The potential of erythropoietin to treat asphyxia in newborns

Figures & data

Figure 1 Comparison of mechanisms of neuroprotection between therapeutic hypothermia and erythropoietin (Epo).

Notes: Mechanisms of brain injury and recovery after injury are listed. Therapeutic hypothermia and Epo have many similar mechanisms of action, but Epo has additional effects of prevention of necrosis and promotion of angiogenesis and neurogenesis beyond hypothermia alone.

Figure 2 Molecular mechanism of erythropoietin (Epo).

Notes: Epo production is upregulated after hypoxia via stimulation of HIF-1, but it can also be given exogenously. Epo binds to the Epo receptor (EpoR) homodimer, causing JAK2 kinase phosphorylation of JAK2 and the EpoR, which triggers a signaling cascade that involves STAT5, NF-κB, PI3K/AKT, and MAPK/ERK. Together, this leads to production of antiapoptotic proteins, including Bcl-2 and Bcl-xL, and also inhibition of proapoptotic proteins, including Bad and Bax. The balance of proapoptotic and antiapoptotic proteins affects release of substances such as cytochrome c from the mitochondria which then leads to apoptosis.

Table 1 Neonatal animal studies of Epo

Animal/age/injury	Dose of Epo/route/timing	Outcomes/results	Reference
Hypoxia/ischemia
Mouse/P7/unilateral carotid ligation, 50 m 10% O2	1,000 or 5,000 U/kg ip 1 hour prior to injury	Reduction in injury at 1 day in cortex, striatum, hippocampus, and thalamus with 5,000 U/kg and at a dose-related reduction in injury at 7 days in cortex, striatum, and thalamus	113
Rat/P7/unilateral carotid ligation, 2.5 h 8% O2	1,000 U/kg ip after hypoxic period complete	Decreased mean infarct volume; decrease in apoptotic cells at 72 hours post-injury	114
Rat/P7/unilateral carotid ligation, 1 h 8% O2	2,000 U/kg ip 10 minutes prior to hypoxia	Reduced brain damage severity and prevented apoptosis at 4 days post-injury; improved short-term functional brain recovery at 24 hours post-injury	115
Rat/P7/unilateral carotid ligation, 2.5 h 8% O2	1,000 U/kg ip after hypoxic period complete	Less macroscopic brain injury, larger brain volume in injured hemisphere, and improvement in hippocampal CA1 neuronal density at 21 weeks; improvement on spatial memory testing at 22 days and 21 weeks	116
Rat/P7/unilateral carotid ligation, 50 m 7.7% O2	10,000 U/kg Epo or 0.08 mg/kg asialoEpo 4 hours prior to injury	Both significantly reduced infarct volumes; asialoEpo reduced activation of ERK	117
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	2,500 U/kg sq at time of injury and 1 and 2 days after injury	Protected dopamine neurons in substantia nigra, pars compacta, and ventral tegmental areas; reduced sensory neglect and prevented rotational asymmetry on behavioral testing at 3 weeks post-injury; no difference in hematocrit, white blood cell, neutrophil, or platelet counts	118
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	300 U ip 24 hours prior to hypoxia and then daily for 2 days	Prevented mortality during hypoxia; increased brain weight at multiple time points; inhibited apoptosis at 24–72 hours post-injury	60
Rat/P7/unilateral carotid ligation, 2.5 h 8% O2	1,000 U/kg ip after hypoxic period complete	Increased glutathione peroxidase activity at 24 hours post-injury	75
Rat/P7/unilateral carotid ligation, 1 h 8% O2	2,000 U/kg ip after hypoxic period complete	Improved recovery of sensorimotor function; reduction in extension of brain injury with preservation of cerebral cortex at 35 days post-injury	119
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	5,000 U/kg ip 24, 48, and 72 hours after injury	Increased brain weight at 1, 2, and 3 weeks post-injury; attenuated HI-induced leukocyte infiltration and increases in IL-1β mRNA and protein levels in first 2 weeks post-injury	79
Rat/P7/unilateral carotid ligation, 2.5 h 8% O2	1,000 U/kg ip after hypoxic period complete	Reversed HI-induced upregulation of proapoptotic BAX and DP5 mRNA at 4 hours post-injury; reversed HI-induced downregulation in antiapoptotic Bcl-2 transcription at 24 hours post-injury; reduced mean infarct volume, decreased number of apoptotic cells, reduced bax, and increased Bcl-2 protein expression at 48 hours post-injury	120
Rat/P7/unilateral carotid ligation, 2 h 8% O2	300 U/kg ip after hypoxic period complete	Attenuated HI-induced impairment in rapid auditory processing and decrease in size of cortex and hippocampus at 2–3 weeks post-injury	121
