Plasticity of the central nervous system (CNS) following perinatal asphyxia: does nicotinamide provide neuroprotection?

We have investigated the idea that nicotinamide, a non-selective inhibitor of the sentinel enzyme Poly(ADP-ribose) polymerase-I (PARP-1), provides neuroprotection against the long-term neurological changes induced by perinatal asphyxia. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. The effect of perinatal asphyxia on neurocircuitry development was studied in vitro with organotypic cultures from substantia nigra, neostriatum and neocortex, platted on a coverslip 3 days after birth. After approximately one month in vitro (DIV 25), the cultures were treated for immunocytochemistry to characterise neuronal phenotype with markers against the N-methyl-D-aspartate receptor subunit 1 (NR1), the dopamine pacemaker enzyme tyrosine hydroxylase (TH), and nitric oxide synthase (NOS), the enzyme regulating the bioavailability of NO. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyctic and caesarean-delivered pups 24, 48 and 72 h after birth. It was found that nicotinamide treatment prevented the effect of perinatal asphyxia on several neuronal parameters, including TH- and NOS-positive neurite atrophy and NOS-positive neuronal loss; supporting the idea that nicotinamide constitutes a therapeutic alternative for the effects produced by sustained energy-failure conditions, as occurring during perinatal asphyxia.

Effects of perinatal asphyxia on cell survival, neuronal phenotype and neurite growth evaluated with organotypic triple cultures.

The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after three days for preparing organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features and cell viability were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for immunocytochemistry using antibodies against the N-methyl-D-aspartate receptor subunit 1 (NR1) and tyrosine hydroxylase (TH). While in vitro survival was similar in cultures from both asphyxiated and control animals, differences were observed when the neuronal phenotype was assessed. Compared to controls, the total number of NR1-positive neurons in substantia nigra, as well as the number of secondary to higher level branching of TH-positive neurites from asphyxiated pups were decreased, illustrating the vulnerability of the dopaminergic systems to perinatal asphyxia.

Perinatal asphyxia impairs connectivity and dopamine neurite branching in organotypic triple culture from rat substantia nigra, neostriatum and neocortex.

The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after 3 days to prepare organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for histochemistry using fast red for cell nucleus labelling and antibodies against tyrosine hydroxylase for dopaminergic neurons. Compared to controls, cultures from asphyxiated pups revealed a diminished integration quantified during 21 DIV. After immunocytochemistry and camera lucida reconstruction, tyrosine hydroxylase-positive neurons showed a decreased number of neurites from secondary and higher level branching, demonstrating a vulnerability of the dopaminergic systems after perinatal asphyxia.

Nicotinamide prevents the long-term effects of perinatal asphyxia on basal ganglia monoamine systems in the rat.

Asphyxia during birth can cause gross brain damage, but also subtle perturbations expressed as biochemical or motor deficits with late onset in life. Thus, it has been shown that brain dopamine levels can be increased or decreased depending upon the severity of the insult, and the region where the levels are determined. In this study, perinatal asphyxia was evoked by immersing pup-containing uterus horns removed by hysterectomy in a water bath at 37 degrees C for various periods of time from 0 to 20 min. After the insult, the pups were delivered, given to surrogate mothers, treated with nicotinamide, further observed and finally, 4 weeks later, killed for monoamine biochemistry of tissue samples taken from substantia nigra, neostriatum and nucleus accumbens. The main effect of perinatal asphyxia was a decrease in dopamine and metabolite levels in nucleus accumbens, and a paradoxical increase in the substantia nigra. Nicotinamide (100 mg/kg i.p., once a day for 3 days, beginning 24 h after the perinatal asphyctic insult) prevented the effect of asphyxia in nucleus accumbens. Furthermore, striatal dopamine levels were increased by nicotinamide in asphyctic animals. No apparent changes were observed in substantia nigra. A prominent unexpected effect of perinatal asphyxia alone was on the levels of the metabolite of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid (5-HIAA), which were increased in substantia nigra and decreased in both neostriatum and accumbens. However, nicotinamide increased 5-HIAA levels in all regions, which appeared to be related to the extent of the asphyctic insult. These results suggest that nicotinamide is a useful treatment against the long-term consequences produced by perinatal asphyxia on brain monoamine systems, and that there is a therapeutic window following the insult, providing a therapeutic opportunity to protect the brain.