Alterations in CD39/CD73 axis of T cells associated with COVID-19 severity.

Purinergic signaling modulates immune function and is involved in the immunopathogenesis of several viral infections. This study aimed to investigate alterations in purinergic pathways in coronavirus disease 2019 (COVID-19) patients. Mild and severe COVID-19 patients had lower extracellular adenosine triphosphate and adenosine levels, and higher cytokines than healthy controls. Mild COVID-19 patients presented lower frequencies of CD4+ CD25+ CD39+ (activated/memory regulatory T cell [mTreg]) and increased frequencies of high-differentiated (CD27- CD28- ) CD8+ T cells compared with healthy controls. Severe COVID-19 patients also showed higher frequencies of CD4+ CD39+ , CD4+ CD25- CD39+ (memory T effector cell), and high-differentiated CD8+ T cells (CD27- CD28- ), and diminished frequencies of CD4+ CD73+ , CD4+ CD25+ CD39+ mTreg cell, CD8+ CD73+ , and low-differentiated CD8+ T cells (CD27+ CD28+ ) in the blood in relation to mild COVID-19 patients and controls. Moreover, severe COVID-19 patients presented higher expression of PD-1 on low-differentiated CD8+ T cells. Both severe and mild COVID-19 patients presented higher frequencies of CD4+ Annexin-V+ and CD8+ Annexin-V+ T cells, indicating increased T-cell apoptosis. Plasma samples collected from severe COVID-19 patients were able to decrease the expression of CD73 on CD4+ and CD8+ T cells of a healthy donor. Interestingly, the in vitro incubation of peripheral blood mononuclear cell from severe COVID-19 patients with adenosine reduced the nuclear factor-κB activation in T cells and monocytes. Together, these data add new knowledge to the COVID-19 immunopathology through purinergic regulation.

Functional interplay between adenosine A2A receptor and NMDA preconditioning in fear memory and glutamate uptake in the mice hippocampus.

N-methyl D-aspartate (NMDA) administered at subtoxic dose plays a protective role against neuronal excitotoxicity, a mechanism described as preconditioning. Since the activation of adenosinergic receptors influences the achievement of NMDA preconditioning in the hippocampus, we evaluated the potential functional interplay between adenosine A1 and A2A receptors (A1R and A2AR) activities and NMDA preconditioning. Adult male Swiss mice received saline (NaCl 0.9 g%, i.p.) or a nonconvulsant dose of NMDA (75 mg/kg, i.p.) and 24 h later they were treated with the one of the ligands: A1R agonist (CCPA, 0.2 mg/kg, i.p.) or antagonist (DPCPX, 3 mg/kg, i.p.), A2AR agonist (CGS21680, 0.05 mg/kg, i.p.) or antagonist (ZM241385, 0.1 mg/kg, i.p.) and subjected to contextual fear conditioning task. Binding properties and content of A2AR and glutamate uptake were assessed in the hippocampus of mice subjected to NMDA preconditioning. Treatment with CGS21680 increased the time of freezing during the exposure of animals to the new environment. NMDA preconditioning did not affect the freezing time of mice per se, but it prevented the response observed after the activation of A2AR. Furthermore, the activation of A2AR by CGS21680 after the preconditioning blocked the increase of glutamate uptake induced by NMDA preconditioning. The immunodetection of A2AR in total hippocampal homogenates showed no significant differences evoked by NMDA preconditioning and did not alter A2AR maximum binding for the selective ligand [3H]CGS21680. These results demonstrate changes in A2AR functionality in mice following NMDA preconditioning.

Adenosine and NMDA Receptors Modulate Neuroprotection-Induced NMDA Preconditioning in Mice.