Rat/P7/unilateral carotid ligation, 130 m 8% O2	500, 1,000, or 2,000 U/kg ip 20 minutes after hypoxia, repeated at 2, 4, and 6 days post-injury	1,000 U/kg reduced tissue volume loss in cortex and striatum at 7 days post-injury without stimulating erythropoiesis (measured at 4 weeks post-injury), enhanced functional revascularization, increased progenitor cell migration and neuronal replacement in ischemic areas, and reversed HI-induced sensorimotor changes seen in 4 weeks post-injury	78
Rat/P7/unilateral carotid ligation, 2 h 8% O2	300 or 1,000 U/kg ip after hypoxic period complete	Both doses decreased histological evidence of brain damage 11 weeks post-injury and improved rapid auditory processing and both spatial and nonspatial learning/memory at different time points from 2–11 weeks post-injury	122
Rat/P7/1.5 h 8% O2 with or without unilateral carotid ligation	2,500 U/kg sq at 0, 24, and 48 hours after hypoxic period complete	Prevented HI-induced impairment of adult memory; protected dopaminergic neurons in substantia nigra, pars compacta, and ventral tegmental area (seen on histology performed in adulthood); prevented hypoxia-induced growth delay at 1 and 10 days post-injury; and improved forelimb strength at 10 days post-injury	123
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	250, 2,500, or 500 U/kg ip or sq within 30 minutes of hypoxia exposure	Intraperitoneal injection produced a faster and higher peak concentration in the plasma than subcutaneous injection. Hypoxia did not induce detectable levels of endogenous Epo, but exogenous Epo did cross the blood–brain barrier in a dose-dependent manner, peaking at 10 hours and remaining detectable for more than 20 hours post-injury	124
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	2,500, 5,000, or 30,000 U/kg sq daily started after hypoxia for 1, 3, or 7 days	Most improvement in gross brain injury, apoptosis, and gliosis at 48 hours and 1 week post-injury with 5,000 U/kg ×3 doses and 30,000 U/kg ×1 dose	62
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	3,000 U/kg ip at 0, 24, and 48 hours after hypoxia	Improvement in HI-induced reduction in hemispheric volume, decrease in expansion of subventricular zone, and forelimb asymmetry on behavioral testing at 2 weeks post-injury	125
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	DFO alone, 1,000 U/kg Epo alone, DFO + 1,000 U/kg Epo at 0, 24, and 48 hours after hypoxia	DFO + Epo decreased apoptotic cells, but neither DFO, Epo, nor DFO-Epo improved gray or white matter damage or dopaminergic neuronal integrity at 72 hours post-injury	126
Rat/P7/unilateral carotid ligation, 2 h 8% O2	1,000 U/kg 30 minutes before or after hypoxia	Attenuated HI-induced decrease in antiapoptotic Bcl-2 expression and increase in proapoptotic Bax, caspase-3, and Bax:Bcl-2 ratio at 7 days post-injury	69
Rat/P7/unilateral carotid ligation, 2.5 h 8% O2	1,000 U/kg ip on days 2, 4, 6, 9, and 13 after hypoxia	No reduction in brain volume loss but did increase oligodendrogenesis, maturation of oligodendrocytes, attenuate white mater injury, enhance neurogenesis, and improve behavioral outcomes between 5 and 14 days after injury	127
Rat/P7 or P8/unilateral carotid ligation, 2 h 8% O2	30,000 U/kg ip between ligation and hypoxia	Decreased size of brain lesion and ADC value but no change in FA value on MRI 30 minutes post-injury and upregulated water channel protein aquaporin-4 within hours after injury, indicating Epo may improve clearance of brain edema	81
Mouse/P9/unilateral carotid ligation, 45 m 10% O2	5,000 or 20,000 U/kg at 0, 24, and 48 hours after hypoxia	5,000 U/kg increased progenitor cell proliferation at 72 hours post-injury and improved sensorimotor function, reduced atrophy of the striatum and volume of hippocampal lesion, and improved myelination at 4 and 9 weeks post-injury in female mice only	128
Rat/P7/unilateral carotid ligation, 2 h 8% O2	1,000 U/kg at 0, 24, and 168 hours after hypoxia with or without hypothermia ×8 hours	No differences in behavioral outcomes or histopathologic brain damage scores at 2 or 6 weeks post-injury other than difference in brain injury score in male rats treated with Epo alone	86
