Rui F. M. Silva

Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid.

BACKGROUND/AIMS Unconjugated bilirubin (UCB) can be neurotoxic in jaundiced neonates and in patients with Crigler-Najjar syndrome. UCB toxicity may culminate in cell death, however, the occurrence of apoptosis has never been investigated. Ursodeoxycholic acid (UDCA) is a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. The aims of this study were to determine whether apoptosis plays a role in neural cell death induced by UCB, and to investigate the ability of UDCA to prevent cell death. METHODS Cultured rat astrocytes were incubated with UCB (17 and 86 microM) plus albumin (5.7 and 28.7 microM) for 4-22 h. In addition, astrocytes and neurones were treated with either UCB, 50 microM UDCA, or their combination for 4 h. Cultures were scored for nonviable cells by trypan blue dye exclusion. Apoptosis was assessed by Hoechst staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assay. RESULTS UCB induced a concentration- and time-dependent decrease in astrocyte viability. Apoptosis was 4- and 7-fold increased after 4 h exposure to 17 and 86 microM UCB, respectively (P < 0.01). UDCA reduced apoptosis to <7%, which represents a appoximately 60% protection (P < 0.01). Cholic acid was not protective, and chenodeoxyholic acid aggravated UCB toxicity (P < 0.05). Finally, neurones showed a 1.5-fold greater sensitivity than astrocytes to UCB, while UDCA was still protective. CONCLUSIONS UCB is toxic to both astrocytes and neurones, causing cell death through an apoptotic process. Moreover, UDCA inhibits UCB-induced apoptosis in neural cells and this could not be mimicked by other bile acids.

Inflammatory signalling pathways involved in astroglial activation by unconjugated bilirubin

During neonatal hyperbilirubinaemia, astrocytes activated by unconjugated bilirubin (UCB) may contibute to brain toxicity through the production of cytokines. As a first step in addressing the signal transduction cascades involved in the UCB‐induced astroglial immunological response, we tested whether tumour necrosis factor (TNF)‐α receptor 1 (TNFR1), mitogen‐activated protein kinase (MAPK) and nuclear factor κB (NF‐κB) would be activated in astrocytes exposed to UCB, and examined the profile of cytokine production. Astrocyte cultures stimulated with UCB showed a rapid rise in TNFR1 protein levels, followed by activation of the MAPKs p38, Jun N‐terminal kinase1/2 and extracellular signal‐regulated kinase1/2, and NF‐κB. Interestingly, the induction of these signal effectors preceded the early up‐regulation of TNF‐α and interleukin (IL)‐1β mRNAs, and later secretion of TNF‐α, IL‐1β and IL‐6. Treatment of astrocytes with UCB also induced cell death, with levels comparable to those obtained after exposure of astrocytes to recombinant TNF‐α and IL‐1β. Moreover, loss of cell viability and cytokine secretion were reduced when the NF‐κB signal transduction pathway was inhibited, suggesting a key role for NF‐κB in the astroglial response to UCB. These results demonstrate the complexity of the molecular mechanisms involved in cell injury by UCB during hyperbilirubinaemia and provide a basis for the development of novel therapeutic strategies.

Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin

High levels of unconjugated bilirubin (UCB) can be neurotoxic. Nevertheless, the mechanism of UCB interaction with neural cells is still unknown. This study investigates whether cultured rat neurons and astrocytes respond differently to UCB exposure. UCB toxicity was evaluated by lactate dehydrogenase release, induction of apoptosis, cytoskeleton degeneration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, and glutamate uptake. Primary cultures of rat brain astrocytes and neurons were incubated at 37°C with 85.5 μM UCB plus 28.5 μM albumin for 4 h. In assays of glutamate uptake, cells were exposed to 80–120 μM UCB plus 100 μM albumin for 15 min. The results showed that after incubation with 85.5 μM UCB, lactate dehydrogenase release was greater in neurons than in astrocytes (38%versus 14%, p < 0.05). Also, levels of apoptosis were markedly enhanced in neurons (29%versus 19%, p < 0.01). In accordance, neuronal cytoskeleton disassembly was evident during incubation with 85.5 μM UCB, whereas equivalent effects on astrocytes required as much as 171 μM. Conversely, inhibition of MTT metabolism and glutamate uptake by UCB was more pronounced in astrocytes than in neurons (74%versus 60%, p < 0.05 and 41% to 56%versus 25% to 33%, p < 0.05, respectively). In conclusion, the study demonstrates that astrocytes are more susceptible to inhibition of glutamate uptake and MTT reduction by UCB, whereas neurons are more sensitive to cell death by necrosis or apoptosis. These results suggest that UCB is toxic to both astrocytes and neurons, although through distinct pathways.

Effects of the vitamin E in catalase activities in hippocampus after status epilepticus induced by pilocarpine in Wistar rats

Experimental manipulations suggest that in vivo administration of exogenous antioxidants agents decreases the concentration of free radical in the brain. Neurochemical studies have proposed a role for catalase in brain mechanisms responsible by development to status epilepticus (SE) induced by pilocarpine. The present study was aimed at was investigating the changes in catalase activities after pilocarpine-induced SE. Animals were treated with vitamin E (VIT E) 200 mg/kg (intraperitoneally (i.p.)) and, 30 min later, they received pilocarpine hydrochloride, 400 mg/kg, subcutaneous (s.c.) (P400). Other three groups received VIT E (200 mg/kg, i.p.), pilocarpine (400 mg/kg, s.c.) or 0.9% NaCl (control) alone. Animals were closely observed for behavioral changes, tremors, stereotyped movements, seizures, SE and death, for 24 h following the pilocarpine injection. The brains were dissected after decapitation. The results have shown that pilocarpine administration and resulting SE produced a significant increase in hippocampal catalase activity of (88%). In the group pre-treated which VIT E in hippocampal catalase activity was increase of 67% and 214% when compared with P400 and control group, respectively. Our results demonstrated a direct evidence of an increase in the activity of the hippocampal catalase of rat adults during seizure activity and after the pre-treated which VIT E that could be responsible by regulation of free radical levels during the establishment of SE.

