Neuroprotective Properties of Food-Borne Polyphenols in Neurodegenerative Diseases.

Fruits and vegetables are the richest source of polyphenols in the regular human diet [...].

Polyphenols from Food and Natural Products: Neuroprotection and Safety.

Polyphenols are naturally occurring micronutrients that are present in many food sources. Besides being potent antioxidants, these molecules may also possess anti-inflammatory properties. Many studies have highlighted their potential role in the prevention and treatment of various pathological conditions connected to oxidative stress and inflammation (e.g., cancer, and cardiovascular and neurodegenerative disorders). Neurodegenerative diseases are globally one of the main causes of death and represent an enormous burden in terms of human suffering, social distress, and economic costs. Recent data expanded on the initial antioxidant-based mechanism of polyphenols' action by showing that they are also able to modulate several cell-signaling pathways and mediators. The proposed benefits of polyphenols, either as protective/prophylactic substances or as therapeutic molecules, may be achieved by the consumption of a natural polyphenol-enriched diet, by their use as food supplements, or with formulations as pharmaceutical drugs/nutraceuticals. It has also been proved that the health effects of polyphenols depend on the consumed amount and their bioavailability. However, their overconsumption may raise safety concerns due to the accumulation of high levels of these molecules in the organism, particularly if we consider the loose regulatory legislation regarding the commercialization and use of food supplements. This review addresses the main beneficial effects of food polyphenols, and focuses on neuroprotection and the safety issues related to overconsumption.

Bioaccessible (poly)phenol metabolites from raspberry protect neural cells from oxidative stress and attenuate microglia activation.

Neuroinflammation is an integral part of the neurodegeneration process inherent to several aging dysfunctions. Within the central nervous system, microglia are the effective immune cells, responsible for neuroinflammatory responses. In this study, raspberries were subjected to in vitro digestion simulation to obtain the components that result from the gastrointestinal (GI) conditions, which would be bioaccessible and available for blood uptake. Both the original raspberry extract and the gastrointestinal bioaccessible (GIB) fraction protected neuronal and microglia cells against H2O2-induced oxidative stress and lipopolysaccharide (LPS)-induced inflammation, at low concentrations. Furthermore, this neuroprotective capacity was independent of intracellular ROS scavenging mechanisms. We show for the first time that raspberry metabolites present in the GIB fraction significantly inhibited microglial pro-inflammatory activation by LPS, through the inhibition of Iba1 expression, TNF-α release and NO production. Altogether, this study reveals that raspberry polyphenols may present a dietary route to the retardation or amelioration of neurodegenerative-related dysfunctions.

Potential for brain accessibility and analysis of stability of selected flavonoids in relation to neuroprotection in vitro.

Natural food sources constitute a promising source of new compounds with neuroprotective properties, once they have the ability to reach the brain. Our aim was to evaluate the brain accessibility of quercetin, epigallocatechin gallate (EGCG) and cyanidin-3-glucoside (C3G) in relation to their neuroprotective capability. Primary cortical neuron cultures were exposed to oxidative insult in the absence and presence of the selected compounds, and neuroprotection was assessed through evaluation of apoptotic-like and necrotic-like cell death. The brain accessibility of selected compounds was assessed using an optimised human blood-brain barrier model. The blood-brain barrier model was crossed rapidly by EGCG and more slowly by C3G, but not by quercetin. EGCG protected against oxidation-induced neuronal necrotic-like cell death by ~40%, and apoptosis by ~30%. Both quercetin and C3G were less effective, since only the lowest quercetin concentration was protective, and C3G only prevented necrosis by ~37%. Quercetin, EGCG and C3G effectively inhibited α-synuclein fibrillation over the relevant timescale applied here. Overall, EGCG seems to be the most promising neuroprotective compound. Thus, inclusion of this polyphenol in the diet might provide an affordable means to reduce the impact of neurodegenerative diseases.

Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.

Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration.

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study.

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells.

Cross-talk between neurons and astrocytes in response to bilirubin: adverse secondary impacts.

