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.

Chromatographic and spectroscopic analysis of heavy crude oil mixtures with emphasis in nuclear magnetic resonance spectroscopy: a review.

The state of the art in the characterization of heavy crude oil mixtures is presented. This characterization can be done by different techniques, such as gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), infrared spectroscopy (IR), Raman spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Nuclear magnetic resonance spectroscopy is the technique of choice due to its capability to provide information on the chemical nature of individual types of hydrogen and carbon atoms in different and complex mixtures of crude oils. The progress made in the interpretation of the NMR spectra with the development of new NMR techniques and different multivariate data analyses could give relevant information about the identification and characterization of hydrocarbons and their physical and chemical properties. These progresses can improve the refining industries operation as a result of the better knowledge on the crude composition that is fed in the refining process, as well as in the prediction of better operating conditions to obtain refined products with desired specifications and in quantities desirable to meet the market demands. The improvement in the refining operation conditions is reflected in economical benefits.

Selective vulnerability of rat brain regions to unconjugated bilirubin.

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin (UCB) encephalopathy, although cerebellum also shows selective yellow staining in kernicterus. We previously demonstrated that UCB induces oxidative stress in cortical neurons, disruption of neuronal network dynamics, either in developing cortical or hippocampal neurons, and that immature cortical neurons are more prone to UCB-induced injury. Here, we studied if immature rat neurons isolated from cortex, cerebellum and hippocampus present distinct features of oxidative stress and cell dysfunction upon UCB exposure. We also explored whether oxidative damage and its regulation contribute to neuronal dysfunction induced by hyperbilirubinemia, considering neurite extension and ramification, as well as cell death. Our results show that UCB induces nitric oxide synthase expression, as well as production of nitrites and cyclic guanosine monophosphate in immature neurons, mainly in those from hippocampus. After exposure to UCB, hippocampal neurons presented the highest content of reactive oxygen species, disruption of glutathione redox status and cell death, when compared to neurons from cortex or cerebellum. In particular, the results indicate that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein implicated in the maintenance of cellular oxidation status. However, longer neuronal exposure to UCB caused down-regulation of DJ-1 expression, especially in hippocampal neurons. In addition, a greater impairment in neurite outgrowth and branching following UCB treatment was also noticed in immature neurons from hippocampus. Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis, protected neurons from UCB-induced oxidative stress and necrotic cell death, preventing DJ-1 down-regulation and neuritic impairment. Taken together, these data point to oxidative injury and disruption of neuritic network as hallmarks in hippocampal susceptibility to UCB. Most importantly, they also suggest that local differences in glutathione content may account to the different susceptibility between brain regions exposed to UCB.

Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin.

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37°C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin=0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α+IL-1ß intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid (tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both L-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α+IL-1ß have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.

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.

Atividade anti-helmíntica dos flavonóides isolados das raízes de Andira anthelmia (Leguminosae) / Anthelmintic activity of flavonoids isolated from roots of Andira anthelmia (Leguminosae)

As cascas e as raízes de A. anthelmia têm sido utilizadas tradicionalmente como vermífugas. Um estudo biomonitorado do extrato metanólico das raízes de A. anthelmia conduziu ao isolamento das isoflavonas biochanina A e genisteína da fração acetato de etila; biochanina A 7-O-β-D-glicopiranosídeo, biochanina A 7-O-α-L-rhamnopiranosil-(1→6)-β-D-glicopiranosídeo e biochanina A 7-O-β-D-apiofuranosil-(1→5)-β-D-apiofuranosil-(1→6)-β-D-glicopiranosídeo da fração n-butanólica e catequina da fração metanólica. Suas estruturas foram elucidadas com base em dados espectrométricos. A atividade anti-helmíntica foi investigada em camundongos naturalmente infectados por Aspiculuris tetraptera. Os compostos isolados aplicados nos animais por via intragástrica na dose de 2,0 mg.kg-1 durante três dias consecutivos revelaram percentuais significativos na eliminação de A. tetraptera, quando comparados com o lote controle.

