RESUMEN
Perinatal asphyxia (PA) is a leading cause of neuronal damage in newborns, resulting in long-term neurological and cognitive deficits, in part due to impairment of mesostriatal and mesolimbic neurocircuitries. The insult can be as severe as to menace the integrity of the genome, triggering the overactivation of sentinel proteins, including poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 overactivation implies increased energy demands, worsening the metabolic failure and depleting further NAD(+) availability. Using a global PA rat model, we report here evidence that hypoxia increases PARP-1 activity, triggering a signalling cascade leading to nuclear translocation of the NF-κB subunit p65, modulating the expression of IL-1ß and TNF-α, pro-inflammatory molecules, increasing apoptotic-like cell death in mesencephalon of neonate rats, monitored with Western blots, qPCR, TUNEL and ELISA. PARP-1 activity increased immediately after PA, reaching a maximum 1-8 h after the insult, while activation of the NF-κB signalling pathway was observed 8 h after the insult, with a >twofold increase of p65 nuclear translocation. IL-1ß and TNF-α mRNA levels were increased 24 h after the insult, together with a >twofold increase in apoptotic-like cell death. A single dose of the PARP-1 inhibitor nicotinamide (0.8 mmol/kg, i.p.), 1 h post delivery, prevented the effect of PA on PARP-1 activity, p65 translocation, pro-inflammatory cytokine expression and apoptotic-like cell death. The present study demonstrates that PA leads to PARP-1 overactivation, increasing the expression of pro-inflammatory cytokines and cell death in mesencephalon, effects prevented by systemic neonatal nicotinamide administration, supporting the idea that PARP-1 inhibition represents a therapeutic target against the effects of PA.
Asunto(s)
Asfixia Neonatal/metabolismo , Asfixia/metabolismo , Mesencéfalo/metabolismo , Niacinamida/administración & dosificación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Asfixia/enzimología , Asfixia Neonatal/enzimología , Humanos , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Mesencéfalo/efectos de los fármacos , Mesencéfalo/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Perinatal asphyxia constitutes a prototype of obstetric complications occurring when pulmonary oxygenation is delayed or interrupted. A primary insult is first produced by the length of the time without oxygenation, leading to hypoxia/ischemia and death if oxygenation is not promptly established. A second insult is produced by re-oxygenation, eliciting a cascade of biochemical events for restoring function, implying, however, improper homeostasis. The effects observed long after perinatal asphyxia can be explained by over-expression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for oxidised nicotinamide adenine dinucleotide (NAD(+)) during re-oxygenation. Asphyxia also induces transcriptional activation of pro-inflammatory factors, including nuclear factor κB (NFκB) and its subunit p65, whose translocation to the nucleus is significantly increased in brain tissue from asphyxia-exposed animals, in tandem with PARP-1 overactivation, leading to the idea that sentinel protein inhibition constitutes a suitable therapeutic strategy. It is proposed that PARP-1 inhibition also down-regulates the expression of pro-inflammatory cytokines.Nicotinamide is a suitable PARP-1 inhibitor, whose effects have been studied in an experimental model of global perinatal asphyxia in rats, inducing the insult by immersing rat foetuses into a water bath for various periods of time. Following asphyxia, the pups are delivered, immediately treated, or given to surrogate dams for nursing, pending further experiments. Systemic administration of nicotinamide 1 h after the insult inhibited PARP-1 overactivity in peripheral and brain tissue, preventing several of the long-term consequences elicited by perinatal asphyxia, supporting the idea that it constitutes a lead for exploring compounds with similar or better pharmacological profiles.
RESUMEN
Oxygen interruption leads to death when re-oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of biochemical events for restoring function at the cost of improper homeostasis. The effects observed long after perinatal asphyxia (PA) have been explained by over-expression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for NAD(+) during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a therapeutic strategy. We studied the effects of nicotinamide and theophylline on PARP-1 activity assayed in brain and peripheral (heart) rat tissue 1-24 h after birth, as well as on changes in behaviour and monoamine neurotransmission in adult rats. PA was induced by immersing rat foetuses into a water bath for 0 or 21 min. After resuscitation, the pups were treated with nicotinamide (0.8 mmol/kg, i.p.), theophylline (0.14 mmol/kg, i.p.) or saline (0.9% NaCl) and nurtured by surrogate dams, pending behavioural and microdialysis experiments, or euthanised after birth for assaying PARP-1 activity. To estimate the in vivo distribution of a single dose of nicotinamide or theophylline into brain and peripheral compartment, a series of animals were implanted with microdialysis probes, one into the brain and other subcutaneously, 1 h after birth, assaying the drugs with a HPLC-UV system. Nicotinamide, but not theophylline prevented the long-term effects induced by PA. Only nicotinamide produced a consistent decrease in PARP-1 activity in brain and heart, whether assayed in control or asphyxia-exposed pups. The present results support the idea that the long-term effects induced by PA imply PARP-1 over-activation.