Neuroprotective effect of the combination therapy of melatonin and URB447 after neonatal hypoxia-ischemia.

Abstract

Background: Perinatal asphyxia leading to neonatal encephalopathy is one of the major causes of death and childhood disability worldwide. As therapeutic hypothermia showed detrimental effects in medium/low-income countries, new therapeutic approaches are urgently needed to treat non-cooled asphyctic newborns. Melatonin and cannabinoids have emerged as possible neuroprotective strategies because of their multiple-acting mechanisms. The objective of this study was to assess the neuroprotective effects of combining melatonin and the cannabinoid URB447 after moderate-to-severe neonatal hypoxia-ischemia in newborn rats.

Methods: On postnatal day (PD7) Sprague-Dawley rats underwent unilateral ligation of the left common carotid artery followed by 2 h exposure to a humidified 92%-nitrogen/8%-oxygen mixture to induce HI. Rat pups received 15 mg/kg of Melatonin (5 min after HI, and repeated at 24 and 48 h; HI + MEL, n = 15: 9 males / 6 females) and/or a single dose of 1 mg/kg of URB447 (3 h after HI; HI + MEL + URB447, n = 15: 8 males / 7 females; HI + URB447, n = 10: 5 males / 5 females). Animals without treatment received the corresponding volume of vehicle (HI-group, n = 15: 9 males / 6 females) and those without ischemia nor hypoxia served as controls (Sham, n = 10: 6 males / 4 females). At PD8 and PD14, a battery of behavioral tests (front limb suspension, righting reflex and negative geotaxis) were evaluated, before the sacrifice of animals for histological analysis of brain infarct and neuropathological damage on PD14. White matter was also assessed at PD14 by immunohistochemistry for myelin basic protein. To compare the five experimental groups, parametric data were analysed using one-way ANOVA with Tukey's multiple comparisons test, whereas non-parametric data were studied with the Kruskal-Wallis test with Dunn's multiple comparisons test. Data considered significantly different if p < 0.05.

Results: The combined therapy of Melatonin + URB447 obtained better results in sensorimotor performances when compared to non-treated HI. Melatonin- or URB447-only therapies did not improve either test. In the histological studies, despite having similar results to URB447-only, Melatonin + URB447 obtained better results in global neuropathological score and white matter injury in the cingulum.

Conclusions: Our data suggest that Melatonin + URB447 improved neurodevelopmental outcomes after HI, a beneficial effect extended to grey and white matter after hypoxia-ischemia in neonatal rats.