Effects Of Dopamine D₂ Receptor And Adenosine A_2A Receptor On The Experimental Myopia In Guinea Pigs

PART 1 To study the expression of dopamine D₂ and adenosine A_2Areceptors in the eyeball and the change of retinal D₂ receptor in experimental myopiaPurpose:To detect the expression of mRNA for dopamine D₂ and adenosine A_2A receptors in the eyeball and to determine the retinal abundance mRNA for dopamine D₂ receptor.Methods:Twenty-two pigmented guinea pigs were randomly assigned to 3 groups including NOR group,FDM group,LIM group.They had been form-deprived or lens-induced for 11 days.All the groups underwent biometric measurement(corneal curvature,refraction and axial length)prior to and after the experiment.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)was performed to detect the expression of mRNA of D₂ and A_2Areceptors in the retina,choroid and sclera.And the retinal abundance of mRNA for dopamine D₂ receptor was determined by Real-time PCR.Results:After 11-day experiment,occlude and lens induced -4.06±2.66D and -3.77±2.80D compared to their contralarteral eyes,respectively.The differences of vitreous length(VL)between two eyes were 0.12±0.04 mm in the FDM group and 0.10±0.03 mm in the LIM group.The differences of axial length(AL)between two eyes were 0.14±0.05 mm in the FDM group and 0.12±0.04 mm in the LIM group.They were all high significantly different with that of NOR groups.But there were no significant changes in intraocular differences of lens thickness(LT)and corneal curvature(CC) between three groups.There were also no significant changes in intraocular differences of refraction error(RE),LT,VL,AL and CC between the FDM and LIM groups.The intraocular difference of RE was related with the differences of VL and AL,but was unrelated with the differences of CC and LT by using the two-variable linear regression analysis.There was mRNA of D₂ and A_2Areceptors in the retina, choroid and sclera by using RT-PCR and mRNA of dopamine D₂ receptor in the retina was decreased in FDM and LIM.Conclusion:These two experimental myopia models are a kind of axial myopia without changing the corneal curvature.Myopia shift and axial length elongation in FDM are little different from LIM(-4D).It is suitable for studying the experimental myopia by using the face-masking and lens.There is mRNA of D₂ and A_2A receptors in the retina,choroid and sclera,and dopamine D₂ receptor in the retina may be related with FDM and LIM.PART 2 Retinal dopamine in the recovery from experimental myopiaPurpose:To address further a possible role for retina dopamine in experimental myopia,we studied the response of retinal dopamine in eyes of guinea pigs in the myopia and recovery process.Methods:Ninety-four guinea pigs(aged 3 weeks)were randomly assigned to 6 groups including FDM group(form deprived for 11 days,n=6),LIM group(lens induced for 11 days,n=6),NOR1(age-matching for 11 days),FDMrec(form deprived for 11 days and then recovering for 7 days,n=7),LIMrec(lens induced for 11 days and then recovering for 7 days,n=7)and NOR2(age-matching for 18 days).All groups underwent biometric measurement(corneal curvature,refraction and axial length)prior to and after the experiment.Retinal levels of dopamine and its principal metabolite 3,4-dihydroxyphenylacetic acid(DOPAC)were assayed by high performance liquid chromatography with electrochemical detection(HPLC-EC).Results:Compared to their contralateral and control eyes,retinas of form-deprivation eyes had reduced levels of dopamine and DOPAC.In eyes recovering from myopia, levels of dopamine and DOPAC showed prominent increases and reached the level of both contralateral and control eyes by 7 days after deprivation removal.There was no change of them in LIM and the recovery process as compared with contralateral and control eyes but they were also increased with age.Conclusion:Form-deprivation myopia may be dependent on dopaminergic pathways in guinea pigs whereas lens induced myopia may be different. PART 3 Apomorphine in control of axial myopia in guinea pigsPurpose:To investigate whether apomorphine was in control of FDM and LIM in guinea pigs.Methods:100 pigmented guinea pigs were conducted in the study which was comprised of two parts.In experiment 1,thirty-nine pigmented guinea pigs were randomly assigned to 6 groups to determine the effect of