Figure 1.

Protection by EPO is concentration and time dependent during diminished expression of the EPOR during NO exposure. (A) Primary hippocampal neuronal cultures were subjected to immunocytochemical detection for EPO (Aa, Ab) and the EPOR (Ac, Ad) by using a rabbit primary polyclonal anti-EPO and anti-EPOR antibodies. For EPO and EPOR detection, representative images are displayed for control cells (untreated neurons) (Aa, Ac) and for cells 24 h following exposure to the NO donor NOC-9 (300 μM) in the adjacent panels (Ab, Ad). (B) Quantitation of the percentage of neurons expressing EPO or the EPOR at 24 h following exposure to either NOC-9 (300 μM) or SNP (300 μM) is shown (^*P<0.01 vs untreated control). (C) Neurons were pretreated with EPO (0.001–100 U ml⁻¹) 1 h prior to exposure to a NO donor (NOC-9 or SNP, 300 μM) and cell survival was assessed 24 h later. Protection of EPO against NO toxicity was evident in cultures with EPO (0.01–10 U ml⁻¹) when compared with cultures exposed to NO alone (^*P<0.01 vs NO treated alone). (D) Protection of EPO was evident in post-treatment paradigms during NO toxicity. Neurons were treated with EPO (1 U ml⁻¹) at 2, 4, 6, and 12 h following NO exposure (NOC-9 or SIN-1, 300 μM). Post-treatment with EPO at 2, 4, and 6 h following NO exposure increased neuronal survival significantly 24 h following NO exposure (^*P<0.01 vs NO treated alone). (E) EPO (1 U ml⁻¹) was pre-administered at 1, 3, 6, 14, and 24 h prior to NO exposure and neuronal survival was assessed 24 h following NO application (NOC-9 or SNP, 300 μM). Administration of EPO at 1, 3, and 6 h prior to NO exposure generated the highest levels of neuronal survival, but EPO applications provided at 14 and 24 h resulted in decreased efficacy for neuroprotection by EPO (^*P<0.01 vs NO treated alone; †P<0.01 vs 24 h pretreated group). In (B, D, E), to simplify the figures, the results for the two NO donors were combined.