Abstract
Background: The pathophysiology of bilirubin neurotoxicity in course of hypoxic–ischemic encephalopathy (HIE) in term and preterm infants is still poorly understood. We hypothesized that oxidative stress may be a common mechanism that link hyperbilirubinemia and HIE.Objectives: The objective of the present study was to evaluate whether unconjugated bilirubin (UCB) may enhance the HI brain injury by increasing oxidative stress and to test pioglitazone and allopurinol as new antioxidant therapeutic drugs in vitro.Methods: The effects of UCB were tested on organotypic hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), used as in vitro model of HIE. The experiments were performed on mature (14 days in culture) and immature (7 days in culture) slices, to mimic the brains of term and preterm infants, respectively. Mature and immature slices were exposed to UCB, human serum albumin (HSA), pioglitazone, and/or allopurinol for 24 h, immediately after 30 min OGD. Neuronal injury was assessed using propidium iodide (PI) fluorescence. ROS formation was quantified by using the 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) method.Results: In mature slices, we found that the neurotoxicity, as well as oxidative stress, induced by OGD were enhanced by UCB. HSA significantly prevented UCB-increased neurotoxicity, but had a slight reduction on ROS production. Allopurinol, but not pioglitazone, significantly reduced UCB-increased neurotoxicity induced by OGD. In immature slices exposed to OGD, no increase of neuronal death was observed, whereas oxidative stress was detected after UCB exposure. HSA, pioglitazone and allopurinol have no protective effects on both OGD-induced neuronal death and on UCB-induced oxidative stress. For this reason, UCB, pioglitazone and allopurinol was also tested on ischemic preconditioning protocol. We found that UCB abolished the neuroprotection induced by preconditioning and increased oxidative stress. These effects were restored by allopurinol but not pioglitazone.Conclusions: UCB characterized a different path of neuronal damage and oxidative stress in mature and immature hippocampal slice model of HIE. Management of hyperbilirubinemia in a complex pathological condition, such as HIE and hyperbilirubinemia, should be very careful. Allopurinol could deserve attention as a novel pharmacological intervention for hyperbilirubinemia and HIE.
Highlights
Hypoxic–ischemic encephalopathy (HIE) is a clinical syndrome that affects newborns following severe or prolonged cerebral hypoxic–ischemic episodes before or during birth
In order to evaluate whether antioxidant pioglitazone and allopurinol could be able to attenuate the neuronal injury and oxidative stress induced by Unconjiugated Bilirubin (UCB) in oxygen-glucose deprivation (OGD) mature slices, we firstly tested separately both pioglitazone and allopurinol in OGD- or UCB-induced neurotoxicity
We observed that the addition of 10 μM pioglitazone decreased cellular death in OGD but not in UCB exposed slices and it was ineffective in decreasing cellular oxidative stress in both experimental setting, suggesting that its neuroprotection was not mediated by reactive oxygen species (ROS) production (Supplementary Figure 1)
Summary
Hypoxic–ischemic encephalopathy (HIE) is a clinical syndrome that affects newborns following severe or prolonged cerebral hypoxic–ischemic episodes before or during birth. Infants affected by HIE have an augmented risk of death and lifelong disability, such as cerebral palsy, cognitive impairment, developmental delay, visual and learning disability, and epilepsy [2, 3]. The pathophysiology of HIE is complicated and involves acute effects, such as cell energy failure caused by hypoxia and low level of glucose, and delayed effects, such as extracellular accumulation of excitatory neurotransmitters, that activating apoptotic or necrotic cascades lead to cell death [6, 7]. Many studies have been described the role of oxidative stress in the pathophysiology of HIE and reactive oxygen species (ROS) are believed to contribute to the degenerative processes of the tissues that lead to acute and chronic brain injury [8, 9]. We hypothesized that oxidative stress may be a common mechanism that link hyperbilirubinemia and HIE
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