The Changes Of Expression Of Melanopsin And Melatonin Receptor-1A For Myopia Induced By Monochromatic Light In Guinea Pig

Part 1 The effect of blue and green light in daytime on the the light-induced melatonin suppression in guinea pigObjective:To investigate the effect of blue and green light in daytime on the the light-induced melatonin suppression in guinea pigs.Method:48 guinea pigs aged 10 days were exposed to blue light(wavelength 480nm),green light(wavelength 530nm) and white light(mixed wavelength) respectively,between 08:00am and 08:00pm for 10 days.The amounts of pineal gland melatonin were measured before and after the light pulse at 10:00am and 10:00pm.Changes observed between before and after the light pulse was compared with a melatonin baseline obtained in the white light group.Results:The amount of pineal gland melatonin was 45.92±1.74pg/ml at 10:00am and 63.27±1.43pg/ml at 10:00pm in blue light group,53.32±2.51 pg/ml and 73.12±2.72 pg/ml in white light group,85.2±2.79 pg/ml and 118.15±4.23 pg/ml in green light group.A significant increase was observed in the red light group(P＜0.05) whereas a significant reduction was observed in the blue light group at night(P＜0.05).Conclusions:Green light means to prevent the light-induced melatonin suppression, blue light enhances the light-induced melatonin suppression,they all change biological rhythms.Part 2 The changes of expression of melanopsin and melatonin receptor -1A for myopia induced by 530nm monochromatic light in guinea pigObjective:To investigate the expression of melanopsin and melatonin receptor 1-A in myopia induced by 530nm monochromatic light in guinea pig and the relationship between the expression of melanopsin and melatonin receptor 1-A and myopia induced by 530nm monochromatic light.Method:Thirty 10-day-old guinea pigs were randomized into3 groups and exposed to green light(530nm),blue light(480nm) and white light for 8 weeks. Measurement of the refractive status was taken after cycloplegia with streak retinoscopy,ocular length and vitreous depth detected by ultrasound biometry were examined before and after the experiment in guinea pigs.Eyes were dissected at 10:00pm,and the ultramicro-structures of the sclear were observed through electromicroscope.The expression of melanopsin and melatonin receptor 1-A were observed by immunocytochemisty.The level of melanopsin and melatonin receptor 1-A and their mRNA were observed through Western-blot and real-time PCR respectively.Result:1) There were no statistically significantly variance of the refractive error,ocular length nd vitreous depth among green light group,white light group and blue light group before experiment.Eye elongation and vitreous depth gain could be observed after 8 weeks of light treatment.The refractive status was 1.37±0.75D in green light group,3.10±0.84D in white light group and 3.25±0.78D in blue light group(F= 17.46,P=0.0032);The vitreous depth was 3.70mm±0.16mm in green light group, 3.24mm±0.21mm in white light group and 3.21mm±0.19mm in blue light group respectively(F=11.04,P=0.0098).Ocular length was 8.46±0.13mm,7.62±0.17mm and 7.56±0.18mm respectively(F=20.86,P=0.0020).Statistical analysis of the three parameter mentioned above indicate statistically significance among them. Guinea pigs in 530nm green light group were in relatively myopic refractive status.2) Compared with those in retina of blue light group,density of melanopsin in retina of green light group decreased,but melatonin receptor 1-A was more abundant in the retina of green light group by immunocytochemisty.3) There was significant difference in expression of melanopsin mRNA and protein between the green light group and blue light group(F=3.61,p=0.016).There was also significant difference in expression of melatonin receptor 1-A mRNA and protein in retina and sclea between the green light group and blue light group(F=0.7945,p= 0.000;F=5.3987,p=0.000)).Green light group expressed higher level of melatonin receptor 1-A mRNA and protein in retina and sclea,while blue light group expressed lower level.Conclusion:Green light(530nm) alter the refractive status of guinea pig,may accelerate myopic development.Monochromatic light with the long-wavelength of 530nm induces the increment of melatonin receptor 1-A and decreasement of melanopsin in myopic guinea pigs.melatonin receptor 1-A and melanopsin might play a role in the forming of 530nm monochromatic induced guinea pig myopia.Part 3 The effect of melatonin injecting periglomerularly on guinea pig myopic shift.Objective:To examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia in guinea pig.Method:30 pigmented guinea pigs aged 4weeks were maintained on a 12-hour lightdark cycle for 14 days.During the 14-day treatment period,guinea pigs were injected periglomerularly on one randomly selected eye during the early dark period(8:00pm) with melatonin 0.2ml(500pg/ml) or 2%ethanol vehicle control 0.2ml each day,and the fellow eye was taken as control. Measurement of the refractive status after cycloplegia with streak retinoscopy,ocular length and vitreous depth detected by ultrasound biometry were examined before experiment and 2 weeks after the treatment of MLT or 2%ethanol in guinea pigs. The eyes were conducted electron microscopy examination to evaluate the pathological changes of the melatonin treated eye and the control eye.Sclera thickness,choroidal thickness and retina thickness were measured in the same site by HE bioscopy.Results:There was no statistically significantly variance of the refractive error,ocular length and vitreous depth between control group and melatonin treated group before treatment.Two weeks later,Eye elongation and vitreous depth gain could be observed.In control group,the refractive status was 3.65D±0.81D,the vitreous depth was 3.28mm±0.49mm,ocular length was 7.6mm±0.29mm,retina thickness was 0.1270mm±0.0057mm,Choroidal thickness was 0.0473mm±0.0016mm,Sclera thickness was 0.1181mm±0.0022mm;In melatonin treated group,the refractive status was 2.76D±0.45D,the vitreous depth was 3.59mm±0.86mm,ocular length was 8.7mm±0.38mm,retina thickness was 0.1028mm±0.0061mm,Choroidal thickness was 0.0449mm±0.0023mm,Sclera thickness was 0.0901mm±0.0012mm respectively.There were statistically significantly variance of the refractive error,ocular length,vitreous depth,retina thickness,choroidal thickness and sclera thickness between control group and melatonin treated group(P＜0.05).Conclusions:The finding that administration of melatonin alters the growth of several ocular tissues in pigmented guinea pig's eyes suggests that melatonin plays a role in ocular growth and myopic development.This conclusion suggests that the action of melatonin is involved in the regulation of the diurnal rhythm of ocular growth。Part 4 Effect of melatonin on rat scleral fibroblasts proliferation in vitroObjective:To investigate the effects of melatonin on rat scleral fibroblasts proliferation in vitro and its mechanisms.Methods:The rat scleral fibroblasts were grown in tissue culture,then were treated with different doses of melatonin((0 vacuity matched control,0 solvent matched control,50pg/ml,100pg/ml,500pg/ml and 1000pg/ml) for 24h,48h,72h individually.The effects of melatonin on scleral fibroblasts proliferation were measured by MTT assay,synthesis of collagen was observed by Chlorozone T Results.After 72h,the expression of melatonin receptor-1A mRNA and protein was detected by real time PCR and immunofluorescent staining respectively.Results melatonin inhibited rat scleral fibroblasts proliferation in vitro significantly,the most efficient concentrations was 500pg/ml;melatonin could inhibit collagen synthesis of scleral fibroblast in concentrations from 100～500pg/ml; Cultured with melatonin,the expression of melatonin receptor-1A mRNA and protein increased.Conclusions:The growth of rat scleral fibroblasts in vitro and collagen synthesis could be inhibited by melatonin.Increased melatonin receptor 1A expression might be involved.