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- Materials and methods
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-1α (HIF-1α) 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-1α 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-1α and EPO for the possible treatment of stroke.
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 is mediated by the intrinsic EPO and its cognate receptor. In this study, we investigated the neuroprotective effect of extrinsic recombinant human EPO (rhEPO) and determined the optimal ‘therapeutic time-window’ with respect to the EPO-induced neuroprotection in hypoxia/ischemia and ischemia/reperfusion injury. EPO was applied before hypoxic stimulation in previous studies (Sinor and Greenberg 2000; Siren et al. 2001b; Ruscher et al. 2002), but the present results demonstrated that not only pre-treatment and post-treatment applications of EPO, but its simultaneous application with the start of hypoxia and after the onset of reoxygenation represents a more relevant approach with respect to possible clinical application of this neuroprotective factor.
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- Materials and methods
The cellular response to hypoxia/ischemia is mainly controlled by HIF-1α. The HIF-1 target gene EPO has been described as neuroprotectant (Digicaylioglu and Lipton 2001; Prass et al. 2003; Jones and Bergeron 2004). The present study showed the magnitude and time course of the induction of HIF-1α and that EPO mRNA expression level paralleled protein up-regulation after hypoxia and the dose–response effects of endogenous EPO on neuronal survival by astrocytic-neuronal signaling pathway in mixed neuronal/astrocytic cultures compared with neuron-rich cultures in which the number of astrocytes was limited by cytosine arabinoside. Most previous in vitro studies used only neuron-rich cultures; however, the mixed culture of neurons and astrocytes might be more physiological for addressing the protective role of intrinsic astrocytic-neuronal signaling in ischemic/hypoxic injury. Furthermore, we also investigated the neuroprotective effect of extrinsic rhEPO and determined the optimal post-hypoxia time-window with respect to the EPO-induced neuroprotection using neuron-rich cultures.
In this study, RT–PCR and immunoblot analysis showed that in response to hypoxia/ischemia, HIF-1α as well as subsequent EPO mRNA and protein levels were significantly higher in mixed cultures compared with neuron-rich cultures after hypoxia. Consistent with the expression level of EPO, neurons cultured together with astrocytes were distinctly rescued from hypoxia/ischemia injury but were significantly damaged in neuron-rich cultures after the same periods of hypoxia (6, 12, 24 h). These findings provide support to previous studies, which provided strong evidence that astrocytes protect cultured neurons from degeneration induced by anoxia (Vibulsreth et al. 1987) or oxygen glucose deprivation (Ruscher et al. 2002). In addition, we demonstrated that the intrinsic neuroprotection is mediated by the intrinsic EPO with its cognate receptor because the neuroprotection could be blocked by anti-EPOR antibody in mixed neuronal/astrocytic cultures during 6–24 h hypoxia. In contrast, co-application of anti-EPOR antibody did not significantly alter neuronal survival compared with hypoxia alone. These results suggest that astrocytes, but not neurons, release sufficient amounts of EPO for paracrine neuroprotection. Although both astrocytes and neurons expressed EPO, we do not exclude the possibility that EPO immunostaining of neurons could be attributed to astrocytic release of EPO, which subsequently bound to neuronal EPOR.
Continuous degradation of HIF-1α protein by oxygen in normoxic cells is prevented during hypoxia, leading to stabilization and activation of HIF-1, which translocates to the nucleus and binds to a conserved sequence (5′-RCGTG-3′) near the 5′ end of the hypoxia-response enhancer of the EPO gene and up-regulates EPO gene transcription (Semenza 2000; Sharp and Bernaudin 2004). In the present study, we found that HIF-1α was up-regulated as early as 30 min after hypoxia and EPO expression subsequently increased at least after 3 h hypoxia. HIF-1α was detected not only in the cytoplasm but also in nuclei of cultured cortical cells, suggesting the translocation of HIF-1α from the cytoplasm to the nucleus in response to hypoxia/ischemia. In contrast to HIF-1α, EPO and EPOR were only expressed in the cytoplasm, as demonstrated by western blot and immunocytochemistry staining. In addition, our results showed that reoxygenation significantly degraded HIF-1α activation, and reduced subsequent transcription of the target gene EPO and the expression of EPOR.
We demonstrated that neuroprotection by rhEPO was time- and concentration-dependent. Interestingly, significant protection with EPO was achieved only in a limited concentration range (0.1–1.0 U/mL) that was also dependent on the temporal exposure of EPO. This concentration range is similar to other injury paradigms in vitro (Chong et al. 2003) and in vivo models (Grasso et al. 2002). In addition, a significant neuronal survival was achieved with administration periods closest to hypoxia exposure and within a 6-h period after the onset of hypoxia before the induction of cellular mechanisms destining a cell to die. In contrast, application times that occur after the induction of specific signal transduction pathways of cell injury appear to render EPO ineffective as a cytoprotectant. This window of opportunity for protection by EPO most likely coincides with the progressive induction of secondary cellular pathways during this 6-h time span, such as cytochrome c release and cysteine protease induction in the cerebral cortex (Lin and Maiese 2001).
For ischemia/reperfusion injury, administration of EPO immediately after the onset of reoxygenation achieved the greatest neuronal protection in contrast to the pre-administration before hypoxia. The time point can be supported by a recent study that demonstrated that treatment with EPO after the onset of reperfusion promotes the greatest protection to the myocardial structure and preserves cardiac function during ischemia/reperfusion (Lipsic et al. 2004). The time point of treatment with EPO, as in the present study, represents a more relevant approach with respect to possible clinical applications of this neuroprotective factor.
Previous studies identified several key pathways by EPO that were critical for protection against neuronal injury and apoptosis (Digicaylioglu and Lipton 2001; Ravati et al. 2001; Chong et al. 2003). The prevention of neuronal apoptosis and microglial phagocytosis by EPO following its binding to the EPOR can occur through cellular pathways that involve enhanced Akt1 activity, Bad phosphorylation, and the maintenance of mitochondrial membrane stability. Alternatively, EPO may act directly upon cytochrome c, caspase 8, caspase 3, or caspase 1 to promote neuronal survival during hypoxic insults (Chong et al. 2003). Although we could not define further the cellular mechanisms underlying neuroprotection by EPO, our detection of neuronal apoptosis generated by hypoxia and the neuroprotection of EPO are consistent with the hypothesis that EPO acts in the CNS primarily as a direct protective factor in neurons via activation of anti-apoptotic pathways (Marti 2004), and we also proved the protective role of astrocytic-neuronal signaling pathway in hypoxic injury.
In conclusion, we present experimental evidence for the functional role of intrinsic EPO in hypoxia injury in vitro, demonstrated the beneficial effects of an optimal therapeutic time-window of extrinsic rhEPO in neuroprotection against in vitro ischemia/reperfusion injury. Extrapolation of these results clinically point to the possible therapeutic application for the treatment of stroke patients. Imitation of brain intrinsic protective mechanisms may be another novel strategy to future successful approaches to provide neuroprotection against hypoxia/ischemia.