The severity score of quinolinic acid (QA)-induced seizures was investigated after N-methyl-D-aspartate (NMDA) preconditioning associated with adenosine receptors. Also, the levels of adenosine A1 and A2A receptors and subunits of NMDA receptors in the hippocampi of mice were determined to define components of the resistance mechanism. Adult CF-1 mice were treated intraperitoneally with saline or NMDA (75 mg/kg), and some mice were treated intracerebroventricularly (i.c.v.) with 0.1 pmol of adenosine receptor antagonists 8-cyclopentyltheophylline (CPT; receptor A1) or ZM241385 (receptor A2A) 0, 1, or 6 h after NMDA administration. These adenosine receptor antagonists were administered to block NMDA's protective effect. Seizures and their severity scores were evaluated during convulsions induced by QA (36.8 nmol) that was administered i.c.v. 24 h after NMDA. The cell viability and content of subunits of the NMDA receptors were analyzed 24 h after QA administration. NMDA preconditioning reduced the maximal severity 6 displayed in QA-administered mice, inducing protection in 47.6% of mice after QA-induced seizures. CPT increased the latency of seizures when administered 0 or 6 h, and ZM241385 generated the same effect when administered 6 h after NMDA administration. The GluN1 content was lower in the hippocampi of the QA mice and the NMDA-preconditioned animals without seizures. GluN2A content was unaltered in all groups. The results demonstrated the components of resistance evoked by NMDA, in which adenosine receptors participate in a time-dependent mode. Similarly, the reduction on GluN1 expression in the hippocampus may contribute to this effect during the preconditioning period.

Guanosine and GMP increase the number of granular cerebellar neurons in culture: dependence on adenosine A2A and ionotropic glutamate receptors.

The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of ß-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.

Production, characterization and toxicology assay of creatine pegylated nanoliposome with polysorbate 80 for brain delivery.

Creatine acts intracellularly as energy buffer and storage, demonstrating protective effects in animal models of neurodegenerative diseases. However, its permeability throught blood-brain barrier (BBB) is reduced. The aim of the present study was developing a carrier to facilitate the delivery of creatine to the central nervous system. Creatine nanoliposomes were produced, characterized and assayed in models of toxicity in vitro and in vivo. Particles showed negative zeta potential (-12,5 mV), polydispersity index 0.237 and medium-size of 105 nm, which was confirmed by transmission electron microscopy (TEM) images. Toxicity assay in vitro was evaluated with blank liposomes (no drug) or creatine nanoliposomes at concentrations of 0.02 and 0.2 mg/mL, that did not influence the viability of Vero cells. The result. of the comet assay that the nanoliposomes are not genotoxic, togeher with cell viability demonstrated that the nanoliposomes are not toxic. Besides, in vivo assays not demonstrate toxicity in hematological and biochemical markers of young rats. Nevertheless, increase content of creatine in the cerebral cortex tissue after subchronic treatment was observed. Altogether, results indicate increase permeability of creatine to the BBB that could be used as assay for in vivo studies to confirm improved effect than free creatine.

Adenosine A1 receptor activation modulates N-methyl-d-aspartate (NMDA) preconditioning phenotype in the brain.

N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.

Mitochondrial respiratory chain and creatine kinase activities following trauma brain injury in brain of mice preconditioned with N-methyl-D-aspartate.

Traumatic brain injury (TBI) induces glutamatergic excitotoxicity through N-methyl-D-aspartate (NMDA) receptors, affecting the integrity of the mitochondrial membrane. Studies have pointed to mitochondria as the master organelle in the preconditioning-triggered endogenous neuroprotective response. The present study is aimed at understanding energy metabolism in the brains of mice after preconditioning with NMDA and TBI. For this purpose, male albino CF-1 mice were pre-treated with NMDA (75 mg/kg) and subjected to brain trauma. Mitochondrial respiratory chain and creatine kinase activities were assessed at 6 or 24 h after trauma. The mice preconditioned and subjected to TBI exhibited augmented activities of complexes II and IV in the cerebral cortex and/or cerebellum. Creatine kinase activity was also augmented in the cerebral cortex after 24 h. We suggest that even though NMDA preconditioning and TBI have similar effects on enzyme activities, each manage their response via opposite mechanisms because the protective effects of preconditioning are unambiguous. In conclusion, NMDA preconditioning induces protection via an increase of enzymes in the mitochondria.

Methylphenidate treatment affects mitogen-activated protein kinase activation in the striatum of young rats.

OBJECTIVE: Methylphenidate (MPD) is a drug prescribed for the treatment of attention deficit/hyperactivity disorder and its therapeutic effect is attributed to the inhibition of dopamine. METHODS: Young male Wistar rats were administered MPD (1, 2, 5, or 10 mg/kg) once a day or an intraperitoneal injection of saline for 28 days (chronic treatment) or for 1 day (acute treatment). Two hours after the last administration the animals were decapitated and their striatum was dissected. RESULTS: In this work, we show that continued treatment with MPD is capable of modifying the levels of phosphorylation of proteins JNK1/2 (c-Jun amino-terminal kinases 1 and 2) and ERK1/2 (extracellular signal-regulated kinases 1 and 2). Whereas the level of phosphorylation of protein ERK increased significantly, that of proteins JNK1/2 diminished. CONCLUSION: The alteration in the level of activation of mitogen-activated protein kinases can be a molecular mechanism through which MPD exerts its therapeutic effect.

