Experimental Study On The Effects Of Lighting Stimulus In Advance On The Expression Of Melanopsin In Mice Retina

Photoadaptation in mammals consists of three predominant types:control of retinal illumination by pupillary light reflex(PLR),direct light modulation of the neuroendocrine system leading to modulation of sleep or activity,and entrainment of the circadian timekeeping system to the ambient light:dark cycle.Intriguingly, photoadaptive responses are left intact or are only partially attenuated in many human patients and in several animal models with complete loss of rod/cone function. Experimental bilateral enucleation in animals,however,abolishes all ocular light responses,suggesting participation of a novel class of ocular photoreceptor(s).Until recently thephotoreceptor(s) have remained elusive.Efforts to understand circadian photoentrainment have led to the discovery of a small population of intrinsically photosensitive retinal ganglion cells(ipRGCs) expressing a novel opsin photopigment, melanopsin(Opn4).According to the different functions,photo reception systems are divided into two types,image-forming system and nonimage-forming system.As the improvement of living quality,people pay increasing attention to diseases caused by the abnormal light entrainment of circadian rhythm.Therefore,in this experiment,the model group of lighting stimulus in advance was established by opening right eye lids of C57BL/6J mice at postnatal 4 days(P4),we investigated the expression of melanopsin in RGCs during the process of retina development in natal mice,and the effects of lighting stimulus in advance on the expression of melanopsin from the cellular and molecular levels,in order to identify the role of melanopsin in the nonimage-forming system.PartⅠModel's establishment of lighting stimulus in advance and morphological features of melanopsin-expression cells in C57BL/6J miceObjectiveTo establish the model of lighting stimulus in advance and investigate the morphological features of melanopsn-expression cells and the expression of melanopsin in RGCs during the process of retina development in natal mice,and the effects of lighting stimulus in advance on the expression of melanopsin.MethodsThe model group of lighting stimulus was established by opening right eye lids of C57BL/6J mice at postnatal 4 days(P4),and left eyes were as the self-control group.The mice were kept on the 12h light:12h dark circle.The distribution and expression of melanopsin expressed cells were observed in experimental group and control group at P5,P6,P7,P14 and P90 by immunohistochemistry.Results1.The animal model of lighting stimulus is established successfully by opening right eye lids of C57BL/6J mice,which facilitates to observe the different expressions of melanopsin in RGCs during the process of retinal development.2.Only a few fractions of retinal ganglion cells express melanopsin by immunohistochemistry.3.As the development of retina,melanopsin expressed RGCs decreased in both the experimental groups and control groups,however,the experimental groups decreased more obviously.Conclusions1.The animal model of lighting stimulus is established successfully by opening right eye lids of C57BL/6J mice,which offers a way to study the expression of melanipsin in condition of lighting stimulus in advance.2.The architecture of melanopsin-expressed RGCs has a relevant relationship with its function.3.Lighting is possibly one of the main reasons to affect the expression of melanopsin during the retinal development.PartⅡEffects of lighting stimulus in advance on the expression of melanopsin during retinal development at the molecular levelObjectiveTo study the expression of melanopsin during the process of retinal development and lighting stimulus in advance at the transcription and translation levels,and investigate the internal mechanism and meaning.MethodsTotal retinal RNA of different postnatal stages were isolated by Trizol reagent.The expression of melanopsin mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR) method in different periods of retinal development from P5, P6,P7,P14 to P90.Total retinal protein of different postnatal stages were isolated,the expression of melanopsin protein were detected by western blotting in different periods of retinal development.Results1.The expression of melanopsin mRNA in the different processes of retinal development by RT-PCR.As the development of retina,the expression of melanopsin mRNA down regulated gradually and expressions in experimental groups are significantly lower than that in control groups respectively before P14(P＜0.05).However,there is no significant difference between experimental group and control group at P90(P＞0.05).2.The expression of melanopsin protein in the different processes of retinal development by western blotting.The expression of melanopsin protein has a similar changeable trend with melanopsin mRNA during different processes of retinal development.ConclusionsIt is a key stage for the development of retinal ganglion cells before P14,the total amount of melanopsin-expression cells are gradually lower,and lighting stimulus by opening eye lids can down regulated melanopsin expression.The results indicate that lighting may be one of circadian rhythm modulating mechanism.