Vulnerability to a Metabolic Challenge Following Perinatal Asphyxia Evaluated by Organotypic Cultures: Neonatal Nicotinamide Treatment.

Abstract

The hypothesis of enhanced vulnerability following perinatal asphyxia was investigated with a protocol combining in vivo and in vitro experiments. Asphyxia-exposed (AS) (by 21min water immersion of foetuses containing uterine horns) and caesarean-delivered control (CS) rat neonates were used at P2-3 for preparing triple organotypic cultures (substantia nigra, neostriatum and neocortex). At DIV 18, cultures were exposed to different concentrations of H2O2 (0.25-45mM), added to the culture medium for 18h. After a 48-h recovery period, the cultures were either assessed for cell viability or for neurochemical phenotype by confocal microscopy. Energy metabolism (ADP/ATP ratio), oxidative stress (GSH/GSSG) and a modified ferric reducing/antioxidant power assay were applied to homogenates of parallel culture series. In CS cultures, the number of dying cells was similar in substantia nigra, neostriatum and neocortex, but it was several times increased in AS cultures evaluated under the same conditions. A H2O2 challenge led to a concentration-dependent increase in cell death (>fourfold after 0.25mM of H2O2) in CS cultures. In AS cultures, a significant increase in cell death was only observed after 0.5mM of H2O2. At higher than 1mM of H2O2 (up to 45mM), cell death increased several times in all cultures, but the effect was still more prominent in CS than in AS cultures. The cell phenotype of dying/alive cells was investigated in formalin-fixed cultures exposed to 0 or 1mM of H2O2, co-labelling for TUNEL (apoptosis), MAP-2 (neuronal phenotype), GFAP (astroglial phenotype) and TH (tyrosine hydroxylase; for dopamine phenotype), counterstaining for DAPI (nuclear staining), also evaluating the effect of a single dose of nicotinamide (0.8nmol/kg, i.p. injected in 100μL, 60min after delivery). Perinatal asphyxia produced a significant increase in the number of DAPI/TUNEL cells/mm3, in substantia nigra and neostriatum. One millimolar of H202 increased the number of DAPI/TUNEL cells/mm3 by ≈twofold in all regions of CS and AS cultures, an effect that was prevented by neonatal nicotinamide treatment. In substantia nigra, the number of MAP-2/TH-positive cells/mm3 was decreased in AS compared to CS cultures, also by 1mM of H202, both in CS and AS cultures, prevented by nicotinamide. In agreement, the number of MAP-2/TUNEL-positive cells/mm3 was increased by 1mM H2O2, both in CS (twofold) and AS (threefold) cultures, prevented by nicotinamide. The number of MAP-2/TH/TUNEL-positive cells/mm3 was only increased in CS (>threefold), but not in AS (1.3-fold) cultures. No TH labelling was observed in neostriatum, but 1mM of H2O2 produced a strong increase in the number of MAP-2/TUNEL-positive cells/mm3, both in CS (>2.9-fold) and AS (>fourfold), decreased by nicotinamide. In neocortex, H2O2 increased the number of MAP-2/TUNEL-positive cells/mm3, both in CS and AS cultures (≈threefold), decreased by nicotinamide. The ADP/ATP ratio was increased in AS culture homogenates (>sixfold), compared to CS homogenates, increased by 1mM of H202, both in CS and AS homogenates. The GSH/GSSG ratio was significantly decreased in AS, compared to CS cultures. One millimolar of H2O2 decreased that ratio in CS and AS homogenates. The present results demonstrate that perinatal asphyxia induces long-term changes in metabolic pathways related to energy and oxidative stress, priming cell vulnerability with both neuronal and glial phenotype. The observed effects were region dependent, being the substantia nigra particularly prone to cell death. Nicotinamide administration in vivo prevented the deleterious effects observed after perinatal asphyxia in vitro, a suitable pharmacological strategy against the deleterious consequences of perinatal asphyxia.