Previous studies using monotypic nerve cell cultures have shown that bilirubin-induced neurological dysfunction (BIND) involves apoptosis and necrosis-like cell death, following neuritic atrophy and astrocyte activation,and that glycoursodeoxycholic acid (GUDCA) has therapeutic efficacy against BIND. Cross-talk between neurons and astrocytes may protect or aggravate neurotoxicity by unconjugated bilirubin (UCB). In a previous work we have shown that bidirectional signaling during astrocyte-neuron recognition attenuates neuronal damage by UCB. Here, we investigated whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB was instead associated with a lower resistance of neurons to UCB toxicity, and if the pro-survival properties of GUDCA were replicated in that experimental model. We have introduced a 24 h adaptation period for neuron-glia communication prior to the 48 h treatment with UCB. In such conditions, UCB induced glial activation, which aggravated neuronal damage, comprising increased apoptosis,cell demise and neuritic atrophy, which were completely prevented in the presence of GUDCA. Neuronal multidrug resistance-associated protein 1 expression and tumor necrosis factor-a secretion, although unchanged by UCB, increased in the presence of astrocytes. The rise in S100B and nitric oxide in the co-cultures medium may have contributed to UCB neurotoxicity. Since the levels of these diffusible molecules did not change by GUDCA we may assume that they are not directly involved in its beneficial effects. Data indicate that astrocytes, in an indirect neuron-astrocyte co-culture model and after homeostatic setting regulation of the system, are critically influencing neurodegeneration by UCB, and support GUDCA for the prevention of BIND.

Cross-talk between neurons and astrocytes in response to bilirubin: early beneficial effects.

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. This condition may lead to the deposition of unconjugated bilirubin (UCB) in the central nervous system, causing nerve cell damage by molecular and cellular mechanisms that are still being clarified. To date, all the studies regarding bilirubin-induced neurological dysfunction were performed in monotypic nerve cell cultures. The use of co-cultures, where astrocyte-containing culture inserts are placed on the top of neuron cultures, provides the means to directly evaluate the cross-talk between these two different cell types. Therefore, this study was designed to evaluate whether protective or detrimental effects are produced by astrocytes over UCB-induced neurodegeneration. Our experimental model used an indirect co-culture system where neuron-to-astrocyte signaling was established concomitantly with the 24 h exposure to UCB. In this model astrocytes abrogated the well-known UCB-induced neurotoxic effects by preventing the loss of cell viability, dysfunction and death by apoptosis, as well as the impairment of neuritic outgrowth. To this protection it may have accounted the induced expression of the multidrug resistance-associated protein 1 and the 3.5-fold increase in the values of S100B, when communication between both cells was established independently of UCB presence. In addition, the presence of astrocytes in the neuronal environment preserved the UCB-induced increase in glutamate levels, but raised the basal concentrations of nitric oxide and TNF-α although no UCB effects were noticed. Our data suggest that bidirectional signalling during astrocyte-neuron recognition exerts pro-survival effects, stimulates neuritogenesis and sustains neuronal homeostasis, thus protecting cells from the immediate UCB injury. These findings may help explain why irreversible brain damage usually develops only after the first day of post-natal life.

Neuritic growth impairment and cell death by unconjugated bilirubin is mediated by NO and glutamate, modulated by microglia, and prevented by glycoursodeoxycholic acid and interleukin-10.

Neuronal oxidative damage and cell death by unconjugated bilirubin (UCB) showed to be mediated by overstimulation of glutamate receptors and nitric oxide (NO) production, which was abrogated by the bile acid glycoursodeoxycholic acid (GUDCA). Microglia, a crucial mediator of CNS inflammation, evidenced to react to UCB by releasing glutamate and NO before becoming senescent. Our studies demonstrated that neurite outgrowth deficits are produced in neurons exposed to UCB and that conditioned media from these UCB-treated neurons further stimulate NO production by microglia. Nevertheless, microglia protective and/or harmful effects in neonatal jaundice are poorly understood, or unrecognized. Here, we investigated the role of microglia, glutamate and NO in the impairment of neurite sprouting by UCB. Therapeutic potential of the anti-inflammatory cytokine interleukin (IL)-10 and GUDCA was also evaluated. By using MK-801 (a NMDA glutamate-subtype receptor antagonist) and L-NAME (a non-specific NO synthase inhibitor) we found that glutamate and NO are determinants in the early and enduring deficits in neurite extension and ramification induced by UCB. Both GUDCA and IL-10 prevented these effects and decreased the production of glutamate and NO. Only GUDCA was able to counteract neuronal death and synaptic changes. Data from organotypic-cultured hippocampal slices, depleted or non-depleted in microglia, supported that microglia participate in glutamate homeostasis and contribute to NO production and cell demise, which were again abrogated by GUDCA. Collectively our data suggest that microglia is a key player in UCB-induced neurotoxicity and that GUDCA might be a valuable preventive therapy in neonates at risk of UCB encephalopathy.

Elevated levels of bilirubin and long-term exposure impair human brain microvascular endothelial cell integrity.

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 µM UCB, in the presence of 100 µM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion at later time points. Upregulation of mRNA expression, particularly by 100 µM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of ß-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and ß-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin.

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1ß promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.

Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation.

Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity.

