Sonic Hedgehog Expression And Role In Form Deprivation Myopic Mice Eye

PrefaceMyopia is now a major public health issue across the world,and more and more research is aimed at finding out its genesis.As visual input dominates the regulation of postnatal eye growth and the development of refractive errors,convincing evidence has identified a central role for the retina in the mechanism linking visual input with refractive development.Sonic hedgehog(Shh),which has been reported to act in the regulation of dorsoventral patterning of the neural tube and somites,is also an important gene in the normal formation and development of the eye.Study has found that Shh gene was differentially expressed in the experimentally induced myopic chicken retinal samples compared with controls.But the role of Shh in the regulation of ocular development and myopia is not clear yet.In this study,we localized the expression of Shh and its downstream mediators in mouse retina and studied the effect of Shh agonist or antagonist on experimental myopia.PARTâ… Establishment of form deprivation myopic model in micePurpose:To compare the efficacy of two methods in the induction of form deprivation myopia(FDM) in C57B/L6 mice.Methods:Eighty C57B/L6 mice of 13 to 14-day-old underwent a 7 to 28-day monocular form-deprivation period by wearing a translucent diffuser(n=44) or suturing the eyelid(n=36).Age-matched untreated mice were used as control(n=16). Retinoscopic refraction was performed at eye reopening.Animals were killed and the eyes enucleated.Axial lengths were measured in highly magnified photos of freshly excised eyes taken under an operating microscope.The sections of the eyes were observed by light and electron microscope.Results:All eyes treated with two methods developed significant myopia and elongated axial length compared with the opened eyes in only one week.Significant difference was neither found in induced myopia nor axial elongation between the two methods following 7 to 14-day deprivation.Greater myopia was found in eyelid suturing group at the end of 28-day deprivation.No significantly histological abnormality was found in the deprived eyes under light microscope.Under electron microscope,the arrangement of rod outer segment in deprived eyes became looser than controls.Conclusions:Form deprivation myopia can be induced in C57B/L6 mice either by translucent diffuser wearing or eyelid suture in one week.This model may be useful to investigate underlying mechanisms of myopia in mammals,because of easier handling and availability of genetically manipulated strains.PARTâ…¡Expression of Sonic hedgehog and its downstream mediators in form deprived myopic mice eyesPurpose:To evaluate changes in the expression of Sonic hedgehog(Shh) and related genes in mice retinal tissues after induction of form deprivation myopia (FDM).Methods:FDM was induced by wearing translucent diffuser in 75 C57B/L6 mice of 13 to 14-day-old.Age-matched untreated mice were used as control(n=75). Expression levels of Shh,its receptor Patched-1(Ptc-1) and transcription factor Gli-3 were evaluated by immunofluorescent staining,real-time fluorescent quantitative reverse transcription polymerase chain reaction(real-time PCR) and Western-blot analysis in FDM or control eyes.Results:Shh,Ptc-1 and Gli-3 were immunohistochemically detected in both the open and the deprived eyes of mice.Gli-3 protein is observed in the cell cytoplasm of normal retinal ganglion cell(RGC),whereas nuclear translocation of Gli-3 was detected in RGC of FDM eyes.The induction of myopia caused significant increase in expression of Shh mRNA and protein,so did Gli-3.In contrast,the expression of Ptc-1 mRNA and protein decreased in the deprived eyes.These changes were most prominent following the first week's deprivation.Conclusions:A change in the expression of Shh and its downstream mediators, as well as the nuclear translocation of Gli-3 in FDM mice retina,suggest an involvement of Shh pathway in the development of experimentally induced myopia.PARTâ…¢Effect of Sonic hedgehog agonist or antagonist on experimental myopiaPurpose:To study the effect of Sonic hedgehog agonist or antagonist on the development of experimental myopia,and test whether Shh is related to the control of experimental myopia.Methods:13 to 14-day-old C57BL/6 mice,some of which wore a unilateral translucent diffuser to induce myopia,received four times' intravitreal injections of Shh or cyclopamine(a specific inhibitor of Shh pathway) every other day. Retinoscopic refraction and axial length measurement were performed on the 11th day of form deprivation.The sections of the eyes were observed by light microscope and immunofluorescent staining.Expression levels of Shh,Ptc-1 and Gli-3 were evaluated by Western-blot analysis.Results:Injection of Shh stimulated the development of myopia and axial growth in both goggled and nongoggled eyes.Cyclopamine slowed the development of FDM and growth of axial length.It also affected nongoggled eyes,inhibiting growth and shifting refraction toward hyperopia.Conclusions:Shh and its downstream mediators modulate eye growth and refractive development.Shh pathway may be involved in the control of postnatal eye growth and the development of myopia.