pirizepine on cell proliferation was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, and distribution of cells along phases of the cell cycle was analyzed by flow cytometry. Effects of pirizepine on type I collagen synthesis was measured by using ELISA.Results Before experiment , the refraction of guinea pigs were +4.50 + 0.35D and the ocular axial length is 7.48±0.13mm.At the end of experiment, in control group the refraction is +4.50 + 0.38D ,the ocular axial length is 7.60 + 0.12mm , and eye weight is 356+5.26mg. In treated group, the refraction is 1.35 + 0.50D ,the ocular axial length is 7.76+0.15mm and eye weight is 369+3.01mg. There is significant difference in refraction, axial length and eye weight between two groups. The form-deprived eye produced -3.15 + 0.45D myopia, became 0.16±0.12mm longer and 13 ± 3.25mg more weigh when compared with the control. Reverse-transcription polymerase chain reaction shows that there is expression of mRNA of Ml receptor in posterior retina and sclera, but not in choroids in guinea pigs. Image analysis shows that the product of mRNA of Ml receptor in posterior sclera is67.52 + 3.25 in control group and is 30.12 + 2.16 in treated group. The expression of mRNA of Ml receptor of posterior sclera in treated group is significantly lower than in control group. The product of mRNA of Ml receptor of posterior retina is 69.52 + 3.32 in control group and is 32.53 + 2.72 in treated group. The expression of mRNA of Ml receptor in posterior retina in treated group is significantly lower than in control group.Cultured cells began to grow out of the sclera after 1-2 weeks, and grew to confluent after 3-4 weeks. The cultured cells was determined to be fibroblasts using immunocytolchemistory methods. Reverse-transcription polymerase chain reaction shows that there is expression of mRNA of Ml receptor in cultured guinea pig scleral fibroblasts. Image analysis shows that the product of mRNA of Ml receptor is 50.65 + 4.02 in the primary passage cells, is 52.02 + 3.96 in the 7rd passage cells, there is no significant difference beteewn two groups. Image analysis also shows that the product of mRNA of Ml receptor is 52.02 + 3.96 in control group , is 68.02 + 3.96 in the 10"3 M pirenzepine treated group. The expression of mRNA of Ml receptor in 10"3M pirenzepine treated group is significantly higher than in control group. MTT assay shows that , after scleral fibroblasts were incubated with 10"2Ms 10*3 M of pirenzepine for 48 h , cells proliferation was significantly inhibited, while 10'4M 10"5M^ 10"6M pirenzepine did not influence cells proliferation . Flow cytometry analysis shows that after scleral fibroblasts were incubated with 10"2M^ 10"3 M of pirenzepine for 48 h ,there is more cells state at G0/G1 phases, and less cells state at S phases, when compared to the control group. ELISA shows that scleral fibroblasts syntheses more type I collagen after being incubated with 10"2M> 10'3 M> 10"4 M pirenzepine for 48 h than the control. 10"5 M> 10"6 M pirenzepine did not influence type I collagen syntheses. Morphologic alterations were observed using a phase contrast microscope and scanning electric microscope in some cells incubated with 10"2M^ 10"3 M of pirenzepine, some cells lose normal profile , and have some hole in the cells surface and dead. |
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