Vulnerability to recurrent metabolic insults following perinatal asphyxia in basal ganglia of rat : effect of neonatal treatment with nicotinamide

Abstract

Perinatal Asphyxia (PA) implies the interruption of oxygen supply, causing energy failure, leading to cell dysfunction and ultimately cell death, affecting in particular neurocircuitries of basal ganglia. The long-term effects occurring in brain after PA worsen the ability of the CNS to cope with stressors occurring along life. Dopaminergic neurons of substantia nigra have been reported to be very sensitive to PA, and vulnerable to any conditions implying oxidative stress, although it has not been studied yet how dopaminergic neurons cope with postnatal stressors once exposed to an initial event of neonatal hypoxia and how that affects basal ganglia neurocircuitry. The enhanced vulnerability of basal ganglia following PA was investigated with a protocol combining in vivo and in vitro experiments. Asphyxia exposed (AS) and caesarean-delivered control (CS) rat neonates were used at P2-3 for preparing triple (substantia nigra, neostriatum and neocortex) or SN-mono organotypic cultures. At DIV 18, cultures were exposed to different concentrations of H2O2 (0.25-45mM), added to the culture medium for 18h. After a 48h recovery period, the cultures were either assessed for cell viability (LIVE/DEAD® Viability/Cytotoxicity assay), or formalin fixed for neurochemical phenotype by confocal microscopy. Energy metabolism (ADP/ATP ratio), oxidative stress (GSH/GSSG), tissue reducing capacity, and lactate dehydrogenase (LDH)- release assays were applied to homogenates or supernatants 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 all regions of AS cultures evaluated under the same conditions. A H2O2 challenge led to a concentration-dependent increase in cell death, 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 DAPI, TUNEL, MAP-2, GFAP, and tyrosine hydroxylase, also evaluating the effect of a single dose of nicotinamide (0.8 mmol/kg, i.p. injected in 100 μL, 60 min after delivery). PA produced a significant increase in the number of DAPI/TUNEL cells/mm3, in substantia nigra and neostriatum. 1 mM of H2O2 increased the number of DAPI/TUNEL cells in all regions of CS and AS cultures. The effect of PA on cellular phenotypes was more prominent in substantia nigra, where the number of MAP-2/TH positive cells/mm3 was decreased in AS compared to CS cultures, also by 1 mM of H2O2, both in CS and AS cultures. Neuronal apoptosis was increased, both in CS (> 2-fold) and AS (> 3 fold) cultures. The disruptive effect of 1 mM of H2O2 was stronger in cultures from AS than that from CS neonates on TH positive cells (decreased by ~70% versus ~30%), while the effect on MAP-2 positive cells was similar in both groups (decreased by ~60% versus ~50%). 1 mM of H2O2 led to a strong increase in the number of TUNEL-positive neurons, both in CS and AS groups. In neocortex, similar MAP-2 positive apoptosis was observed. The ADP/ATP ratio was increased in AS culture homogenates compared to CS, and increased by 1 mM of H2O2, both in CS and AS cultures. The GSH/GSSG ratio was decreased in AS compared to CS cultures, meanwhile 1 mM of H2O2 decreased that ratio in both groups. AS samples showed a decreased total reducing capacity, and high LDH levels compared to CS. Similar effects were observed comparing mono and triple organotypic cultures, suggesting that the vulnerability of basal ganglia neurocircuitries after PA relies mainly, but not exclusively on substantia nigra. The present results demonstrate that PA induces long-term changes in metabolic pathways, implying energy failure and oxidative stress, priming cell vulnerability to both neuronal and astroglial phenotypes, affecting the coping capacity of neurons and astroglia against further postnatal challenges. The observed effects were region dependent, the substantia nigra being more prone to cell death than other regions. Nicotinamide administration in vivo prevented the deleterious effects observed after PA and after a recurrent metabolic insult in vitro