Neuroprotective Effects Of Erythropoietin (EPO) On Rat Retinal Neurons Hypoxia Injuries In Vitro

Objective Recent studies indicates that common character of some eye diseases,such as age related macular degeneration, retinitis pigmentosa, glaucoma,retinal detachment inherited retinal dystrophies are photoreceptor and nueron death by apoptosis leads to loss of vision. In the current study, apart from its hematopoietic function, erythropoietin(EPO) exerts neuroprotective activity upon reduced oxygenation or ischemia of brain . To examine whether EPO has neuroprotection on the hypoxic injuries of retinal neuron cells, We establish a culture method of retinal neuron cells for further research on neurons cells and study the protective effect s of recombinant human erythropoietin (rhEPO ) pretreatment on rat retinal neurons injuries induced by defect of oxygenation in vitro.Methods Retina of postnatal Wistar rats was dissected into cell suspension by using trypsin and EDTA digestion. Immuniocytochemistry identified neuron cells using anti-rat neurofilament( NF) antibody after 3days. rat retinal neuron cells were subjected to C0CL2 to develop hypoxic preconditioning , following acute hypoxia model. The expression of EPO and EPOR genes were detected with semi-quantitative RT-PCR method and immunocytochemisty. After being treated with rhEPO, the protective effects of the drug on neuronal cells injury and apoptosis were observed.Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay demonstrated the neuron apoptosis iuduced by hypoxia.Results MTT assay indicated that hypoxic preconditioning could increase neuronal cells viability, the mechanism may be related with up regulation EPO and EPOR genes expression. Normal retinal neuron cells only expressed EPOR gene faintly, the expression of EPO and EPOR genes were elevated in hypoxic preconditioning and control group from lh to 24h, reached the peak at lh. The mRNA expression of EPO and EPOR genes were significantly increased as compared with control group (P<0. 01). Immunocytochemisty assay indicated the protein of EPO and EPOR located in neuronalcell's body and neurite . After hypoxic preconditioning, the protein were expressed strongly. Being treated with rhEPO(2.5-40 U/ml) the hypoxia injuries of neuronal cells relieved effectively, rhEPO significantly protected retinal neurons from cell death. TUNEL assay indicated the apoptosis lowered markedly after rhEPO pretreatment. For further research and determine the expressions of erythropoietin (EPO) gene in retinal MUller cells and the influence of hypoxia on the expressions of EPO gene , MUller cells were purified with the mechanically dissociated and strike repeatedly method , retinal Muller cells were cultured successfully , which showed more than 95% cells were glial fibrillary acidic protein(GFAP) positive by using immunostaining. Rat retinal Mtlller cells expressed EPO protein and mRNA faintly. After hypoxia, the expression of EPO mRNA and protein in retinal Muller cells was elevated obviously, which was positively related to the time of hypoxia.Contusions Successful culture of retinal neuron cells and well developed neurons in vitro is helpful in the research of retinal diseases. Trypsin combined ethylene diamion tet raacetic acid (EDTA) is better in digestioning epithelium of neurosensory retina. rhEPO pretreatment could effectively protect the neuronal cells from injuries induced by hypoxia., which was positively related to the time and dosage.Hypoxia can increase the expression of EPO mRNA and protein, which may play an important neuronal protection.role in retinal hypoxia diseases.