Evaluation of the neuroprotective effects of synthetic erythropoietin derivatives in a mouse model of mild and moderate hypoxic-ischemic encephalopathy

Introduction: Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal brain injury and long-term neurodevelopmental impairment, and current therapies only partially prevent adverse outcomes. Although recombinanterythropoietin is neuroprotective via EPOR–CD131 (βcR), its use is limited by hematopoietic side effects, motivating evaluation of non-hematopoietic peptide analogs such as Ara-290 and Epobis in HIE. Materials and Methods: Neonatal hypoxia-ischemia was induced in 9-day-old CD-1 mice (n = 204) using a modified Rice–Vannucci model, and animals were stratified into mild and moderate severity groups 3 hours later by laser speckle contrast imaging (RFLSI-ZW) before assignment to treatment. EPO (5000 IU/kg) and Epobis (20 mg/kg) were administered subcutaneously every 24 hours and Ara-290 (30 µg/kg) intraperitoneally every 12 hours for 7 days, after which motor-coordination performance, macroscopic lesion volume, and expression of IL-4, IL-1b, IL-6, and TNF-α were assessed. Results: In both mild and moderate HIE, Epobis provided the most consistent neuroprotection, improving motor performance and neurological outcomes and being the only treatment to significantly reduce macroscopic lesion volume. EPO produced moderate functional benefits, whereas Ara-290 showed a less stable efficacy profile, with limited or absent effects in several behavioral and morphological endpoints. Conclusion: In mild HIE, Epobis showed the strongest neuroprotection, improving motor-coordination performance, reducing neurological deficits, and producing the greatest reduction in macroscopic lesion volume, whereas EPO and Ara-290 had only moderate effects. In moderate HIE, Epobis and EPO most consistently improved motor outcomes, but only Epobis significantly reduced lesion volume and neurological symptom severity and produced the most pronounced immunomodulation.