Oxidative Imbalance, Nitrative Stress, and Inflammation in C6 Glial Cells Exposed to Hexacosanoic Acid: Protective Effect of N-acetyl-L-cysteine, Trolox, and Rosuvastatin.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder caused by disfunction of the ABCD1 gene, which encodes a peroxisomal protein responsible for the transport of the very long-chain fatty acids from the cytosol into the peroxisome, to undergo ß-oxidation. The mainly accumulated saturated fatty acids are hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in tissues and body fluids. This peroxisomal disorder occurs in at least 1 out of 20,000 births. Considering that pathophysiology of this disease is not well characterized yet, and glial cells are widely used in studies of protective mechanisms against neuronal oxidative stress, we investigated oxidative damages and inflammatory effects of vesicles containing lecithin and C26:0, as well as the protection conferred by N-acetyl-L-cysteine (NAC), trolox (TRO), and rosuvastatin (RSV) was assessed. It was verified that glial cells exposed to C26:0 presented oxidative DNA damage (measured by comet assay and endonuclease III repair enzyme), enzymatic oxidative imbalance (high catalase activity), nitrative stress [increased nitric oxide (NO) levels], inflammation [high Interleukin-1beta (IL-1ß) levels], and induced lipid peroxidation (increased isoprostane levels) compared to native glial cells without C26:0 exposure. Furthermore, NAC, TRO, and RSV were capable to mitigate some damages caused by the C26:0 in glial cells. The present work yields experimental evidence that inflammation, oxidative, and nitrative stress may be induced by hexacosanoic acid, the main accumulated metabolite in X-ALD, and that antioxidants might be considered as an adjuvant therapy for this severe neurometabolic disease.

Urinary biomarkers of oxidative stress and plasmatic inflammatory profile in phenylketonuric treated patients.

Oxidative stress has been proposed as an important pathophysiologic feature of various inborn errors of metabolism, including phenylketonuria (PKU). Considering that there are few studies relating oxidative stress and inflammation directly in PKU disease, the aim of this study was to evaluate and correlate oxidative damage to biomolecules, antioxidant defenses, pro-inflammatory cytokines, phenylalanine (Phe) and its metabolites (phenyllactic acid--PLA and phenylacetic acid--PAA) levels in urine and plasma from patients with PKU under dietary treatment. We observed a marked increase of isoprostanes, which is a lipid peroxidation biomarker, in urine from these treated patients. Next, we demonstrated that protein oxidative damage, measured by di-tyrosine formation, was significantly increased in urine from PKU treated patients and that decreased urinary antioxidant capacity was also observed. Our findings concerning to the inflammatory cytokines interleukin-6 and interleukin-1ß, both significantly increased in these patients, provide evidence that the pro-inflammatory state occurs. Besides, interleukin-1ß was positively correlated with isoprostanes. We observed a negative correlation between interleukin-6 and interleukin-10, an anti-inflammatory cytokine. Di-tyrosine was positively correlated with Phe, which indicates oxidative damage to proteins, as well as with PAA. These findings may suggest that the protein damage may be induced by Phe and its metabolite PAA in PKU. Our results indicate that pro-oxidant and pro-inflammatory states occur and are, in part, correlated and protein oxidation seems to be induced by Phe and PPA in PKU patients.

Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT (n=17) and healthy age-matched controls (n=10-15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress.

Purple grape juices prevent pentylenetetrazol-induced oxidative damage in the liver and serum of Wistar rats.

Oxidative damages in hepatocytes may be caused by epilepsy and/or anticonvulsant drugs. Epilepsy is one of the most common neurological disorders, characterized by recurrent seizures, which may increase the content of reactive oxygen species. Organic and conventional grape juices are rich in polyphenols, compounds with important antioxidant activity. It is hypothesized that organic and conventional purple grape juices may have protective effect against oxidative damage induced by pentylenetetrazole (PTZ) (a standard convulsant drug) in the liver and serum of Wistar rats. Animals (n = 16 in each group) received, by gavage, saline, organic grape juice or conventional grape juice (10 µL/g of body weight) for 17 days. Subsequently, half of the rats in each group received PTZ (60 mg/kg). After 30 minutes, the animals were euthanized by decapitation. Liver and blood samples were isolated to evaluate oxidative parameters (lipid and protein oxidation, nitric oxide metabolite content, antioxidant defenses, and protein sulfhydryl content). The results of this study showed that although organic juice contains higher polyphenol content than conventional juice, both juices conferred protection against lipid and protein oxidative damage and limited the increase in PTZ-induced nitric oxide metabolite content in the liver and serum. In addition, both juices inhibited the PTZ-induced reduction in enzymatic antioxidant defenses (superoxide dismutase and catalase activities) and sulfhydryl protein content in the liver and serum. In summary, both organic and conventional grape juices were able to reduce oxidative damage induced by PTZ in the liver and serum of Wistar rats.