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	5,000 U/kg at 0, 24, and 48 hours after hypoxia with or without hypothermia ×3 hours	Epo and hypothermia improved sensorimotor function at 2 and 5 weeks post-injury with more improvement in females; hypothermia interacted with sex in affecting outcomes, with only hypothermia and not Epo improving histological outcomes at 5 weeks post-injury	87
Rat/P7/unilateral carotid ligation, 2 h 8% O2	1,000 U/kg ip at 0, 60, or 180 minutes after hypoxia	Immediate dosing improved defects in rapid auditory processing in juveniles and adults and reduced ventriculomegaly seen on histology in adults associated with HI, but dosing delayed 180 minutes did not, and 60-minute delayed dosing had intermediate effects	129
Rat/P10/unilateral carotid ligation, 2.5 h 8% O2	Epo or nanoEpo 30, 100, 300 U/kg, or Epo 5,000 U/kg ip 1 hour after hypoxia, repeated at 24 and 48 hours post-injury	For Epo, only 5,000 U/kg significantly decreased infarct volume at 72 hours post-injury, whereas 100 and 300 U/kg nanoEpo both significantly decreased infarct volume, with 300 U/kg approximating the effect of 5,000 U/kg Epo and both approximating sham surgery animals; similar effects seen on neurobehavioral testing at 3 weeks post-injury and brain weight analysis 4 weeks post-injury	130
Rat/P7/unilateral carotid ligation, 1.5 h 8% O2	1,000 U/kg ip after hypoxia	Improved hearing threshold to similar to that of control animals; reduced apoptotic changes in brain stem structures, organ of Corti, spiral ganglion cells, and temporal lobe neurons at 6 weeks post-injury	67
Ischemia/stroke
Rat/P7/MCAO	Either 1,000 U/kg ip ×1 15 minutes after injury or 100, 1,000, or 5,000 U/kg/dose daily ×3 starting 15 minutes after injury	Decreased infarction volume and area at 1 and 3 days post-injury, with 1,000 U/kg being the most effective dose. 1,000 U/kg ×3 regimen reduced number of apoptotic cells in ischemic cortex. No effect on Epo receptor expression in the ischemic cortex. Prevented injury-induced decrease in phosphorylated Jak2 and Stat5 expression; increased antiapoptotic Bcl-xL expression; and had no effect on NF-κB at 1 and 3 days post-injury	52
Rat/P10/MCAO	5,000 U/kg ip immediately after injury	Improved percentage preserved tissue volume, decreased injury-induced subventricular zone enlargement, and improved paw preference asymmetry at 2 weeks post-injury	131
Rat/P7/MCAO	1,000 U/kg ip at 15 minutes, 1 day, and 2 days post-injury	Increased brain weight at 6 and 12 weeks post-injury, decreased infarct volume at 6 weeks (greater effect in females than males), increase in size of lesion by 12 weeks in Epo-treated males but not females, improved neurobehavioral outcomes at 6 and 12 weeks post-injury (again greater effect in females)	132
Rat/P10/MCAO ×45 minutes	Epo 5,000 U/kg ×1 after injury	Decreased tissue loss overall in brain, decreased number of astrocytes, and increased neurogenesis in damaged striatum, without changes in subventricular zone volume or density of newly generated cells in injured striatum at 6 weeks post-injury	133
Rat/P10/MCAO ×45 minutes	Epo, either 5,000 U/kg ×1 after injury or 1,000 U/kg ×3 (after injury and at 1 and 7 days post-injury)	Three-dose regimen of Epo significantly improved spatial memory and vision and motor-based function and showed nonsignificant trends toward improvement in other behavioral tests at 3 months post-injury; reduced tissue loss and increased neocortex, visual cortex, striatum, and hippocampus volumes at 3–4 months post-injury	134
Rat/P7/MCAO after injection of Lentivirus to label subventricular zone cells at P1	1,000 U/kg at reperfusion and 1 and 7 days later	Increased subventricular zone neural stem cell lineage cells in injured striatum and neural stem cell-derived neurogenic activity, increased oligodendrocyte precursors, and decreased astrocytes in striatum at 72 hours and 2 weeks post-injury	135
In utero ischemia (prenatal or term)
Monkey/P0 (term)/umbilical cord occlusion	Either 3,500 U/kg at 30 minutes of life followed by 2,500 U/kg at 24, 48, and 168 hours or 1,000 U/kg at 30 minutes and 24, 48, and 168 hours, all IV, plus hypothermia ×72 hours	Prevented death and moderate-to-severe cerebral palsy (assessed through 9 months post-injury); improved motor and cognitive responses, cerebellar growth, and DTI measures on MRI without adverse drug reactions or changes in hematology, liver, or kidney laboratory values	49