Bioactivity-guided fractions of the methanol extract from the roots of Andira anthelmia led the isolation of the isoflavones biochanin A and genistein from the ethyl acetate fraction; biochanin A 7-O-β-D-glucopyranoside, biochanin A 7-O-α-L-rhamnopyranosyl-(1→6)β-D-glucopyranoside and biochanin A 7-O-β-D-apiofuranosyl-(1→5)-β-D-apiofuranosyl-(1→6)β-D-glucopyranoside from the n-butanol fraction and catechin from the methanol one. Their structures were elucidated on the basis of spectroscopy data. The anthelmintic activity was investigated in mice naturally infected by Aspiculuris tetraptera. The compounds administered in the animals by intragastric route in doses of 2.0 mg.kg-1, were effective in the removal of the total number of the A. tetraptera when compared with the control group.

[Evaluation of the anthelmintic activity of extracts from Luxemburgia octandra St. Hill. in mice naturally infected with Aspiculuris tetraptera and Vampirolepis nana]. / Avaliação da atividade anti-helmíntica de extratos de Luxemburgia octandra St. Hill. em camundongos naturalmente infectados com Aspiculuris tetraptera e Vampirolepis nana.

The anthelmintic activity of the extracts obtained from Luxemburgia octandra was evaluated naturally infected mice with Aspiculuris tetraptera and Vampirolepis nana. The leaves extracts were obtained through maceration and given to the animals by gavage in doses 8 and 20 mg/kg during three days. The ethanolic and ethyl acetate extracts presented significant increase of the V. nana elimination, but did not present the nematicide effect against A. tetraptera.

Ação anti-helmíntica de extratos brutos de andira anthelmia (Vell.) Macbr. e Andira fraxinifolia Benth., camundongos naturalmente infectados por vampirolepis nana e aspiculuris tetraptera vampirolepis nana e aspiculuris tetraptera / Anthelmintic action of the crude extracts of andira anthelmia (Vell.) Macbr. and andira fraxinifolia Benth., in mice vampirolepis nana and aspiculuris tetraptera naturally infected by vampirolepis nana and aspiculuris tetraptera

A atividade anti-helmíntica dos extratos brutos da folha fresca e seca, casca do tronco e da raiz da espécie Andira anthelmia e A. fraxinifolia, foi avaliada sob as espécies Vampirolepis nana e Aspiculuris tetraptera. Foram utilizadas as doses de 8 g/kg/dia para folhas e casca do tronco, e de 1, 2, 4 e 8 g/kg/dia para a casca da raiz de A. anthelmia, e as doses de 8 g/kg/dia para folhas e casca da raiz, e de 4, 8 e 16 g/kg/dia para a casca do tronco de A. fraxinifolia. A dose de 8 g/kg/dia do extrato bruto da folha fresca e seca, da casca do tronco e da raiz, quando comparadas, não apresentaram diferença significativa no percentual de eliminação de V. nana, 14,9 per cent, 16,4 per cent, 6,3 per cent, 17,9 per cent , respectivamente, o mesmo ocorreu quando comparadas ao grupo controle (7,4 per cent). As doses de 2 e 4 g/kg/dia, 49,4 per cent e 42,7 per cent, respectivamente, foram significativa-mente mais ativas para V. nana, em comparação com as demais partes anatômicas e o grupo controle, não apresentando diferença em relação a dose de 1 g/kg/dia da casca da raiz e do mebendazol. A dose 2 g/kg/dia foi significati-vamente mais ativa para A. tetraptera (63,2 per cent), em comparação com as folhas, a casca da raiz nas doses de 1 e 4 g/kg/dia, e o grupo controle, 1,2 per cent, 1,7 per cent, 31,7 per cent, 15,3 percent, 0,1 per cent, respec-tivamente. Contudo, não apresentou diferença significativa em relação ao nitroscanato, sendo menos ativa do que o mebendazol. Embora a dose de 8 g/kg/dia da casca do tronco de A. fraxinifolia tenha apresentado um percentual de eliminação de V. nana maior (37,5 per cent), não foi observado diferença estatística, quando comparado com as demais doses, partes anatômicas, grupo controle e o mebendazol. Similarmente, não foi observado diferença significativa nos percentuais de eliminação de A. tetraptera. Os resultados sugerem o extrato bruto da casca da raiz de A. anthelmia como anti-helmíntico promissor, necessitando de estudos complementares, devido a alta toxicidade apresentada.