NMDA preconditioning prevents object recognition memory impairment and increases brain viability in mice exposed to traumatic brain injury.

Recent studies have focused on the role of N-methyl-d-aspartate (NMDA) in brain injury. The present study is aimed at verifying memory, anxiety/depression parameters, and cellular viability in the brain of mice preconditioned with NMDA and subjected to the model of mild traumatic brain injury. For this purpose, male albino CF-1 mice were pre-treated with NMDA (75 mg/kg) and subjected to brain trauma, and after 24h submitted to memory tasks and anxiety and depression-like behavioral tests. The memory tests were evaluated at 1.5h, 24h, and 7 days after the training. In addition, the cellular viability was evaluated in the cerebral cortex and hippocampus 96 h after the trauma. It was observed that the cellular viability was reduced in the hippocampus of the mice subjected to trauma and the preconditioning with NMDA was able to protect this damage. All mice learnt the task in the habituation test, but in the object recognition task the mice preconditioned with NMDA were protected against impairment induced by TBI in both short and long-term memory. On the other hand, in the step-down inhibitory avoidance test, only the mice treated with NMDA showed impairment of long-term memory (7 days after training session). The evaluation of anxiety/depression behavior showed no changes after TBI. In conclusion, NMDA preconditioning induced impairment of the long-term memory; however, it was able to protect against the novel recognition memory impairment and increase the cellular survival in the hippocampus of mice exposed to traumatic brain injury.

Anti-HIV drugs nevirapine and efavirenz affect anxiety-related behavior and cognitive performance in mice.

Nevirapine (NVP) and efavirenz (EFV) belong to the class of anti-HIV drugs called non-nucleoside reverse transcriptase inhibitors (NNRTIs), commonly used as part of highly active antiretroviral therapy (HAART). Although the HAART is able to bring down viral load to undetectable levels and restore immune function, their prolonged use causes several adverse effects. It has been demonstrated that both NVP and EFV are able to cross the blood-brain barrier, causing important central nervous system-related side effects. Thus, this study investigated the effects of chronic administration of EFV (10 mg/kg) and NVP (3.3 mg/kg) in mice submitted to two distinct series of experiments, which aimed to evaluate: (1) the emotional behavior (elevated plus-maze, forced swimming, and open-field test) and (2) the cognitive performance (object recognition and inhibitory avoidance test) of mice. Our results demonstrated that EFV, but not NVP, reduced the exploration to open arms in the elevated plus-maze test. Neither NVP nor EFV altered mouse behavior in the forced swimming and open-field tests. Both drugs reduced the recognition index in the object recognition test, but only EFV significantly impaired the aversive memory assessed in the inhibitory avoidance test 24 h after training. In conclusion, our findings point to a genuine anxiogenic-like effect to EFV, since it reduced exploration to open arms of elevated plus-maze test without affecting spontaneous locomotion. Additionally, both drugs impaired recognition memory, while only the treatment with EFV impaired significantly aversive memory.

N-methyl-D-aspartate preconditioning improves short-term motor deficits outcome after mild traumatic brain injury in mice.

Traumatic brain injury (TBI) causes impairment of fine motor functions in humans and nonhuman mammals that often persists for months after the injury occurs. Neuroprotective strategies for prevention of the sequelae of TBI and understanding the molecular mechanisms and cellular pathways are related to the glutamatergic system. It has been suggested that cellular damage subsequent to TBI is mediated by the excitatory neurotransmitters, glutamate and aspartate, through the excessive activation of the N-methyl-D-aspartate (NMDA) receptors. Thus, preconditioning with a low dose of NMDA was used as a strategy for protection against locomotor deficits observed after TBI in mice. Male adult mice CF-1 were preconditioned with NMDA (75 mg/kg) 24 hr before the TBI induction. Under anesthesia with O(2)/N(2)O (33%: 66%) inhalation, the animals were subjected to the experimental model of trauma that occurs by the impact of a 25 g weight on the skull. Sensorimotor gating was evaluated at 1.5, 6, or 24 hr after TBI induction by using footprint and rotarod tests. Cellular damage also was assessed 24 hr after occurrence of cortical trauma. Mice preconditioned with NMDA were protected against all motor deficits revealed by footprint tests, but not those observed in rotarod tasks. Although mice showed motor deficits after TBI, no cellular damage was observed. These data corroborate the hypothesis that glutamatergic excitotoxicity, especially via NMDA receptors, contributes to severity of trauma. They also point to a putative neuroprotective mechanism induced by a sublethal dose of NMDA to improve motor behavioral deficits after TBI.