Abbreviations: ADC, apparent diffusion coefficient; asialoEpo, asialoerythropoietin; bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; DFO, desferoxamine; DTI, diffusion tensor imaging; Epo, erythropoietin; ERK, extracellular signal-related kinase; FA, fractional anisotropy; HI, hypoxia-ischemia; IL-1β, interleukin-1 beta; ip, intraperitoneal; IV, intravenous; Jak2, Janus kinase 2; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; nanoEpo, nanoerythropoietin; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B-cells; sq, subcutaneous; Stat5, signal transducer and activator of transcription 5 ; P7, postnatal day 7; m, minutes, h, hours.

Table 2 Human infant studies of Epo

Subjects/treatment groups/type	Dose/timing	Outcomes	Reference
153 infants with moderate-to-severe HIE (73 given one of two different doses of Epo and 80 controls); randomized trial	Either 300 U/kg or 500 U/kg given every other day for 2 weeks starting <48 hours after birth	Overall improvement in rate of death/moderate-to-severe disability in infants with moderate HIE in Epo groups at 18 months; improvement in behavior as early as day 7; no negative hematopoietic side effects; no difference in outcomes between doses	90
30 infants with HIE (15 controls and 15 given Epo) plus 15 controls; case-control study	2,500 U/kg given subcutaneously daily for 5 days	Improvement in blood NO concentrations and EEG background; fewer neurologic and developmental abnormalities; no difference in MRI findings	91
24 infants undergoing hypothermia for HIE given Epo at varying doses (Phase I safety and pharmacokinetics study)	250 U/kg, 500 U/kg, 1,000 U/kg, or 2,500 U/kg given every 48 hours for six doses starting at <24 hours of age	No deaths or serious adverse events; nonlinear pharmacokinetics; plasma concentrations that are neuroprotective in animals seen at 1,000 U/kg dosing	92
30 infants with perinatal hypoxia (randomized as: ten to supportive care, ten given moderate hypothermia, ten given Epo) and 15 healthy infants; case-control study	Single 1,500 U/kg dose given on day 1 of life	Infants given hypothermia had the best survival, followed by infants given Epo and then the control group, though differences not statistically significant; significantly higher brain-derived neurotrophic factor in hypothermia and Epo groups than in supportive care group	94

Abbreviations: EEG, electroencephalography; Epo, erythropoietin; HIE, hypoxic-ischemic encephalopathy; MRI, magnetic resonance imaging; NO, nitric oxide.