As
therapeutic hypothermia is only partially protective for neonatal
encephalopathy, safe and effective adjunct
therapies are urgently needed.
Melatonin and
erythropoietin show promise as safe and effective neuroprotective
therapies. We hypothesized that
melatonin and
erythropoietin individually augment 12-h
hypothermia (double
therapies) and
hypothermia +
melatonin +
erythropoietin (triple
therapy) leads to optimal brain protection. Following carotid artery occlusion and
hypoxia, 49 male piglets (<48 h old) were randomized to: (i)
hypothermia + vehicle (n = 12), (ii)
hypothermia +
melatonin (20 mg/kg over 2 h) (n = 12), (iii)
hypothermia +
erythropoietin (3000 U/kg bolus) (n = 13) or (iv) triple
therapy (n = 12).
Melatonin,
erythropoietin or vehicle were given at 1, 24 and 48 h after
hypoxia-ischaemia.
Hypoxia-ischaemia severity was similar across groups. Therapeutic levels were achieved 3 hours after
hypoxia-ischaemia for
melatonin (15-30 mg/l) and within 30 min of
erythropoietin administration (maximum concentration 10 000 mU/ml). Compared to
hypothermia + vehicle, we observed faster amplitude-integrated EEG recovery from 25 to 30 h with
hypothermia +
melatonin (P = 0.02) and
hypothermia +
erythropoietin (P = 0.033) and from 55 to 60 h with triple
therapy (P = 0.042). Magnetic resonance spectroscopy
lactate/
N-acetyl aspartate peak ratio was lower at 66 h in
hypothermia +
melatonin (P = 0.012) and triple
therapy (P = 0.032). With
hypothermia +
melatonin,
terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick-end labelled-positive cells were reduced in sensorimotor cortex (P = 0.017) and
oligodendrocyte transcription factor 2 labelled-positive counts increased in hippocampus (P = 0.014) and periventricular white matter (P = 0.039). There was no reduction in
terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick-end labelled-positive cells with
hypothermia +
erythropoietin, but increased
oligodendrocyte transcription factor 2 labelled-positive cells in 5 of 8 brain regions (P < 0.05). Overall,
melatonin and
erythropoietin were safe and effective adjunct
therapies to
hypothermia.
Hypothermia +
melatonin double
therapy led to faster amplitude-integrated EEG recovery, amelioration of
lactate/
N-acetyl aspartate rise and reduction in
terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick-end labelled-positive cells in the sensorimotor cortex.
Hypothermia +
erythropoietin double
therapy was in association with EEG recovery and was most effective in promoting oligodendrocyte survival. Triple
therapy provided no added benefit over the double
therapies in this 72-h study.
Melatonin and
erythropoietin influenced cell death and oligodendrocyte survival differently, reflecting distinct neuroprotective mechanisms which may become more visible with longer-term studies. Staggering the administration of
therapies with early
melatonin and later
erythropoietin (after
hypothermia) may provide better protection; each
therapy has complementary actions which may be time critical during the neurotoxic cascade after
hypoxia-ischaemia.