Non‐additive Effects of Adjunct Erythropoietin Therapy with Therapeutic Hypothermia after Global Cerebral Ischaemia in Near‐term Fetal Sheep

Key points Recombinant human erythropoietin (rEpo) is neuroprotective in immature animals, but it is unclear whether the combination of high‐dose rEpo therapy with therapeutic hypothermia can further improve outcomes. Hypothermia and rEpo independently improved neuronal survival, with greater improvement with hypothermia, and similarly reduced numbers of caspase‐3 positive cells and reactive microglia after 7 days recovery. Hypothermia, but not rEpo, was associated with markedly improved EEG power, whereas both interventions improved recovery of EEG frequency. There was no significant improvement in any outcome after combined rEpo and hypothermia compared with hypothermia alone, and of concern, the combination was associated with increased numbers of cortical caspase‐3‐positive cells compared with ischaemia‐hypothermia. These data suggest that the mechanisms of neuroprotection with hypothermia and rEpo overlap and, thus, high‐dose rEpo infusion does not appear to be an effective adjunct therapy for therapeutic hypothermia. AbstractTherapeutic hypothermia for hypoxic‐ischaemic encephalopathy (HIE) provides incomplete neuroprotection. Recombinant human erythropoietin (rEpo) is neuroprotective in immature animals, but it is unclear whether adjunct rEpo therapy with therapeutic hypothermia can further improve outcomes. Near‐term fetal sheep received sham‐ischaemia (n = 9) or global cerebral ischaemia for 30 min (ischaemia‐vehicle, n = 8), followed by intravenous infusion of rEpo (ischaemia‐Epo, n = 8; 5000 U/kg loading dose, then 833.3 U/kg/h), cerebral hypothermia (ischaemia‐hypothermia, n = 8), or rEpo plus hypothermia (ischaemia‐Epo‐hypothermia, n = 8), from 3 to 72 h post ischaemia. Fetal brains were collected 7 days after cerebral ischaemia. Cerebral ischaemia was associated with severe neuronal loss and microglial induction in the parasagittal cortex and subcortical regions. Hypothermia reduced overall neuronal loss, cortical caspase‐3 and reactive microglia in the striatum and cortex, with greater recovery of electroencephalographic (EEG) power and spectral edge (SEF) from 48 h onwards. rEpo independently improved neuronal survival in the parasagittal cortex, hippocampal CA4 and thalamus, and reduced cortical caspase‐3 and activated microglia in striatal and cortical areas, with greater SEF from 120 h onwards. However, ischaemia‐Epo‐hypothermia did not further improve outcomes compared with ischaemia‐hypothermia and was associated with increased numbers of cortical caspase‐3‐positive cells. These findings suggest that although delayed, prolonged treatment with both hypothermia and rEpo are independently neuroprotective, they have overlapping anti‐inflammatory and anti‐apoptotic mechanisms, such that the delayed, high‐dose rEpo infusion for 3 days did not materially augment neuroprotection with therapeutic hypothermia.