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 micromol/L UCB, in the presence of 100 micromol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3',5'-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.

Bilirubin injury to neurons: contribution of oxidative stress and rescue by glycoursodeoxycholic acid.

It is well established that high levels of unconjugated bilirubin (UCB) can be toxic to the central nervous system, and oxidative stress is emerging as a relevant event in the mechanisms of UCB encephalopathy. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been reported as a cytoprotective and antioxidant molecule. In this study, we investigated if exposure of rat neurons in primary culture to clinically relevant concentrations of UCB leads to oxidative injury. The contribution of oxidative stress in UCB neurotoxicity was further investigated by examining whether the reduction of NO production by NAME, an inhibitor of nitric oxide synthase, prevents the disruption of the redox status and neuronal damage. Moreover, we evaluated the ability of glycoursodeoxycholic acid (GUDCA), the most relevant conjugated derivative in the serum of patients treated with UDCA, to abrogate the UCB-induced oxidative damage. Cultured rat neurons were incubated with 50 or 100microM UCB in the presence of 100microM human serum albumin, alone or in combination with 100microM NAME or with 50microM GUDCA, for 4h at 37 degrees C. Protein carbonyls, 4-hydroxy-2-nonenal-protein adducts, intracellular glutathione content and cell death were determined. The results obtained showed that UCB induces protein oxidation and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were correlated with the extent of cell death. Moreover, these events were counteracted by NAME and abrogated in the presence of GUDCA. Collectively, this study shows that oxidative stress is one of the pathways associated with neuronal viability impairment by UCB, and that GUDCA significantly prevents such effects from occurring. These findings corroborate the antioxidant properties of the bile acid and point to a new therapeutic approach for UCB-induced neurotoxicity due to oxidative stress.

Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells.

We investigated whether nerve cell damage by unconjugated bilirubin (UCB) is mediated by oxidative stress and ascertained the neuronal and astroglial susceptibility to injury. Several oxidative stress biomarkers and cell death were determined following incubation of neurons and astrocytes isolated from rat cortical cerebrum with UCB (0.01-1.0 microM). We show that UCB induces a dose-dependent increase in neuronal death in parallel with the oxidation of cell components and a decrease in the intracellular glutathione content. Comparison of the results obtained in both cell types demonstrates that neurons are more vulnerable than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells. Moreover, neuronal oxidative injury is prevented by supplementation with N-acetylcysteine, a glutathione precursor, whereas astroglial sensitivity to UCB is enhanced by inhibition of glutathione synthesis, using buthionine sulfoximine. Collectively, we demonstrate that oxidative stress is involved in UCB neurotoxicity and depict a new therapeutic approach for UCB-induced oxidative damage.

Glycoursodeoxycholic acid and interleukin-10 modulate the reactivity of rat cortical astrocytes to unconjugated bilirubin.

The pathogenesis of bilirubin encephalopathy seems to result from accumulation of unconjugated bilirubin (UCB) within the brain. We have recently demonstrated that UCB causes astroglial release of proinflammatory cytokines and glutamate, as well as cell death. The bile acid glycoursodeoxycholic acid (GUDCA) and the anti-inflammatory cytokine interleukin (IL)-10 have been reported to modulate inflammation and cell survival. In this study we investigated the effect of these therapeutic agents on the astroglial response to UCB. Only GUDCA prevented UCB-induced astroglial death. The secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta elicited by UCB in astrocytes was reduced in the presence of GUDCA and IL-10, whereas the suppression of IL-6 was only counteracted by GUDCA. Neither GUDCA nor IL-10 modulated the accumulation of extracellular glutamate. Additionally, IL-10 markedly inhibited UCB-induced nuclear factor-kappaB nuclear translocation and cytokine mRNA expression, whereas GUDCA only prevented TNF-alpha mRNA expression. Moreover, GUDCA inhibited TNF-alpha- and IL-1beta-converting enzymes, preventing the maturation of these cytokines and their consequent release. Collectively, this study shows that IL-10 action is restricted to UCB-induced release of TNF-alpha and IL-1beta from the astrocytes, whereas GUDCA presents a more ubiquitous action on the astroglial reactivity to UCB. Hence, GUDCA may have potential benefits over an IL-10 therapeutic approach in reducing UCB-induced astrocyte immunostimulation and death.

MAPKs are key players in mediating cytokine release and cell death induced by unconjugated bilirubin in cultured rat cortical astrocytes.

