Protective Effects And Mechanism Of C-Ski On Optic Never Injury In Glaucoma

Part oneEstablishment of N-methyl-D-aspartate（NMDA）-induced normal-tension glaucoma rat modelObjective:To explore the optimal scheme of N-methyl-D-aspartate（NMDA）-induced normal-tension glaucoma rat model.Methods:Thirty-six Sprague-Dawley male rats were randomly distributed into 4groups.Nine rats were in normal group（group A）.The rest of 27 rats were equally divided into 3 groups（groups B,C,D）to receive intravitreal injection of 3 different concentrations（5μl）of NMDA（20 nmol,40 nmol,and 80 nmol）into the right eyes.The left eyes received intravitreal injection of 5μl PBS used as a control（group E）.Three days,seven days and fourty days after the intravitreal injection of NMDA,all rats were killed and the eyes were enucleated.The ganglion cell complex（GCC）thickness was measured by Hematoxylin/eosin staining.The apoptosis index（AI）of cells in the ganglion cell layer（GCL）was detected by TUNEL staining.Results:1.Intravitreal injection of NMDA（groups B,C,D）caused GCC become thin around the optic disc in the rat retina,compared with un-injected rats in group A and E（P<0.05）,and group C is thinner than group B（P<0.05）.The thickness of GCC in group B,C,D on 14d is thinner than those on 3d and 7d（P<0.05）.There was no significant difference between group A and group E,between group B,C,D on 3d and 7d,and between group C and group D（P>0.05）.2.It is obvious that rats in groups B,C and D had a dramatically higher of cells AI in GCL,compared with those in group A and group E（P<0.05）,cells AI in GCL in group C is higher than that in group B（P<0.05）.The cells AI in GCL of group B,C,D on 14d is higher than those on 3d and 7d（P<0.05）.There was no obvious difference between group A and group E,group B,C,D on 3d and 7d,as well as group C and group D（P>0.05）.Conclusion:The optimal dosage of NMDA was 40nmol to establish normal-tension glaucoma rat model,and the best time of testing was 14 days after intravitreal injection of NMDA.Part twoConstruction of recombinant type II adeno-associated viruses-mediated c-Ski overexpression and interferenceObjective:To construct recombinant type II adeno-associated viruses（rAAV2）-mediated c-Ski overexpression and interference vectors and obtain the effective concentration of gene therapy.Methods:To pack recombinant type II adeno-associated viruses（rAAV2-c-Ski/GFP）,we overexpressed c-Ski and GFP gene at the same time and confirmed that the purpose protein expression in cell level.The recombinant type II adeno-associated viruses（rAAV2-GFP）only expressed GFP gene used as control.The virus titer was detected by Real-time PCR.We used the same methodtobuildrecombinanttypeIIadeno-associatedviruses（rAAV2）-mediated c-Ski interference of expression vectors.Results:The recombinant type II adeno-associated viruses（rAAV2）-mediated c-Ski overexpression vectors were constructed and confirmed by sequencing.Recombinant adeno-associated viruses rAAV2-c-Ski/GFP and rAAV2-GFP were produced in 239T cells and the titer of both viruses was 1.55×10¹²² v.g./ml.We packed type II adeno-associated viruses-mediated c-Ski interference virus（rAAV2-c-Ski-shRNA/GFP and rAAV2-NC-shRNA/GFP）with the same method,and the titers were 2.03×10¹³³ v.g./ml and 1.56×10¹³³ v.g./ml.Conclusion:We constructed recombinant type II adeno-associated viruses-mediated c-Ski overexpression and interference vectors successfully and confirmed the efficiency of virus titers in the 293T cells.Part three To study the effect of recombinant type II adeno-associated virus-mediated c-ski gene overexpression and interference on glaucomal optic nerve injury.Objective:To observe the transfection of recombinant type II adeno-associated virus（rAAV2）-mediated the c-Ski gene in the retina and explore the protective effects and possible mechanism of c-Ski on glaucomal optic nerve injury.Methods:Sixty Sprague-Dawley male rats were distributed into 5 groups:normal group（group A,n=12）;glaucoma group（group B,n=12）accepting intravitreal injection of 40 nmol NMDA;glaucoma+rAAV2-c-Ski/GFP group（group C,n=12）accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-c-Ski/GFP;glaucoma+rAAV2-GFP group（group D,n=12）accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-GFP and normal+rAAV2-c-Ski/GFP group（group E,n=12）accepting intravitreal injection of rAAV2-c-Ski/GFP.Another 60 rats were randomly distributed into 5 groups:normal group（group a,n=12）;glaucoma group（group b,n=12）accepting intravitreal injection of 40 nmol NMDA;glaucoma+rAAV2-c-Ski–shRNA/GFP group（group c,n=12）accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-c-Ski-shRNA/GFP;glaucoma+rAAV2-NC-sh RNA/GFP group（group d,n=12）accepting intravitreal injection of NMDA plus intravitreal injection of rAAV2-GFP and normal+rAAV2-c-Ski-sh