Organic and Conventional Yerba Mate (Ilex paraguariensis A. St. Hil) Improves Metabolic Redox Status of Liver and Serum in Wistar Rats.

Organic and conventional yerba mate (Ilex paraguariensis) is widely used in South America to prepare nonalcoholic drinks rich in polyphenols. These compounds are able to prevent the generation of reactive species, thus minimizing the incidence of several diseases. In this perspective, we hypothesized that yerba mate may have protective effects against pentylenetetrazol (PTZ)-induced oxidative damage in liver and serum of rats. Animals (n = 42) received distilled water (control) or yerba mate (organic or conventional) for fifteen days. Then, half of the rats of each group received 60 mg/kg PTZ intraperitoneally or saline solution. After 30 min the animals were euthanized and the liver and blood were collected. The results showed that organic and conventional yerba mate avoided PTZ-induced oxidative damage and nitric oxide production in the liver and serum of the rats. Moreover, both kinds of yerba mate prevented the decrease in enzymatic (superoxide dismutase and catalase) and non-enzymatic (sulfhydryl protein content) defenses in the liver and serum. In addition, histopathologic analysis of the liver showed that yerba mate reduced PTZ-induced cell damage. These findings indicate that yerba mate provides hepatoprotection and improves antioxidant status in the serum, which may contribute to the development of new therapeutic strategies using nutraceuticals drinks.

Controle de esterilidade de produtos de células progenitoras hematopoéticas do sangue periférico / Sterility control of hematopoietic progenitor cells from peripheral blood products

A taxa de contaminação microbiana dos produtos de células progenitoras hematopoéticas do sangue periférico é baixa. Neste estudo pesquisou-se a prevalência de hemoculturas positivas em células progenitoras hematopoéticas do sangue periférico (CPHSP) no Serviço de Hemoterapia do Hospital de Clínicas de Porto Alegre. Do total de 618 coletas realizadas no período de 2000 a 2007, 26 (4,2 por cento) apresentaram contaminação por bactérias. O Staphylococcus coagulase-negativo foi predominantemente isolado nas hemoculturas. A antibioticoterapia pré e pós-infusão foi estabelecida de acordo com o microorganismo e seu antibiograma, sendo que, em cinco das doze infusões contaminadas realizadas, não foram administrados antimicrobianos profilaticamente. Episódios febris foram observados em sete pacientes (58 por cento), enquanto cinco (42 por cento) não apresentaram febre. Das doze infusões contaminadas realizadas, seis (50 por cento) apresentaram hemocultura pós-descongelamento positivas, enquanto as restantes (50 por cento) foram negativas. Isto se deve às propriedades bactericidas do DMSO, de células fagocitose-ativas e de temperaturas muito baixas atingidas na criopreservação. Autores têm relatado sucesso neste procedimento após a infusão desses produtos contaminados com o mínimo de consequências clínicas.

The rate of microbial contamination of hematopoietic progenitor cell products from peripheral blood is low. In this study, we investigated the prevalence of positive blood cultures of hematopoietic progenitor cells from peripheral blood in a hemotherapy service. Of a total of 618 samples taken during the period from 2000 to 2007, 26 (4.2 percent) were contaminated by bacteria. Staphylococcus coagulase-negative was the predominant bacterium isolated in blood cultures. Pre- and post-infusion antibiotic therapy was established depending on the microorganism and antibiogram, whereas in five out of twelve contaminated infusions, no antibiotics were administered prophylactically. Febrile episodes were observed in seven patients (58 percent), while five (42 percent) did not suffer from fever. Of the twelve contaminated infusions performed, six (50 percent) of the samples had positive blood cultures after thawing, while the others (50 percent) were negative. This is due to the bactericidal properties of DMSO, phagocytosis-active cells and the extremely low temperatures during cryopreservation. Authors have reported success in the procedure after the infusion of contaminated products with minimal clinical consequences.

Short-term memory formation and long-term memory consolidation are enhanced by cellular prion association to stress-inducible protein 1.

