Mitochondrial Oxidative Stress In A Rat Model Of Chronic Intraocular Pressure Elevation And Manganese Superoxide Dismutase Gene Therapy

Purpose:to investigate factors of oxidative stress in a rat model of chronic intraocular pressure(IOP) elevation,and injury to retinal ganglion cell(RGC) and mitochondria;deliver manganese superoxide dismutase (MnSOD) to retina then produce IOP elevation,and to find the effects on retinal ganglion cells and the possible mechanism.Methods:1. experimental glaucoma was induced unilaterally using a diode laser with wavelength of532nm aimed at the trabecular meshwork and episcleral veins,finding the histologic examination of retina in1month,2month,3month and6month,and rats grouped to4groups:G1,G2,G3,G6, using TUNEL Assay to detect the apoptosis of RGCs,detection of RGCs by immunolabeling Brn-3b;2. superoxide assays to messure Mn-sod and catalase activity, realtime-PCR to detect caspase3,manganese superoxide dismutase and catalase mRNA expression in rats'retina; using Western Blot method to detect caspase-3;3. and to contract all the factors to the groups of post intraocular injections of AAV-SOD in left eyes and then after21days produce IOP elevation1,2,3and6months, Western Blot to detect MnSOD expression to make sure the transmission succeed;Western Blot to detect nuclear gene OPA1and DRP1protein expression,and using realtime-PCR to test the mRNA expression.4. detect optic atrophy type1protein(OPA1) and dynamin-related protein1(Drp-1) expression in glaucoma group and transmission group.Results:1. pathology examination and transmission electron microscopy of the retina confirmed the loss of RGCs and mitochondrion getting edema and cracking in glaucoma rats'retina,Brn-3b positive retinal ganglion cells getting decreased as the time went by; TUNEL shows the number of apoptosis cells in retinal getting increased as the time went by;2. In glaucoma group,SOD and CAT activity getting decreased with the time went by(p<0.01),but we find the mRNA of SOD and CAT getting increased(p<0.05),the MDA levels getting increased compared to control group(p<0.01);when the intraocular pressure getting normal,that is in the6th month,RGCs'numbers still getting decrease (p<0.05), TUNEL shows the number of apoptosis cells in retinal still getting increased, SOD and CAT activity still getting decreased(p<0.01),and caspase-3expression still getting up (p<0.05); OPA1in mitochonrial decreased and drp1increased in glaucoma groups (p<0.05);3. pathology changes in therapy groups are less significant change than glaucoma group; Brn-3b positive retinal ganglion cells getting increased and TUNEL shows the number of apoptosis cells in retinal decreased significantly;caspase-3increased than normal group,but less than glaucoma group;with time went by (p<0.05),the therapy group shows up-express OPA1L and OPA1S,and down express Drp-l;but at the6th month,OPA1L expression has a little down expression in therapy group;Conclusions:1. we modified the delivery of laser energy to the anterior structures of the rat eye to develop a model of glaucoma in rats,and IOP getting higher than normal lasting for12weeks;2. Antioxidant activity was significantly lower in glaucoma group,and when IOP getting normal,we still find damage to retina;3. Antioxidant activity was significantly lower in glaucoma group,but mRNA up-express,maybe protein translation be blocked by some reason;4. a significant change of OPA1and Drp-1expression shows Drp-1translocation into mitochondria,and OPA1translocation from mitochondria to cytoplasm,contributes to the mechanism of mitochondrial fission in retina after elevated intraocular pressure.5. expression of MnSOD increasing can protect retinal ganglion cells from oxidative stress after elevated intraocular pressure,and MnSOD can improve mitochondrial fussion,reduce mitochondrial fission,but MnSOD can't diminish all the damage in retina.