No abstract

With the current practice of therapeutic hypothermia for neonatal encephalopathy, disability rates and the severity spectrum of cerebral palsy are reduced. Nevertheless, safe and effective adjunct therapies are needed to optimize outcomes. This study's objective was to assess if 18 mg/kg melatonin given rapidly over 2 h at 1 h after hypoxia-ischemia with cooling from 1-13 h was safe, achieved therapeutic levels within 3 h and augmented hypothermic neuroprotection. Following hypoxia-ischemia, 20 newborn piglets were randomized to: (i) Cooling 1-13 h (HT; n = 6); (ii) HT+ 2.5% ethanol vehicle (Ht+V; n = 7); (iii) HT + Melatonin (Ht+M; n = 7). Intensive care was maintained for 48 h; aEEG was acquired throughout, brain MRS acquired at 24 and 48 h and cell death (TUNEL) evaluated at 48 h. There were no differences for insult severity. Core temperature was higher in HT group for first hour after Hi. comparing Ht+M to HT, aEEG scores recovered more quickly by 19 h (p < 0.05); comparing HT+V to HT, aEEG recovered from 31 h (p < 0.05). Brain phosphocreatine/inorganic phosphate and NTP/ exchangeable phosphate were higher at 48 h in HT+M versus Ht (p = 0.036, p = 0.049 respectively). Including both 24 h and 48 h measurements, the rise in Lactate/N-acetyl aspartate was reduced in white (p = 0.030) and grey matter (p = 0.038) after HI. Reduced overall TUNEL positive cells were observed in Ht+M (47.1 cells/mm 2 ) compared to HT (123.8 cells/mm 2 ) (p = 0.0003) and HT+V (97.5 cells/mm 2 ) compared to Ht (p = 0.012). Localized protection was seen in white matter for HT+M versus Ht (p = 0.036) and internal capsule for HT+M compared to Ht (p = 0.001) and HT+V versus Ht (p = 0.006). Therapeutic melatonin levels (15-30mg/l) were achieved at 2 h and were neuroprotective following HI, but ethanol vehicle was partially protective.Intrapartum-related neonatal encephalopathy (NE) is a major healthcare problem. Worldwide in 2010, NE accounted for 287,000 deaths and 400,000 survivors with impairment 1 . NE cannot be prevented in most cases and therapies are limited. The incidence of NE in Western Europe is 1-3/1000 term births and in low-and mid-resource settings the incidence is ~10 times higher 1,2 . Over the last 2 decades, in settings with neonatal intensive care facilities, therapeutic hypothermia (HT) is routinely used for moderate-to-severe NE, improving survival and reducing disability 3 . However, although the severity of cerebral palsy has reduced with HT 4 , survivors have significantly lower cognitive scores which are on average 14 IQ points lower than matched peers even in the absence of cerebral palsy at school-age 5 . Further adjustments to HT protocols do not improve outcome 6,7 , therefore adjunct therapies to augment HT protection are urgently needed.Pre-clinical studies suggest that melatonin (N-acetyl-5-methoxytryptamine) in pharmacologic levels is safe and neuroprotective for hypoxic-ischemic injury in the adult 8 and neonatal 9 brain, mediated by anti-oxidant, anti-apoptotic and anti-inflammatory properties 10,11 . Ex...

Co-existing infection/inflammation and birth asphyxia potentiate the risk of developing neonatal encephalopathy (NE) and adverse outcome. In a newborn piglet model we assessed the effect of E. coli lipopolysaccharide (LPS) infusion started 4 h prior to and continued for 48 h after hypoxia on brain cell death and systemic haematological changes compared to LPS and hypoxia alone. LPS sensitized hypoxia resulted in an increase in mortality and in brain cell death (TUNEL positive cells) throughout the whole brain, and in the internal capsule, periventricular white matter and sensorimotor cortex. LPS alone did not increase brain cell death at 48 h, despite evidence of neuroinflammation, including the greatest increases in microglial proliferation, reactive astrocytosis and cleavage of caspase-3. LPS exposure caused splenic hypertrophy and platelet count suppression. The combination of LPS and hypoxia resulted in the highest and most sustained systemic white cell count increase. These findings highlight the significant contribution of acute inflammation sensitization prior to an asphyxial insult on NE illness severity.

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-hour hypothermia (double therapies) and hypothermia + melatonin + erythropoietin (triple therapy) leads to optimal brain protection. Following carotid artery occlusion and hypoxia, 49 male piglets (&lt;48 hours old) were randomized to: (i) hypothermia + vehicle (n = 12), (ii) hypothermia + melatonin (20 mg/kg over 2 hours) (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 hours after hypoxia-ischemia. Hypoxia-ischemia severity was similar across groups. Therapeutic levels were achieved 3 hours after hypoxia-ischemia for melatonin (15-30mg/L) and within 30 minutes of erythropoietin administration (maximum concentration 10,000 mU/mL). Compared to hypothermia + vehicle, we observed faster amplitude integrated EEG recovery from 25-30 hours with hypothermia + melatonin (p = 0.02) and hypothermia + erythropoietin (p = 0.033) and from 55-60 hours with triple therapy (p = 0.042). Magnetic Resonance Spectroscopy Lactate/N-acetyl aspartate peak ratio was lower at 66 hours 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 &lt; 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 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-hour 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-ischemia.