Study On The Mechanism Of Retinal Neurons' Apoptosis And The Retinal Neuroprotection In Early Diabetic Rats

Objective1.Observe the pathology change of the retinal structure,the ultrastructure change of the retinal neurons,and the apoptosis of the neurons prolong with the diabetic process in the STZ-induced diabetic rats.2.Study the activity and gene expression of MnSOD and Cu/ZnSOD,observe the expression of capase-3,and investigate their relationship with the neurons' apoptosis,to discover the apoptosis mechanism of the retinal neurons in the early diabetic rats.3.Observe the activity and gene expression of MnSOD and Cu/ZnSOD, caspase-3 mRNA expression and apoptosis of the neurons in the diabeitc rats with the melatonin treament,to discover the neuroprotection mechanism in the diabetic retina.Methods1.The rats were injected with a single dose of streptozotocin to induce the diabetic model.Observe the pathological change of the retinal structure,and the ultrastructure of the retinal neurons by electron microscope,and detected the apoptosis of the retinal neurons by TUNEL assay.2.Xanthine oxidase method were used to measure the activity of the total SOD,MnSOD and Cu/ZnSOD,MnSOD mRNA,Cu/ZnSOD mRNA and caspase-3 mRNA expression were determined by SYBR Green Realtime PCR Master Mix,the protein expression of caspase-3 were detected by immnohistochemistry.3.The diabetic rats were treated with melatonin(10mg/kg/d,gastric perfusion),observe the activity of T-SOD,MnSOD,Cu/ZnSOD and gene expression of MnSOD,Cu/ZnSOD,and caspase-3 mRNA expression and the apoptosis of the retinal neurons by above methods. Results1.In diabetic rats,the pathological change of the retinal structure displayed the decrease of the retinal ganglion cells' number,and the signs of the apoptosis in different degrees could be found in the altrastructure of retinal neurons,including:cell shrinkage,chromatin margination and nuclear collapse,and so on.The apoptosis of the retinal neurons located in the rentinal ganglion cells layer(GCL)and the inner nuclear layer(INL),the apoptosis degree of the retinal neurons increased prolong with the process of diabetes mellitus.2.In diabetic rats,the activity and of the total SOD,MnSOD,Cu/ZnSOD were also declined,and accorded with the change of their gene expression.In early stage,the change of MnSOD was obvious,especially on 4 week,the change of the Cu/ZnSOD happened late,mainly on 12 week.The opsitive expression of caspase-3 protein were located in GCL and INL,the gene expression of caspase-3 enhanced prolong with the diabeitc process,and the change displayed obviously on 4 week and 8 week,the change degree were declined on 12 week.3.Comparing Mel groups with DM groups,the activity and gene expression of the retinal MnSOD and Cu/ZnSOD increased in different degrees,while the gene expression of caspase-3 and the apoptosis of the retinal neurons decreased,in early stage(4w),the melatonin mainly affected MnSOD,its effect on caspase-3 and the apoptosis of neurocytes happened on 8 week,and on Cu/ZnSOD happened on 12 week.Conclusion1.In diabetic rats,the apoptosis of the retinal neurons mainly located in the inner nuclear layer and ganglion cell layer,and were more serious prolong with the process of diabetes melltus.2.In the early diabetic stage,the activation of caspase-3 and the apoptosis of the retinal neurons increased,which may relate with the activity and gene expression of MnSOD and Cu/ZnSOD decreased,and may close relate with the change of MnSOD. 3.Melatonin could enhance the activity and the gene expression of retinal MnSOD and Cu/ZnSOD,then decrease the activation of caspase-3 and the apoptosis of retinal neurons to protect retinal neurons in diabetic rats,its effect on the MnSOD happened more earlier and obvious than Cu/ZnSOD.