The Role Of Sonic Hedgehog Signaling In Regulating From-deprivation Myopic Development Of Guinea Pigs

PrefaceThe mechanism of myopic development still remains unknown. One of the generally acknowledged pathogenesis in myopia involves the stimulation of external factors on retinal signals firstly and subsequently,the breaken balance between transforming growth factor-P(TGF-β) and basic-fibroblast growth factor(b-FGF).Then the matrix metalloproteinases(MMP) in sclera will be activated and as a result, causes scleral degradation, elongation of axial lenths and development of myopia. Now, the most attention should be paid to look for the exact signal pathway in order to figure out the myopic mechanism. Previous studies indicate that Sonic hedgehog (Shh) play a key role in regulating myopic development. Moreover, studies in tumor find out that Shh pathway can modulating the expression of MMPs and TGF-β, which also shows us the possible downstream pathways with Shh in myopia. Thus, we aim to explore the role of Shh signaling pathway and its regulating approach in myopic development by upregulating with exogenous Shh and downregulating with cyclopamine, in order to provide evidence for myopic prevention and treatment.PART I The expression of retinal Shh signaling and scleral MMP-2/TGF-β in guinea pigs with form deprivation myopiaPurpose:To observe the expression of retinal Shh signaling and scleral MMP-2/TGF-P in guinea pigs with form deprivation myopia. Methods:Fifty-six healthy guinea pigs of2to3-week-old were randomly assigned to the FDM group(n=40) and the control group(n=16). The right eyes of guinea pigs in the FDM group were wore a translucent diffuser. Ten and four guinea pigs were separately choosen from the FDM group and the control group for retinoscopic refraction and A-scan measurement at0.1,2an4weeks. Then, animals were killed and the eyes were encleated for detection of retinal Shh, Ptc-1and Gli-3immunohistochemically, mRNA of retinal Shh, Ptc-1and Gli-3by real-time PCR and protein of retinal Shh, scleral MMP-2/TGF-β with Western-blot.Results:The right eyes in the FDM group developed significant myopia of-2.35±1.10D、-5.13±1.73D and-6.78+1.04D compared with the left eyes at1,2and4weeks. And the myopia progressed with the prolonging of diffuser wearing. The right eyes in the FDM group had an elongation of0.1±0.05mm、0.11±0.04mm and0.37±0.19mm in axial lengths compared with the contralateral eyes at1.2and4weeks, which were statistically significant. And The right eyes in the FDM group had an elongation of0.07±0.04mm、0.10±0.07mm and0.36±0.18mm in lengths of the vitreous compared with the contralateral eyes at1.2and4weeks, which were statistically significant. Shh was immunohistochemically detedcted in retina while Ptc-1and Gli-3were observed in cell cytoplasm of retinal ganglion cell. The induction of myopia caused significant increase in expression of Shh mRNA(P=0.043) and Ptc-1mRNA (P=0.024) at week4compared with the contralateral eyes. Significant increase of retinal Shh protein and scleral MMP-2as well as notable decrease of scleral TGF-β were seen in the right eyes of the FDM group since week2and continued to week4.Conclusions:The changes in the expression of Shh and its downstream mediators in retina of guinea pigs with form deprivation myopia suggests an involvement of Shh signaling pathway in the development of FDM.PART II Effect of intravitreal injection of Sonic hedgehog on the myopic development in guinea pigs Purpose:To investigate the effect of intravitreal injection of Sonic hedgehog on the myopic development and its associated expression of scleral MMP-2/TGF-β in guinea pigs.Methods:Fourty healthy guinea pigs of2to3-week-old were randomly assigned into two groups (n=20,each). The right eyes in each group were intravitreally injected with Shh-N/0.1%BSA and the other eyes were intravitreally injected with0.1%BSA. The injection was performed every2days for4times with a volume of10μl for each eye. The concentrations of Shh-N for the two groups were20μg/ml (the group of Shh20) and50μg/ml (the group pf Shh50).separately. Retinoscopic refraction and A-scan measurement were performed at week2. Then, animals were killed and the eyes were encleated for pathological examination as well as scleral MMP-2/TGF-β detection with Western-blot. The results were compared with those in group of FDM2W and Control2W.Results:Intravitreal injection of Shh-N at concentration of20μg/ml and50g/ml can induce significant development of myopia of-1.54±0.75D and-4.04±1.48D, elongation of axial lengths of0.11±0.09mm and0.14±0.03mm and elongation of vitreous lengths of0.1±0.09mm and0.13±0.03mm, separately when compared with the left eyes. Of which, the myopia progressed (P<0.001) and the axial lengths elongated (P=0.0019) with the higher concentration of Shh-N. Upregulation of MMP-2in sclera was observed with the Shh-N injection from the Western-blot results. However, there was no significant difference in MMP-2between these two groups.Conclusions:Shh signaling pathway modulates eye growth and refractive development by regulating the expression of MMP-2in guinea pigs with form deprivation myopia.PART Ⅲ Effect of intravitreal injection of Cyclopamine in guinea pigs with form deprivation myopia Purpose:To investigate the effect of intravitreal injection of cyclopamine in guinea pigs with form deprivation myopia and its associated expression of scleral MMP-2/TGF-β in guinea pigs.Methods:Sixty healthy guinea pigs of2to3-week-old were randomly assigned into three groups (n=20,each). Both eyes in each group were intravitreally injected with cyclopamine at the same concentration while the right eyes in each group were wore a translucent diffuser. The injection was performed every2days for4times with a volume of10μl for each eye. The concentrations of cyclopamine for the three groups were50μg/ml (FDM50group),100μg/ml(FDM100group) and200μg/ml (FDM200group),separately. Retinoscopic refraction and A-scan measurement were performed at week2. Then, animals were killed and the eyes were encleated for pathological examination as well as scleral MMP-2/TGF-β detection with Western-blot. The results were compared with those in group of FDM2W and Control2W.Results:The right eyes in the group of FDM50、FDM100and FDM200induce relative myopia of-2.69±1.15D.-1.46±0.91D and-0.38±0.81D, separately. Intravitreal injection of cyclopamine at each concentration can significantly slow the development of myopia, among which less myopia was developed in the FDM200group compared with that in the FDM50group (P<0.0001). The right eyes in the group of FDM50. FDM100and FDM200induce relative axial elongation of0.09±0.05mm、0.06±0.04mm and0.04±0.02mm, separately. Cyclopamine of100μg/ml and200μg/ml can slow the axial growth induced by FDM. The relative elongations of vitreous lengths were0.09±0.05mm、0.07±0.05mm and0.04±0.05mm for the FDM50、FDM100and FDM200group, separately. Cyclopamine of200μg/ml can slow the axial growth induced by FDM to a greater degree than cyclopamine of100μg/ml and50μg/ml. Downregulation of MMP-2in sclera was observed in the FDM100and FDM200group compared with that in the FDM2W group.Conclusions:Intravitreal injection of cyclopamine can slow the development of myopia and the axial growth by downregulating the expression of MMP-2in sclera. Cyclopamine, the inhibitor of Shh signaling pathway, may be one of the ways for myopic prevention and treatment.