Blockade of adenosine A2A receptor counteracts neuropeptide-S-induced hyperlocomotion in mice.

Neuropeptide S (NPS) is the endogenous ligand of a G-protein-coupled receptor named as NPSR. Behavioral effects have been recently attributed to NPS, i.e. hyperlocomotion, anxiolysis, and wakefulness. However, little is known about the mechanisms by which NPS evokes such biological actions. The present study aimed to investigate the role played by the adenosine A(2A) and A(1) receptors in hyperlocomotion induced by NPS. Spontaneous locomotion was assessed in an activity cage for 30 min in mice acutely treated with caffeine (a nonselective adenosine receptor antagonist), ZM241385 (a selective A(2A) receptor antagonist), or CPT (a selective A(1) receptor antagonist) before NPS challenge (0.1 nmol, i.c.v.), which induce hyperlocomotion in mice. The pretreatment with caffeine (3 mg/kg, i.p.), in an inactive dose per se, prevented the increase in locomotion evoked by NPS. The co-administration of NPS (0.1 nmol, i.c.v.) and ZM241385 (0.1 pmol, i.c.v.) counteracted hyperlocomotion evoked by NPS. The co-administration of NPS and CPT (0.1 pmol, i.c.v.) slightly facilitated the increase in locomotion evoked by NPS alone. In summary, the pharmacological blockade of A(2A) receptors significantly attenuated the stimulatory effects of NPS. By contrast, the antagonism of A(1) receptors facilitated NPS-induced hyperlocomotion in mice, but we cannot rule out a merely additive effect of two stimulatory systems in the brain. Altogether, this is the first evidence of a putative role played by A(2A) and A(1) receptors in modulating hyperlocomotion induced by NPS.

Superoxide production after acute and chronic treatment with methylphenidate in young and adult rats.

The prescription of methylphenidate (MPH) has dramatically increased in this decade for attention deficit hyperactivity disorder (ADHD) treatment. The action mechanism of MPH is not completely understood and studies have been demonstrated that MPH can lead to neurochemical adaptations. Superoxide radical anion is not very reactive per se. However, severe species derived from superoxide radical anion mediate most of its toxicity. In this study, the superoxide level in submitochondrial particles was evaluated in response to treatment with MPH in the age-dependent manner in rats. MPH was administrated acutely or chronically at doses of 1, 2 or 10 mg/kg i.p. The results showed that the acute administration of MPH in all doses in young rats increased the production of superoxide in the cerebellum and only in the high dose (10mg/kg) in the hippocampus, while chronic treatment had no effect. However, acute treatment in adult rats had no effect on production of superoxide, but chronic treatment decreased the production of superoxide in the cerebellum at the lower doses. Our data suggest that the MPH treatment can influence on production of superoxide in some brain areas, but this effect depends on age of animals and treatment regime with MPH.

Exercise training provides cardioprotection via a reduction in reactive oxygen species in rats submitted to myocardial infarction induced by isoproterenol.

Exercise training has demonstrated cardioprotection effects. However, the exact mechanism behind this effect is not is clear. The present study evaluated the effects of 12 weeks of previous treadmill training on the levels of oxidative damage, antioxidant enzyme activity and injury in the myocardium of rats submitted to infarction induced by isoproterenol (ISO). Isoproterenol treatment (80 mg/kg given over 2 days in two equal doses) caused arrhythmias and 60% mortality within 24 h of the last injection in the control group (C + ISO) group when compared with the saline control group (saline). Creatine Kinase--MB levels were markedly increased in hearts from ISO-treated animals in the C + ISO group. Twelve weeks of treadmill training reduced superoxide production, lipid peroxidation levels and protein carbonylation in these animals, as well as increasing the activities and expressions of SOD and CAT. Previous training also reduced CK-MB levels and numbers of deaths by 40%, preventing the deleterious effects of ISO. Based on the data obtained in this study, it is suggested that 12-week treadmill training increases antioxidant enzymes, decreases oxidative damage and reduces the degree of infarction induced by ISO in the hearts of male rats.