Vitamin C antioxidant effects in hippocampus of adult Wistar rats after seizures and status epilepticus induced by pilocarpine.

Vitamin C (VIT C) is an exogenous antioxidant able to alter the brain oxidative stress. Antioxidant properties have been showed in seizures and status epilepticus (SE) induced by pilocarpine in adult rats. This present study was aimed at was investigating the VIT C effects on latency to first seizure, in percentage of seizures, mortality rate, as well as hippocampal lipid peroxidation levels and catalase activity after seizures and SE. The VIT C effects were investigated after the pretreatment with dose 250 mg/kg, i.p., 30 min before pilocarpine administration (400mg/kg, s.c., pilocarpine group (P400)). The VIT C increase the latency to first seizure and decrease the mortality rate and lipid peroxidation levels. In P400+VIT C and VIT C groups were observed an increase in hippocampal catalase activity. Our results suggests that the vitamin C can exert antioxidant and anticonvulsive effects in adult rats, suggesting that this vitamin can be able by reduction of lipid peroxidation content and increased of catalase enzymatic activity which cerebral compensatory mechanisms in free radical formation during SE.

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.

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‐α, IL‐1β 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 κB (NF‐κB) 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‐α and IL‐6, while IL‐1β secretion was only reduced by JNK1/2 and ERK1/2 inhibitors. In addition, activation of the NF‐κB 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‐κB 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‐κB 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.

Unconjugated bilirubin activates and damages microglia.

Microglia are the resident immune cells of the brain and are the principal source of cytokines produced during central nervous system inflammation. We have previously shown that increased levels of unconjugated bilirubin (UCB), which can be detrimental to the central nervous system during neonatal life, induce the secretion of inflammatory cytokines and glutamate by astrocytes. Nevertheless, the effect of UCB on microglia has never been investigated. Hence, the main goal of the present study was to evaluate whether UCB leads to microglial activation and to the release of the cytokines tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL‐6. Additionally, we investigated the effects of UCB on glutamate efflux and cell death. The results showed that UCB induces morphological changes characteristic of activated microglia and the release of high levels of TNF‐α, IL‐1β, and IL‐6 in a concentration‐dependent manner. In addition, UCB triggered extracellular accumulation of glutamate and an increased cell death by apoptosis and necrosis. These results demonstrate, for the first time, that UCB is toxic to microglial cells and point to microglia as an important target of UCB in the central nervous system. Moreover, they suggest that UCB‐induced cytokine production, by mediating cell injury, can further contribute to exacerbate neurototoxicity. Interestingly, microglia cells are much more responsive to UCB than astrocytes. Collectively, these data indicate that microglia may play an important role in the pathogenesis of encephalopathy during severe hyperbilirubinemia.

Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state.

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. The increased vulnerability of premature infants to unconjugated bilirubin (UCB)-induced brain damage may be due to a proneness of immature nerve cells to UCB-toxic stimulus. Thus, in this study, we evaluated UCB-induced cell death, glutamate release and cytokine production, in astrocytes and neurons cultured for different days, in order to relate the differentiation state with cell vulnerability to UCB. The age-dependent activation of the nuclear factor-κB (NF-κB), an important transcription factor involved in inflammation, was also investigated. Furthermore, responsiveness of neurons and astrocytes to UCB were compared in order to identify the most susceptible to each induced effect, as an approach to what happens in vivo. The results clearly showed that immature nerve cells are more vulnerable than the most differentiated ones to UCB-induced cell death, glutamate release and tumour necrosis factor (TNF)-α secretion. Moreover, astrocytes seem to be more competent cells in releasing glutamate and in producing an inflammatory response when injured by UCB. Activation of NF-κB by UCB also presents a cell-age-dependent pattern, and values vary with neural cell type. Again, astrocytes have the highest activation levels, which are correlated with the greater amount of cytokine production observed in these cells. These results contribute to a better knowledge of the mechanisms leading to UCB encephalopathy by elucidation of age- and type-related differences in neural cell responses to UCB.

Bilirubin-induced inflammatory response, glutamate release, and cell death in rat cortical astrocytes are enhanced in younger cells.

Unconjugated bilirubin (UCB) encephalopathy is a predominantly early life condition resulting from the impairment of several cellular functions in the brain of severely jaundiced infants. However, only few data exist on the age-dependent effects of UCB and their association with increased vulnerability of premature newborns, particularly in a sepsis condition. We investigated cell death, glutamate efflux, and inflammatory cytokine dynamics after exposure of astrocytes at different stages of differentiation to clinically relevant concentrations of UCB and/or lipopolysaccharide (LPS). Younger astrocytes were more prone to UCB-induced cell death, glutamate efflux, and inflammatory response than older ones. Furthermore, in immature cells, LPS exacerbated UCB effects, such as cell death by necrosis. These findings provide a basis for the increased susceptibility of premature newborns to UCB deleterious effects, namely when associated with sepsis, and underline how crucial the course of cell maturation can be to UCB encephalopathy during moderate to severe neonatal jaundice.