Non‐additive effects of adjunct erythropoietin therapy with therapeutic hypothermia after global cerebral ischaemia in near‐term fetal sheep

Abstract

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. Abstract Therapeutic 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.

The Effect of Size, Maturation, Global Asphyxia, Cerebral Ischemia, and Therapeutic Hypothermia on the Pharmacokinetics of High-Dose Recombinant Erythropoietin in Fetal Sheep

“…Erythropoietin is a hematopoietic cytokine used for treatment and prevention of anemia in preterm neonates [1]. Preclinical and clinical studies have examined the potential neuroprotective and neuroreparative effect of human recombinant erythropoietin (rEPO) after hypoxic-ischemia (HI) [2][3][4][5][6][7]. Neuroprotection with rEPO treatment after HI is associated with anti-apoptotic, anti-inflammatory and neuroreparative mechanisms [3,8,9].…”

“…A better understanding of the pharmacokinetics of high-dose rEPO is important for further clinical development and for designing preclinical studies to compare various treatment regimens of rEPO. We have recently completed studies in preterm (0.7 gestation, brain maturation equivalent to 28-32 week preterm neonates) and near-term (0.87 gestation, equivalent to term neonates) fetal sheep examining neuroprotection with rEPO treatment administered as prolonged continuous infusion independently or as a combined treatment with therapeutic hypothermia after HI [7,19]. Collectively, these studies showed that treatment with a prolonged continuous infusion of rEPO after HI reduced neuronal loss in both preterm and near-term fetal sheep [7,19].…”

“…We have recently completed studies in preterm (0.7 gestation, brain maturation equivalent to 28-32 week preterm neonates) and near-term (0.87 gestation, equivalent to term neonates) fetal sheep examining neuroprotection with rEPO treatment administered as prolonged continuous infusion independently or as a combined treatment with therapeutic hypothermia after HI [7,19]. Collectively, these studies showed that treatment with a prolonged continuous infusion of rEPO after HI reduced neuronal loss in both preterm and near-term fetal sheep [7,19]. Here, we report an analysis of pharmacokinetic models of combined first-order and mixed-order elimination, first-order and saturable EPO-R-mediated clearance and rEPO treatment-induced changes in EPO-R to describe rEPO elimination.…”

“…Here, we report an analysis of pharmacokinetic models of combined first-order and mixed-order elimination, first-order and saturable EPO-R-mediated clearance and rEPO treatment-induced changes in EPO-R to describe rEPO elimination. We examined the effect of an increase in fetal size, maturation, exposure to global asphyxia, cerebral ischemia, therapeutic hypothermia and exogenous rEPO treatment on the pharmacokinetics of high-dose rEPO in well-established fetal sheep models [7,[19][20][21][22]. Finally, we compared the pharmacokinetics of high-dose rEPO in preterm fetal sheep given an i.v.…”

The Function of the NMDA Receptor in Hypoxic-Ischemic Encephalopathy

“…In addition, EPO can also promote post-damage repair by regulating the differentiation of neurons (Lee et al, 2007;Zhu et al, 2009;Wu et al, 2012). However, although clinical trial studies have shown that EPO combined with hypothermia therapy is safe and effective (Hua et al, 2017;Nonomura et al, 2019), a recent study revealed that the therapeutic function of EPO may overlap with hypothermia, since these two treatments share intracellular signaling cascades (Wassink et al, 2020).…”