Early long-term exposure with caffeine induces cross-sensitization to methylphenidate with involvement of DARPP-32 in adulthood of rats.

Chronic ingestion of caffeine causes dependence and sleep disturbance in children and adolescents. In rodents, the administration of caffeine may produce behavioral cross-sensitization to some psychostimulants, such as dopaminergic psychoactive drugs. Methylphenidate (MPH; Ritalin) is a psychostimulant used in pediatric- and adult human populations to manage the symptoms associated with attention-deficit hyperactivity disorder (ADHD). Previous studies have suggested that dopamine- and cAMP-regulated phosphoproteins of 32 kDa (DARPP-32) participate in the manifestation of behavioral activity following ingestion of caffeine or MPH. The aim of the present study was to evaluate whether long-term administration of low doses of caffeine in rodents during their adolescence induces cross-sensitization to MPH challenge in their adulthood and investigate the involvement of DARPP-32 in this model. Young rats (P25) consumed water or caffeine (0.3 g/L; mean consumption was 7.5 mg/day/kg) for 28 days. The caffeine consumption was then suspended for 14 days (washout period) when the animals received saline solution or MPH (1, 2, or 10 mg/kg) (P67) intraperitoneally. The locomotor activity of these rats was assessed using the open-field test, following which the immunocontent of DARPP-32 was evaluated in samples of their prefrontal cortex, striatum, or hippocampus. Rats chronically exposed to caffeine in their adolescent period and to inactive doses of MPH (1mg/kg) in adulthood showed augmented locomotor activity. The behavioral effect observed was accompanied by increased levels of DARPP-32 in the striatum and prefrontal cortex compared to control groups (saline or caffeine). However, no alteration caused by these treatments was noted in the hippocampus. In conclusion, chronic caffeine exposure induces likely long-term cross-sensitization to MPH in a DARPP-32-dependent pathway.

Atorvastatin prevents hippocampal cell death due to quinolinic acid-induced seizures in mice by increasing Akt phosphorylation and glutamate uptake.

Statins are cholesterol-lowering agents due to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Recent studies have shown statins possess pleiotropic effects, which appear to be independent from its cholesterol-lowering action. In this study, we investigated whether atorvastatin would have protective effects against hippocampal cell death promoted by quinolinic acid (QA)-induced seizures in mice. Mice were pretreated with Atorvastatin (1 or 10 mg/kg) or vehicle (saline, 0.9%), orally, once a day for 7 days before the intracerebroventricular (i.c.v.) QA infusion (36.8 nmol/site). Atorvastatin treatment with 1 mg/kg/day did not significantly prevent QA-induced seizures (13.34%). However, administration of atorvastatin 10 mg/kg/day prevented the clonic and/or tonic seizures induced by QA in 29.41% of the mice. Additionally, administration of atorvastatin 10 mg/kg/day significantly prevented QA-induced cell death in the hippocampus. Atorvastatin treatment promoted an increased Akt phosphorylation, which was sustained after QA infusion in both convulsed and non-convulsed mice. Moreover, atorvastatin pretreatment prevented the reduction in glutamate uptake into hippocampal slices induced by QA i.c.v. infusion. These results show that atorvastatin attenuated QA-induced hippocampal cellular death involving the Akt pathway and glutamate transport modulation. Therefore, atorvastatin treatment might be a useful strategy in the prevention of brain injury caused by the exacerbation of glutamatergic toxicity in neurological diseases such as epilepsy.

Physical exercise increases mitochondrial function and reduces oxidative damage in skeletal muscle.

The present study investigated mitochondrial adaptations and oxidative damage after 4 and 8 weeks of running training in skeletal muscle of mice. Twenty-one male mice (CF1, 30-35 g) were distributed into the following groups (n = 7): untrained (UT); trained-4 weeks (T4); trained-8 weeks (T8). Forty-eight hours after the last training session the animals were killed by decapitation and quadriceps (red portion) were removed and stored at -70 degrees C. Succinate dehydrogenase (SDH), complexes I, II, II-III and IV, lipoperoxidation (TBARS), protein carbonyls (PC) and total thiol content were measured. Results show that endurance training (8-wk) increases the SDH activity and complexes (I, II, III, IV), decreases oxidative damage (TBARS, CP) and increases total thiol content in skeletal muscle when compared to untrained animals. In conclusion, eight weeks of running training are necessary for increases in mitochondrial respiratory chain enzyme activities to occur, in association with decreased oxidative damage.