Cellular prion protein (PrP(C)) is a cell surface glycoprotein that interacts with several ligands such as laminin, NCAM (Neural-Cell Adhesion Molecule) and the stress-inducible protein 1 (STI1). PrP(C) association with these proteins in neurons mediates adhesion, differentiation and protection against programmed cell death. Herein, we used an aversively motivated learning paradigm in rats to investigate whether STI1 interaction with PrP(C) affects short-term memory (STM) formation and long-term memory (LTM) consolidation. Blockage of PrP(C)-STI1 interaction with intra-hippocampal infusion of antibodies against PrP(C) or STI1 immediately after training impaired both STM and LTM. Furthermore, infusion of PrP(C) peptide 106-126, which competes for PrP(C)-STI1 interaction, also inhibited both forms of memory. Remarkably, STI1 peptide 230-245, which includes the PrP(C) binding site, had a potent enhancing effect on memory performance, which could be blocked by co-treatment with the competitive PrP(C) peptide 106-126. Taken together, these results demonstrate that PrP(C)-STI1 interaction modulates both STM and LTM and suggests a potential use of ST11 peptide 230-245 as a pharmacological agent.

The interaction between prion protein and laminin modulates memory consolidation.

Cellular prion protein (PrPc) has a pivotal role in prion diseases. PrPc is a specific receptor for laminin (LN) gamma1 peptide and several lines of evidence indicate that it is also involved in neural plasticity. Here we investigated whether the interaction between PrPc and LN plays a role in rat memory formation. We found that post-training intrahippocampal infusion of PrPc-derived peptides that contain the LN binding site (PrPc163-182 and PrPc173-192) or of anti-PrPc or anti-LN antibodies that inhibit PrPc-LN interaction impaired inhibitory avoidance memory retention. The amnesic effect of anti-PrPc antibodies and PrPc173-192 peptide was reversed by co-infusion of a LN gamma1 chain-derived peptide containing the PrPc-binding site, suggesting that PrPc-LN interaction is indeed crucial for memory consolidation. In addition, PrPc173-192 peptide and anti-PrPc or anti-LN antibodies also inhibited the activation of hippocampal cAMP-dependent protein kinase A (PKA) and extracellular regulated kinase (ERK1/2), two kinases that mediate the up-regulation of signaling pathways needed for consolidation of inhibitory avoidance memory. Our findings show that, through its interaction with LN, hippocampal PrPc plays a critical role in memory processing and suggest that this role is mediated by activation of both PKA and ERK1/2 signaling pathways.

Retrograde amnesia induced by drugs acting on different molecular systems.

The gamma aminobutyric acid-A (GABA-sub(A)) agonist, muscimol, the glutamate N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoic acid (AP5), and the inhibitor of the extracellularly regulated kinases (ERKs), UO 126, cause retrograde amnesia when administered to the hippocampus. In the present study, the authors found that they all cause retrograde amnesia for 1-trial inhibitory avoidance, not only when infused into the dorsal CA1 region of the hippocampus, but also when infused into the basolateral amygdala or the entorhinal, parietal, and posterior cingulate cortices. The posttraining time course of the effect of each drug was, however, quite different across brain structures. Thus, in all of them, NMDA receptors and the ERK pathway are indispensable for memory consolidation, and GABA-sub(A) receptor activation inhibits memory consolidation: but in each case, their influence is interwoven differently.

Role of the hippocampus and amygdala in the extinction of fear-motivated learning.

Fear-motivated learning is at the root of phobias, panic, generalized anxiety and the posttraumatic stress disorder. This makes the inhibition of fear-motivated behavior a therapeutic desideratum in these diseases. The simplest way to accomplish this is by extinction, a procedure by which a given association between a conditioned stimulus or context (CS) and a fearsome event is replaced by a new association between the CS and the lack of the fearsome stimulus. This is a new learning for the subject and, in rats, it requires gene expression and protein synthesis both in the hippocampus and the basolateral amygdala, alongside with the activation of various metabolic signaling pathways. These requirements are similar to, but not identical with those for consolidation of the original memory. In addition, some systems uninvolved in original consolidation appear to be involved in extinction, namely, the endocannabinoid system. Extinction can be enhanced by prolonging the exposure to the lack of fearsome stimulation; e.g., in rats, by increasing the time of permanence in the compartment where the animals no longer receive a footshock. Further research into the possibilities of enhancing extinction at the expense of the original fearsome learning is desirable.

Cellular prion protein ablation impairs behavior as a function of age.

