Study On Neuroprotective Effect Of Erythopoietin Pretreatment On Cerebral Ischemia Injury In Vitro

Background: Direct and indirect oxygen-glucose deprivation,which is caused by edema compres sion and mass effect,could bring about the damage of brain tissue’s neurons and nerv e fibers,resulting in motion, sensory, mental, cognitive disorders.The potential mechanis ms are energy burn-out,free radical formation, calcium overload,cells apoptosis and so on.Neurons of the hippocampus and cortex are ones of the most important functioni ng cells of central nervous system and a strict extracellular environment is needed fo r their energy requirement and function sustentation. Thus reaserchs on how to increa se these cell’s tolerance to hypoxia ischemia are very meaningful and promising.As f or erythopoietin pretreatment,the best application conditions does not reach unanimity o f opinion.Besides, most current studys on preventive treatment of ischemic cerebrovas cular disease with erythopoietin are animal-model-based.However, erythopoietin lacks ability to penetrate the blood brain barrier and may be disturbed in vivo.Objective: To study the protective effect of recombinant human erythopoietin(rhEPO) pretrea tment on cerebral ischemia injury and to explore the suitable dosage range and time win dow in vitro.Methods: Isolate and culture cerebral cortex cells from neonatal Wistar rats born in 24 h.By using specified medium for neurons to promote cell growth and adding 5-fu to s uppress the proliferation of gliocyte,the primary cultured cerebral cortex neurons of hi gh purity could be achived,tested by streptavidin-perosidase(SP). Then the neurons we re damaged by OGD to establish cerebral ischemia models in vitro,which was identifie d with MTT assay.Further more,the primary cultured cerebral cortex neurons of high purity at stable growth phase were divided into thee groups,namely the normal contro l group,oxygen-glucose deprivation group and rhEPO pretreation group. RhEPO in different concentration(0.01U/ml、0.1U/ml、1U/ml、10U/ml、100U/ml) were given at di fferent time points(24h,48 h,72h before OGD) in rhEPO pretreation group.The viabil ity of cells was determined by MTT assay and cell morphology was observed under light microscopy.Results: 1.Cortical neurons of neonatal Wistar rats could be primarily cultured separately with specified medium for neurons, and the medium was mixed with 5-fu so as to s uppress the proliferation of gliocyte. Identified by SP, the percentage of NSE positive cells could reach up to 92.33%±2.88%. 2.The neurons grew stably from the sevent h to the eleventh day. 3.Compared with the other groups,the optimum oxygen-glucose-deprivation time was 15 h and accordingly the survival rate was 63.25±8.56(P <0.05). 4.Compared with the OGD group,rhEPO pretreation at the concentration of 0.01,0.1,1,10 U·ml-1 could significantly increase the viability of neurons damaged by OG D(P<0.05) repectively and when the concentration of rhEPO was 1U·ml-1,the protective effect was strongest(P<0.05). 5.Compared with the OGD group,before OGD preincub ation of neurons with 1U/ml rhEPO for 24 h or 48 h induced a prolonged neuroprote ct(P<0.05).The maximum of protection was observed after a pretreatment period of 48h(P<0.05). 6.These findings could also be proven by the improved morphology of n eurons.Conclusions: 1.Neurons with relatively high purity can be cultured in vitro by using specified medium for neurons and adding 5-fu to suppress the proliferation of gliocyte, which can be used for the study of their relative features from the seventh to eleventh day. 2. OGD for 15 h was appropriate to establish cerebral ischemia models in vitro. 3.Our studies had revealed the markedly neuroprotective effect of rhEPO pretreatment on the cerebral cortical neurons in OGD.Its effective concentration was 0.01-10U/ml and the most effective concentration was 1U/ml. Further more,the effective time windows was 48 h or 24 h before OGD,of which 48 h before OGD was better.4. RhEPO pretreatment had a dual regulation effect on cell viability. The up-regulated effect was observed in the concentration of 0.01-1U/ml.When the concentration exceeded 1U/ml,it negatively moderated the cell viability as the concentration increased.