Neuroprotective effects of erythropoietin posttreatment against kainate‐induced excitotoxicity in mixed spinal cultures

Abstract Although the neuroprotective effects of erythropoietin (EPO) preconditioning are well known, the potential of postapplied EPO to protect neurons against excitotoxic injury has not been clearly established. Here we show that kainate (KA)‐induced excitotoxicity, which plays a key role in secondary spinal cord injury, decreased neuron survival, inhibited neurite extension, and significantly reduced the expression of erythropoietin receptors (EpoR) in cultured spinal neurons. Posttreatment with EPO for 48 hr protected neurons against KA‐induced injury, opposing KA‐induced apoptosis and promoting regrowth of motoneuron neurites. These neuroprotective effects were paralleled by a restoration of EpoR expression. The importance of the EpoR signaling pathway was demonstrated using an EpoR blocking antibody, which neutralized the neuroprotective action of EPO posttreatment and prevented EPO‐induced increases in EpoR expression. We also found that up‐regulated EpoR stimulated the Janus kinase 2 (JAK2) pathway, which is known to facilitate neuronal growth and neurite regeneration. Although EPO posttreatment modestly attenuated KA‐induced reactive gliosis in mixed neuron–glial cultures, blocking EpoR activity did not alter glial fibrillary acidic protein expression or astrocyte proliferation. In conclusion, 48 hr treatment with EPO following KA exposure induced EpoR‐dependent protection against excitotoxic injury, demonstrating that preconditioning is not a prerequisite for neuroprotection by EPO. The neuroprotective effects of EPO posttreatment were mediated by an EpoR‐dependent signaling pathway that possibly involves JAK2. The neuroprotective effect of EPO posttreatment against KA excitotoxicity appears to reflect direct effects on neurons and not indirect effects mediated by astrocytes. © 2008 Wiley‐Liss, Inc.