Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro

Abstract

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1a (HIF-1a) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1a induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1a and EPO for the possible treatment of stroke. Keywords: erythropoietin, hypoxia-inducible factor-1, hypoxia/ischemia injury, neuroprotection. Erythropoietin (EPO) has emerged as a potent neuroprotectant in vivo and in vitro (Morishita et al. 1997;Bernaudin et al. 1999;Siren et al. 2001a). In the brain, EPO gene expression is regulated by the transcription factor hypoxiainducible factor-1 (HIF-1), which is activated by a variety of stressors, including hypoxia (Semenza 2000). EPO-induced neuroprotection is mediated by interaction with the cognate receptor EPOR (Chong et al. 2002;Marti 2004). The main cellular source of intrinsic EPO in the brain appears to be astrocytes (Masuda et al. 1994;Marti et al. 1996). In addition Bernaudin et al. (2000) provided direct evidence that not only astrocytes but also neurons express and produce EPO after hypoxia.However, there are no studies that compared the EPO expression levels and the neuroprotective effect between mixed neuronal/astrocytic cultures and neuron-rich cultures. In the present study, we determined EPO expression levels and its neuroprotective effect using both mixed neuronal/astrocytic cultures and neuron-rich cultures exposed to hypoxia and reoxygenation. We considered that the in vitro model of mixed culture might be more physiological than separate cultures for addressing the protective role of intrinsic astrocytic-neuronal signaling in hypoxic/ischemic injury. Furthermore, we compared neuronal survival in the presence or absence of antibodies against EPOR to examine whether neuroprotection i...

Cdk5 interacts with Hif-1α in neurons: A new hypoxic signalling mechanism?

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AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease

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Neuroprotection by erythropoietin administration after experimental traumatic brain injury

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