The progressive brain damage observed following perinatal asphyxia is sustained by a long-term impairment of redox homeostasis : effect of nicotinamide

Abstract

Perinatal asphyxia (PA) is a clinical condition characterized by oxygen deprivation, following PA brain damage progresses, leading to behavioural cognitive disabilities affecting surviving neonates. There is still lack of consensus on suitable therapeutic approaches and protocols preventing the progression of neuronal damage. Reduction of oxidative stress is one of the identified molecular targets, based on the fact that the immature brain is highly susceptible to free radicals, showing scarcely developed antioxidant defences. In the present thesis it was proposed the hypothesis that progression of brain damage during a delayed cell death period in vulnerable brain areas of animals exposed to perinatal asphyxia is associated with sustained oxidative stress along development, impairing redox homeostasis. Nicotinamide enhances the response of glutathione-dependent enzymes by a pentose phosphate dependent pathway, preventing asphyxia-dependent brain damage. The hypothesis was evaluated using a rat model of severe perinatal asphyxia. The first part of the present Thesis evaluated the effect of global perinatal asphyxia on several parameters of oxidative stress and cell death in rat brain tissue sampled at an extended neonatal period of 14 days. Brain samples (mesencephalon, telencephalon and hippocampus) were assayed for glutathione (reduced and oxidized levels; spectrophotometry), tissue reducing capacity (potassium ferricyanide reducing assay, FRAP), catalase (the key enzyme protecting against oxidative stress and reactive oxygen species, Western blots and ELISA) and cleaved caspase-3 (the key executioner of apoptosis, Western blots) levels. It was found that global PA produced a regionally specific and sustained increase in GSSG/GSH ratio, a regionally specific decrease in tissue reducing capacity and a regionally and time specific decrease of catalase activity and increase of cleaved caspase-3 levels, indicating a long-term impairment in redox homeostasis. Multivariate analysis demonstrated that progression of brain damage along postnatal days is importantly influenced by changes on glutathione ratio, cleaved caspase-3 levels and catalase activity. In the second part of the Thesis, the experimental approach focused on the damage induced in hippocampus at P1 and P14. Glutathione, glutathione reductase (GR), glutathione peroxydase (GPx) (all by spectrophotometry), catalase (Western blots and ELISA), TIGAR (Western blots), calpain (fluorescence), and XRCC1 (Western blots) were assayed for characterizing the glutathione-dependent redox pathway and for determining the effect of nicotinamide; a precursor of NAD+ and NADP+, on death cell mechanisms in hippocampus. It was found that global PA produced (i) a sustained increase of GSSG levels and GSSG/GSH ratio at P1 and P14; (ii) a decrease of GR, GPx, and catalase activity at P1 and P14; (iii) a decrease at P1, followed by an increase at P14 of TIGAR levels; (iv) an increase of calpain activity at P14; and (v) an increase of XRCC1 levels, but only at P1. (vi) Nicotinamide prevented the effect of PA on GSSG levels and GSSG/GSH ratio, and on GR, GPx, and catalase activity, also on the enhancement of TIGAR levels and calpain activity observed at P14. The present thesis demonstrates that PA induces a sustained oxidative stress along development in mesencephalon and hippocampus, both vulnerable brain areas of animals exposed to PA, whereas telencephalon, a resistant brain area, displayed an increasing of antioxidant response mediated by glutathione and FRAP. The progression of brain damage during a delayed cell death period is associated mainly with glutathione ratio, cleaved caspase-3 levels and catalase activity. Nicotinamide enhanced the response of glutathione-dependent enzymes by a pentose phosphate dependent pathway, preventing asphyxia-dependent brain damage by a reduction of death cell mechanisms mediated by calpain. In conclusion the present thesis demonstrates that changes in redox environment induced by PA are sustained along the time, contributing to progression of brain damage, but also resistance to further damage during the delayed death cell period. The changes in redox environment can be modulated, controlling the cell metabolism induced by pentose phosphate dependent pathways