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 21 min 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 (0.25–45 mM), added to the culture medium for 18 h. 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 0.25 mM of H₂O₂) in CS cultures. In AS cultures, a significant increase in cell death was only observed after 0.5 mM of H₂O₂. At higher than 1 mM of H₂O₂ (up to 45 mM), 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 1 mM 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 (0.8 nmol/kg, i.p. injected in 100 μL, 60 min 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 1 mM 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 1 mM 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 1 mM 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 1 mM 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.

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