apomorphine at 250 ng subconjunctival injection on FDM.Adding fifteen animals to randomly design another 2 groups including 2.5 ng and 25 ng,divided into logarithmic(base 10)steps to study the dose-dependent fashion.In experiment 2,thirty pigmented guinea pigs were randomly assigned to 3 groups equally to study the effect of apomorphine at 250 ng subconjunctival injection on LIM.Another 2 groups covered the higher dose ranging from 2500 ng to 25000 ng to estimate the dose-dependent effect.Myopia was induced monocular by fitting either an opaque latex balloon(FDM)or a -4 D spectacle lens(LIM)over one eye randomly of each animal.Normal eye without any treatment was as normal control.Injection of injective water contained 0.1% ascorbic acid on FDM or LIM was as vehicle control.A subconjunctival injection of 100μl apomorphine was performed daily for 11 days.All the groups underwent biometric measurement(corneal curvature,refraction and axial length)prior to and after the experiment.At the end of the experiment,normal control,vehicle or drug combinations with the visual treatments were analyzed for pathological examination.Results:Prior to occluded,there were no significant changes in refraction,corneal curvature and axial components between treated eyes and the contralateral eyes and there were also no difference between treated groups.At the end of the 11-day treatment period,apomorphine(250 ng)subconjunctival injection inhibited the myopia development and lessened the vitreous and axial elongation on FDM almost completely and at a dose-dependent fashion.But it couldn't inhibit LIM even it was used in higher dose than that on FDM.Moreover,in all groups,there were all no significant changes in refraction,corneal curvature and axial components between the contralateral eyes and normal control eyes prior to and after the experiment.Under light microscopy,there were no significant differences of morphology in the experimental groups and the control groups.But under transmission electron microscope(TEM),we detected characteristic ultra structural morphologic change in ganglion cells and photoreceptor.Conclusion:Subconjunctival injection of apomorphine is an effective way to prevent FDM and it has no influence on LIM even in higher doses in guinea pigs.PART 4 The adenosine A_2Areceptor antagonist SCH58261 inhibits form-deprivation myopia in guinea pigsPurpose:To study the effect of SCH58261,a specific antagonist of adenosine A2A receptor,on FDM in guinea pigs and to measure the content of adenosine in retina in order to investigate the effect of adenosine system on FDM in guinea pigs.Methods:Ninety-four guinea pigs(aged 3 weeks)were randomly assigned to 10 groups including form-deprivation myopia(FDM)and control groups.A subconjunctival injection of 100μl SCH58261 at a range of doses(0.1 to 100μmol) was performed daily for 11 days.Refraction,corneal curvature and axial components of the eye were measured prior to and 11 days after the experiment. Retinal levels of dopamine and its principal metabolite 3,4-dihydroxyphenylacetic acid(DOPAC)were assayed by high performance liquid chromatography(HPLC) with ultraviolet detection(HPLC-UV).Morphology changes were evaluated under light microscopy.Results:There were all myopia shift and axial elongation in different SCH58261-FDM groups when compared to their contralataral eyes.But there were significant changes in intraocular differences of RE,VL,AL between different SCH58261-FDM groups and DMSO-FDM group,especially the highliest inhibiting effect was at dose of 1μol,but they were not in a dose-dependent fashion.It estimated subconjunctival injection of SCH58261 could prevent FDM.By using HPLC,it found that there were no significant changes in the retinal adenosine between the experimental and the contralateral eyes in FDM and SCH58261-FDM groups.Moreover,there were no significant changes between different groups.It showed that form-deprivation and subconjunctival injection of SCH58261 could not change adenosine in the retina. Under light microscopy,there were no significant differences of morphology in the subconjunctival injection of SCH58261 and the normal control eyes.It showed that it wasn't pathologic change but pharmacologic action.Conclusion:Adenosine A_2Areceptors may be a therapeutic target for preventing FDM. But it need further to he studied.