When activated by unconjugated bilirubin (UCB), astrocytes are important sources of inflammatory mediators such as TNF-alpha, IL-1beta and IL-6, which may contribute for the neurotoxicity observed during severe neonatal hyperbilirubinemia. In the present study, we have addressed the role of the mitogen-activated protein kinases (MAPKs) p38, Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)1/2 pathways and their relation with the nuclear factor kappaB (NF-kappaB) cascade in the signalling events involved in cytokine release and cell death caused by UCB in primary cultures of rat astrocytes. Stimulation of astrocytes with UCB in the presence of all the MAPK inhibitors prevented UCB-induced release of TNF-alpha and IL-6, while IL-1beta secretion was only reduced by JNK1/2 and ERK1/2 inhibitors. In addition, activation of the NF-kappaB transcription factor, needed for cytokine release by UCB-stimulated astrocytes, was shown to be dependent on JNK1/2 and ERK1/2 phosphorylation. Moreover, all MAPK inhibitors prevented astroglial apoptosis triggered by UCB. Interestingly, UCB-induced lactate dehydrogenase release was prevented by blockade of JNK1/2, ERK1/2 and NF-kappaB cascades but enhanced by p38 inhibition. Taken together, our data demonstrate for the first time that MAPK transduction pathways are key players in the UCB-induced inflammatory response and cell death in astrocytes, probably also involving NF-kappaB modulation. These findings contribute to unraveling the complex mechanisms of astrocyte reactivity to UCB and may ultimately prove useful in the development of new therapeutic strategies to prevent nerve cell damage during acute bilirubin encephalopathy.

Apoptosis and impairment of neurite network by short exposure of immature rat cortical neurons to unconjugated bilirubin increase with cell differentiation and are additionally enhanced by an inflammatory stimulus.

Nerve cell injury induced by unconjugated bilirubin (UCB) has been implicated in brain damage during severe neonatal hyperbilirubinemia, although the molecular mechanisms underlying UCB neurotoxicity are still not clarified. It has been suggested recently that there is an association between hyperbilirubinemia and long-term neurologic dysfunctions. We incubated immature neurons with UCB to evaluate the short- and long-term effects of UCB on apoptotic death and on neuritic outgrowth and ramification. We also evaluated whether mature neurons, exposed previously to UCB in an early stage of differentiation, are more sensitive to apoptosis or to neuritic breakdown when treated with inflammatory agents, such as lipopolysaccharide and tumor necrosis factor-alpha. Results show that exposure of immature neurons to UCB increased apoptosis and provoked a reduction of both neurite extension and number of nodes. These injurious effects observed in immature cells treated with UCB were increasingly perpetuated along cell differentiation, as compared to neurons incubated in the absence of UCB. In addition, neurons that were exposed to UCB when immature showed an increased susceptibility to death by apoptosis, as well as an additional decrease in neurite outgrowth when incubated with an inflammatory agent afterward. This work shows, for the first time, that UCB induces neurite changes consistent with neurodevelopment abnormalities. Furthermore, pre-exposure to UCB followed by an inflammatory stimulus leads to an enhanced susceptibility to long-term apoptosis, as well as a greater neuritic breakdown. These data support the association between neonatal hyperbilirubinemia and the later development of mental illness, such as schizophrenia.

Influence of hypoxia and ischemia preconditioning on bilirubin damage to astrocytes.

Hypoxia-ischemia in the perinatal period is a common cause of neurologic disability in children and is often associated with neonatal morbidity and mortality. Another frequent condition of the newborn is hyperbilirubinemia and it is well known that deposition of unconjugated bilirubin (UCB) in the central nervous system can damage nerve cells and cause encephalopathy. Interestingly, some studies report the onset of cerebral hypoxia-ischemia as a risk factor for UCB encephalopathy, since that condition often precedes neonatal hyperbilirubinemia. However, the cellular mechanisms triggered by hypoxia-ischemia that may enforce UCB deleterious effects are not well elucidated. Therefore, we designed this study to investigate whether hypoxia (HP) or combined oxygen-glucose deprivation (OGD) followed by reoxygenation, modifies glial cell susceptibility to UCB injury. Thus, cultured astrocytes were exposed to HP or OGD for 4 h and returned to normoxic conditions for another 12 h prior to incubation with UCB for 4 h. HP and OGD effects in UCB toxicity were compared to normoxic conditions. Our results demonstrate that HP and OGD preconditioning increase the vulnerability of glial cells to UCB damage by enhancing some of the deleterious effects of UCB, namely cell death by both apoptosis and necrosis. This preconditioning also augments the UCB-induced stimulation of an inflammatory response by an effect that involves the activation of the nuclear factor kappaB activation. These findings provide a novel basis for the increased risk of brain damage in jaundiced newborns that were previously exposed to hypoxia or ischemia during the perinatal period, namely during delivery.