The Role And Mechanism Of KCNQ5 Gene And Channel In The Occurrence And Development Of Myopia

Part 1 The effect of form-deprivation myopia on the expression of KCNQ5 and the potassium ion microenvironment in the retina of guinea pigsObjectives:The aim of this study was to investigatethe expression of retinalpotassium channel gene KCNQ5 in form deprivation myopia(FDM)guinea pigs.Methods:A totalof 54 guinea pigs were randomly divided into the normal control(NC)group,theself-control(SC)group,and the form-deprivation(FD)group for different treatments.Refraction and axial length were measured at 5 time points(0,1,2,3,and 4 weeks after form deprivation).Molecular assays and immunohistochemistry(IHC)were conducted to measure theexpression and distribution of KCNQ5-related gene and protein in the retina.Results:The FD group exhibited more myopic refractionand longer AL.The results of IHC showed that KCNQ5 was expressed in the retinal ganglion cell layer,the inner and outer plexiform layers,the inner and outer segments of photoreceptors,and near the RPE.The results of Real-Time PCR and Western Blot showed that the mRNA level(all P<0.01)and protein level(all P<0.01)of KCNQ5 were significantly decreased in FDM.Conclusions:The expressionof KCNQ5 was significantly downregulated in mRNA and protein levels in the retina of guinea pigs with FDM.Itsuggested that the retinal potassium channel gene KCNQ5 may play a role in the occurrence and development of myopia.Part 2 The effect of form deprivation myopia on potassium microenvironment in theretina ofguinea pigObjectives:The aim of this study was to investigate the effect of form deprivation myopia(FDM)on K+ homeostasis in retina and M-type K+current(IK)in retinal pigment epithelial(RPE)cells guinea pig.Methods:A totalof 36 guinea pigs were randomly divided into the normal control(NC)group,theself-control(SC)group,and the form-deprivation(FD)group for different treatments.Refraction and axial length were measured at 5 time points(0,1,2,3,and 4 weeks after form deprivation).After 4 weeks of form deprivation,the K+ concentration detection kit was used to detect the difference of K+ concentration inretina.Whole-cell patch clamp technique was used to study the effect of FDM on IK in RPE cells,and the pharmacological sensitivity of KCNQ5activator Retigabine and inhibitor XE991.Results:The K+concentration level of retina in the FD group was higher than the NCand the SC group(all P<0.01).The Patch-clamp electrophysiological results showed that the IK density decreased in RPE cellsofthe FD group(all P<0.01),and were activated or inhibited in a concentration-dependent manner due to the addition of Retigabine or XE991.Conclusion:The K+ homeostasis in the retina of guinea pigs with FDM was unbalanced,and the IK activity was blocked.In addition,the activation and inhibition of KCNQ5 channel can further affect the changes of IK.Part 3 Theeffect of KCNQ5 channel intervention on the development of form deprivation myopia in guinea pigsObjective:To observe the effect of activation and inhibition of KCNQ5 channel on KCNQ5 expression and the development of guinea pig myopia by intravitreal injection of KCNQ5 channel activator Retigabine and inhibitor XE991.Methods:Eighty-four guinea pigs(3-week-old)were randomly divided into 5 groups,normal control group(NC),form deprivation group(FD),solvent group(FD+DMSO),Retigabinegroup(FD+Retigabine)and XE991 group(FD+XE991).The injection concentrations of the Retigabine group were 100μM and 500μM,and the injection concentrations of the XE991 group were 20μM,50μM and 100μM,and the solvent group was injected with normal saline containing 10%DMSO,and the control group was not treated.Injections were given every 3 days at a fixed time.Refraction and axial length were measured at 5 time points(0,1,2,3,and 4 weeks after form deprivation),and IOP was examined after every injection.After 4 weeks of form deprivation,eyeballs were taken to detect the expression of KCNQ5 at transcription and protein levels,and TUNEL staining was used to detect the cell apoptosis.Results:After 2 weeks of form deprivation,the Retigabine group with different concentrations developed lower myopiaand shorter axial length than the FD group(all P<0.05),and the differences were statistically significant;the XE991 group with different concentrations developed highermyopia andlonger axial length than the FD group,and the difference between the FD group and the 20μM concentration group was not statistically significant(P=0.73,P=0.82,respectively),and the differences between the 50μM(all P<0.05)and 100μM concentration groups(all P<0.01)were statistically significant.There was no significant difference in intraocular pressure among the groups(all P>0.05).The results of Real-Time PCR and Western Blot showed that the mRNA level(P<0.05,P<0.01,respectively)and protein level(all P<0.01)of KCNQ5 in Retigabine groups were higher than the FD group,and these XE991 groups were lower than the FD group(P=0.16,P<0.05,P<0.05;P=0.07,P<0.01,P<0.01,respectively).The results of TUNEL staining showed that there were no significant differences in the positive cell rates of all retinal layers.Conclusions:Intravitreal injection of KCNQ5 activators and inhibitors can play a certain positive and negative role in the development of form deprivation myopia in guinea pigs,and KCNQ5 channel may be involved in the occurrence and development of myopia.