Selenium compounds counteract the stimulation of ecto-nucleotidase activities in rat cultured cerebellar granule cells: putative correlation with neuroprotective effects.

Glutamate is the main excitatory neurotransmitter in brain involved in pathophysiology of several brain injuries. In this context, glutamate showed to stimulate ecto-nucleotidase activities in cerebellar granule cells increasing extracellular adenosine levels, an important neuromodulator in the CNS able to prevent cell damage. The organoselenium compounds, such as ebselen and diphenyl diselenide [(PhSe)(2)], display neuroprotective activities mediated at least in part by their antioxidant and anti-inflammatory properties. Ebselen was described to prevent glutamate-induced lipid peroxidation and cell death in cerebellar granule cells and (PhSe)(2) modify glutamatergic synapse parameters in vitro and in vivo. In the present study, we investigated the effects of ebselen or (PhSe)(2) on glutamate-induced stimulation of ecto-nucleotidase activities in rat cultured cerebellar granule cells. Glutamate increased nucleotide hydrolysis at lower concentrations (10 and 100 microM) than described in the literature and this effect was counteracted by both organoselenium compounds tested. Based on these results, we investigated the association of organoselenium effects with their antioxidant properties searching for redox site modulation by using the alkylant agent N-ethylmaleimide (NEM). Our results suggest that selenium compounds, as well as the well-known antioxidant trolox, can avoid the increase on glutamate-induced stimulation of ecto-nucleotidase activities probably due to their antioxidant properties.

Effect of N-acetylcysteine and/or deferoxamine on oxidative stress and hyperactivity in an animal model of mania.

Studies have consistently reported the participation of free radicals in Bipolar Disorder. Administration of d-amphetamine (d-AMPH) is a relevant animal model of mania and it increases oxidative stress in rat brain. Evidences indicate that the antioxidants N-acetylcysteine (NAC) and Deferoxamine (DFX) exert protective effects in the brain. The present study was designed to evaluate the effects of NAC, DFX or their combination on AMPH-induced hyperactivity. The protein oxidation levels were analyzed in prefrontal cortex and hippocampus. In the first animal model (reversal treatment), adult male Wistar rats received saline or d-AMPH for 14 days, and from the 8th to the 14th day, they were treated with saline, NAC, DFX, or NAC plus DFX. In the second animal model (prevention treatment), rats were pretreated with saline or antioxidant regime, and from the 8th to the 14th day, they also received saline or d-AMPH. In the prefrontal cortex, the protein carbonyls were not affected by the treatment with antioxidants alone but it was increased by treatment with NAC plus DFX. At the same model, NAC plus DFX reversed the protein damage in the hippocampus, but NAC alone increased this damage. In the prevention treatment, it was observed that the protein damage in the prefrontal cortex was prevented by DFX or NAC plus DFX. In the hippocampus, the pretreatment with all antioxidant regime prevented protein damage induced by d-AMPH. At both treatments (reversal or prevention) the antioxidants did not present any effect against d-AMPH-induced hyperactivity. In conclusion, NAC or DFX and the combination of NAC plus DFX reverse and protect against d-AMPH-induced oxidative protein damage. Using these protocols we could not observe affects on locomotion, however this effect varies depending on the brain region and the treatment regime.

Antioxidant enzyme activities following acute or chronic methylphenidate treatment in young rats.

Methylphenidate (MPH) is psychostimulants used to treat Attention-Deficit/Hyperactivity Disorder and can lead to a long-lasting neurochemical and behavioral adaptations in experimental animals. In the present study, the cerebral antioxidant enzymatic system, superoxide dismutase (SOD) and catalase (CAT) was evaluated at in different age following MPH (1, 2 or 10 mg/kg MPH, i.p.) treatment in young rats. In the acute treatment the SOD activity decreased in the cerebral prefrontal cortex with opposite effect in the cerebral cortex; and the CAT activity decreased in hippocampus. In the chronic treatment the SOD activity increased in the hippocampus and cerebral cortex and decreased in the striatum. The observed changes on the enzyme activities in rat brain were dependent on the structure brain region and duration of treatment with MPH. Probably, the activity of enzymes was not be enough to prevent MPH-induced oxidative damage in specific regions from brain, such as observed for us in another recent study.