Objective
Recently, Erythropoietin (EPO) has emerged as a potent neuroprotectant against ischemic/hypoxic injury. In this study, we attempt to detect the expression of EPO regulated by hypoxia-inducible factor-1α (HIF-1α) in astrocytes and neurons, and investigate the neuroprotective effect of endogenous EPO and exogenous recombinant human Erythropoietin (rhEPO) against in vitro ‘ischemia’-hypoxia by use of primary pure neuronal culture and mixed neuronal/astrocytic culture models.
Methods
Primary pure neuronal culture and mixed neuronal/astrocytic culture were obtained from embryos (E 16∼17) of wistar rats. On Day 8, cultures were placed into a hypoxic incubator (2% O2) for 30 min∼ 48 h. We detected the localization of EPO and EPO receptor (EPOR) in astrocytes or neurons by immunocytochemical staining and double-immunofluorescence staining. The expression of HIF-1α and EPO between mixed cultures and pure neuronal cultures compared by western blot and reverse transcription–polymerize chain reaction (RT-PCR). After hypoxia, neuronal survival between mixed and pure neuronal culture was assessed by means of staining with the dye presidium iodide (PI) and fluoresce indiacetate (FDA). Recombinant human Erythropoietin (rhEPO) was applied 1∼48 h before hypoxia, at the start of hypoxia and after onset of re-oxygenation, and the neuronal survival was assessed.
Results
Immunoreactive EPO was prominent in astrocytes, especially in the cytoplasm, and also observed in neurons with weaker staining after 3 to 48 h hypoxia. EPOR was strongly detected in neurons. Western Blot analysis showed that the EPO band of neuron cultures was weaker than that of mixed neuronal/astrocytic cultures. HIF-1α mRNA expression was observed after 30 minutes hypoxia preceding EPO expression coincident with protein level. Re-oxygenation completely degraded the expression of HIF-1α and EPO. Neurons cultured together with astrocytes were significantly rescued by endogenous EPO compared with pure neuronal cultures. Both EPO and EPOR antibody (2.5 μg/ml) were able to reduce the endogenous neuroprotection (P<0.0001). Administration of rhEPO (0.1 U/ml) within 6 h before hypoxia or after onset of reoxygenation significantly increased neuronal survival compared with hypoxia or hypoxia-reoxygenation alone (p<0.0001).
Conclusion
Our findings indicated that HIF-1á target gene EPO released by astrocytes acts as an essential mediator of neuroprotection, suggested a critical role of EPO in cerebral ischemia and promote the possible therapeutic application and beneficial effect for the treatment of stroke patients. Furthermore, imitation of brain endogenous protective mechanisms may be another novel strategy to future successful approaches to neuroprotection against hypoxic/ischemic injury.