Cellular prion protein (PrPc) has been associated with some physiological functions in recent reports. Here we investigate behavioral parameters in 3- and 9-month-old mice lacking PrPc protein (Prnp0/0) and in rats after intrahippocampal administration of affinity purified anti-PrPc IgG (0.09 microg/side). No differences were observed between 3-month-old animals. However, 9-month-old Prnp0/0 mice and rats infused with anti-PrPc antibody showed a clear impairment of short- and long-term memory retention of a step-down inhibitory avoidance task. A decreased locomotor activity during exploration of an open field was also observed. These results suggest that systems involved in memory formation become more susceptible to mechanisms that require PrPc between the ages of 3 and 9 months in both mice and rats.

Memory extinction requires gene expression in rat hippocampus.

Rats with cannulae in the dorsal CA1 region of the hippocampus were trained in one-trial step-down inhibitory avoidance, and submitted to four consecutive daily test sessions without the footshock. This produced extinction of the conditioned response in control animals. The bilateral infusion into the CA1 region of the dorsal hippocampus of two different inhibitors of gene transcription, DRB (80 microg/side) or alpha-amanitin (25 pg/side), or of the protein synthesis inhibitor, anisomycin (80 microg/side) blocked extinction of the CR. The treatments were effective when given 15 min before, but not 1 or 3h after the first test session. Retrieval itself was not affected by the drugs. The treatments did not affect general activity in an open field or anxiety levels measured in an elevated plus maze. The data indicate that gene transcription and protein synthesis are necessary at the time of the first test session in order to generate extinction. These requirements are to be expected from learning that involves new synaptic associations.

Decreased hyperlocomotion induced by MK-801, but not amphetamine and caffeine in mice lacking cellular prion protein (PrP(C)).

The cellular prion protein (PrP(C)) has been involved in several neurodegenerative disorders however it has been proposed that it is also be implicated in psychotic disorders. We investigated the effect of three psychotropic drugs in locomotor activity of PrP(C) knockout (Prnp(O/O)) and wild-type mice. The NMDA receptor channel blocker MK-801 (0.25 mg/kg), the indirect dopamine agonist amphetamine (1 mg/kg) and the adenosine receptor antagonist caffeine (10 mg/kg) were administered i.p. after 60 min of habituation and locomotion was monitored for 3 h. Prnp(O/O) mice presented a diminished hyperlocomotor response to MK-801 treatment but normal response to amphetamine and caffeine compared to wild type mice. These results suggest that lack of PrP(C) leads to a functional alteration in the glutamatergic system, whereas the regulation of both dopaminergic and adenosinergic systems are preserved. Finally, lack of PrP(C) seems not to exacerbate the response to these psychotropic drugs, which modulate neurotransmitter systems possibly involved in schizophrenia and psychotic disorders.

Intrahippocampal infusion of ebselen impairs retention of an inhibitory avoidance task in rats.

Ebselen is a seleno-compound used in the treatment of neurological disorders involving the glutamatergic system. Although ebselen is currently used in clinical trials, the physiological effects of this seleno-compound are poorly known. In this study, we investigated the effects of intrahippocampal infusion of ebselen (0.1-3 nmol) in rats submitted to an inhibitory avoidance task. Ebselen (1-3 nmol) infused after the training session impaired retention of inhibitory avoidance, tested 90 min or 24 h after the training session. Moreover, ebselen also impaired the retention when infused 30 min prior to training or 10 min prior to test sessions. In summary, ebselen impaired memory consolidation, acquisition and retrieval. This amnesic effect of ebselen could be related to oxidant activity at N-methyl-D-aspartate (NMDA) receptors. Our results indicate that more studies must be performed to investigate the mechanisms of this amnesic effect and whether ebselen has a cognition-impairing effect when administered chronically.

Memory retrieval and its lasting consequences.

Many, if not all psychiatric diseases are accompanied by memory disturbances, in particular, the dementias, schizophrenia, and, to an extent, mood disorders. Anxiety and stress, on the other hand, cause important alterations of memory, particularly its retrieval. Here we discuss several new findings on the basic mechanisms of consolidation, retrieval and extinction of a prototype form of episodic memory in the rat: conditioned fear. The findings point the way for investigations on the pathology of these aspects of memory in health and disease. Emphasis is placed on the parallel processing of retrieval in several cortical areas, on the links between retrieval and the onset of extinction, on the fact that extinction involves new learning requiring gene expression, and on the differences between the retrieval of recent or remote long-term memories.