RNA/GFP group（group e,n=12）accepting intravitreal injection of rAAV2-c-Ski-shRNA/GFP.Another 8 healthy Sprague-Dawley male rats were randomly divided into 2 groups:group G accepting intravitreal injection of rAAV2-GFP and group S accepting intravitreal injection of rAAV2-c-Ski/GFP.All rats in group C,E,S received intravitreal injection of 5μl rAAV2-c-Ski/GFP of 1.55×10¹²² v.g./ml in titer in right eyes.All rats in group c and group e received intravitreal injection of 5μl rAAV2-c-Ski-shRNA/GFP of 2.03×10¹³³ v.g./ml in titer in right eyes.All rats in group D and G received intravitreal injection of 5μl rAAV2-GFP of 1.55×10¹²v.g./ml in titer in right eyes.All rats in group d received intravitreal injection of5μl rAAV2-NC-shRNA/GFP of 1.56×10¹³³ v.g./ml in titer in right eyes.After two weeks,all rats in group B（b）,C（c）and D（d）received intravitreal injection of 5μl 40 nmol NMDA in right eye.At 4 weeks,Flash-visual evoked potential（F-VEP）and electroretinogram（ERG）were recorded under scotopic conditions to assess the retinal function.We observed the transfection of recombinant type II adeno-associated virus（rAAV2）–mediated the c-Ski gene in the retina by frozen section of the retina.Hematoxylin/eosin staining was performed on retina cross-sections for measurement of ganglion cell complex（GCC）thickness.Apoptosis index（AI）of cells in the ganglion cell layer（GCL）was assessed by TUNEL on retina cross-sections.The protein level of c-Ski,TGF-β2,Bax,Bcl-2,Smad2/3,Smad4,p-Smad2/3,FN,LN were detected by Western blotting.Results:1.Fluorescent microscopy found that rAAV2 mediated c-Ski genes transfected successfully in the retina（in groups G and S）.Most GFP+-transfected cells were located in the GCL.2.Compared with rats in group A（a）and group E（e）,there was a significant reduction in the amplitudes of the F-VEP amplitudes and b-waves of the ERG in the rats of intravitreal injection of NMDA（P<0.05）).Administration of rAAV2-c-Ski/GFP mitigated the reduction of the F-VEP amplitude and the b-waves amplitude of ERG in the glaucomatous eyes（P<0.05）.However,administration of rAAV2-c-Ski-shRNA/GFP aggravated the reduction of the b-waves amplitude of ERG and the F-VEP amplitude in the glaucomatous eyes（P<0.05）.3.Intravitreal injection of NMDA caused GCC become thinn in the rat retina,compared with normal rats in group A（a）and group E（e）（P<0.05）.Administration of rAAV2-c-Ski/GFP mitigated the changes and significantly increased the thickness of GCC（P<0.05）,compared with rats in group B and D.But,administration of rAAV2-c-Ski-shRNA/GFP aggravated the changes and significantly thinned the thickness of GCC（P<0.05）,compared with rats in group b and d.4.It was evident that rats of group B（b）,group C（c）and group D（d）had a distinctly higher of TUNEL cells number of RGC,compared with rats in group A（a）and group E（e）（P<0.05）.Administration of rAAV2-c-Ski/GFP mitigated the changes and significantly reduced the TUNEL cells in RGCs（P<0.05）,compared with ratsingroupBandgroupD.However,administrationof rAAV2-c-Ski-shRNA/GFP aggravated the changes and significantly increased the TUNEL cells in RGCs（P<0.05）,compared with rats in group b and d.5.The protein level of c-Ski,TGF-β2,Bax,Bcl-2,Smad2/3,Smad4,p-Smad2/3,FN,LN was detected by Western blotting.The expression of c-Ski was increased in the eyes that received intravitreal injection of rAAV2-c-Ski/GFP（P<0.05）.However,the expression of c-Ski was decreasedintheeyesthatreceivedintravitrealinjectionof rAAV2-c-Ski-shRNA/GFP（P<0.05）.There was a marked decrease in the level of Bcl-2 with a concomitant increase in the level of Bax protein in the eyes of group B（b）,group C（c）and group D（d）,relative to the rats in group A（a）and group E（e）（P<0.05）.Moreover,the expression of TGF-β2,p-Smad2/3,FN,LN in group B（b）,group C（c）and group D（d）was significant increased,compared with rats in group A（a）and group E（e）（P<0.05）.In group C,the eyes received intravitreal injection of rAAV2-c-Ski/GFP caused the protein level of TGF-β2,p-Smad2/3,FN,LN reduced,relative to rats in group B and group D（P<0.05）.However,the eyes of group c received intravitreal injection of rAAV2-c-Skish-RNA/GFP caused the protein level of TGF-β2,p-Smad2/3,FN,LN further increased,compared with the rats in group b and group d（P<0.05）.Conclusion:1.The rAAV2 successfully mediated the expression of c-Ski gene in the retina,and the positive transfection cells were mainly located in the retinal of GCL.2.Intravitreal injection of rAAV2-c-Ski/GFP could reduce the RGC apoptosis induced by NMDA.It may be a candidate therapy for neuroprotective in the treatment of glaucoma.The possible mechanism is that c-Ski inhibits TGF-β/Smad signaling pathway via disturbing Smad2 and Smad3phosphorylation,causing the retinal ECM deposition reduction to control the RGC apoptosis.