RESUMO
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.
Assuntos
Asfixia Neonatal/tratamento farmacológico , Neocórtex/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Niacinamida/farmacologia , Substância Negra/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Feminino , Peróxido de Hidrogênio/metabolismo , Masculino , Neocórtex/metabolismo , Neocórtex/patologia , Neostriado/metabolismo , Neostriado/patologia , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos Wistar , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
Perinatal asphyxia (PA) is associated to delayed cell death, affecting neurocircuitries of basal ganglia and hippocampus, and long-term neuropsychiatric disabilities. Several compensatory mechanisms have been suggested to take place, including cell proliferation and neurogenesis. There is evidence that PA can increase postnatal neurogenesis in hippocampus and subventricular zone (SVZ), modulated by dopamine, by still unclear mechanisms. We have studied here the effect of selective dopamine receptor agonists on cell death, cell proliferation and neurogenesis in organotypic cultures from control and asphyxia-exposed rats. Hippocampus and SVZ sampled at 1-3 postnatal days were cultured for 20-21 days. At day in vitro (DIV) 19, cultures were treated either with SKF38393 (10 and 100 µM, a D1 agonist), quinpirole (10 µM, a D2 agonist) or sulpiride (10 µM, a D2 antagonist) + quinpirole (10 µM) and BrdU (10 µM, a mitosis marker) for 24 h. At DIV 20-21, cultures were processed for immunocytochemistry for microtubule-associated protein-2 (MAP-2, a neuronal marker), and BrdU, evaluated by confocal microscopy. Some cultures were analysed for cell viability at DIV 20-21 (LIVE/DEAD kit). PA increased cell death, cell proliferation and neurogenesis in hippocampus and SVZ cultures. The increase in cell death, but not in cell proliferation, was inhibited by both SKF38393 and quinpirole treatment. Neurogenesis was increased by quinpirole, but only in hippocampus, in cultures from both asphyxia-exposed and control-animals, effect that was antagonised by sulpiride, leading to the conclusion that dopamine modulates neurogenesis in hippocampus, mainly via D2 receptors.