Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke.

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.

Methotrexate up-regulates ecto-5'-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment.

Glioblastoma multiforme (GBM) is a deadly cancer characterized by a pro-tumoral immune response. T-regulatory (Treg) lymphocytes suppress effector immune cells through cytokine secretion and the adenosinergic system. Ecto-5'-nucleotidase/CD73 plays a crucial role in Treg-mediated immunosuppression in the GBM microenvironment (GME). Methotrexate (MTX) is an immunosuppressive drug that can increase the extracellular concentration of adenosine. In this manuscript, C6 GBM cells were treated with 1.0 µM MTX, and ecto-5'-nucleotidase/CD73 expression and extracellular AMP metabolism were analyzed in vitro. For in vivo studies, rats with implanted GBM were treated for 10 days with MTX-loaded lipid-core nanocapsules (MTX-LNCs, 1 mg/kg/day). The activity of ectonucleotidase and the expression of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 were measured. The frequencies of T lymphocytes (CD3(+)CD4(+), CD3(+)CD8(+), and CD4(+)CD25(high)CD39(+)) were quantified. In vitro, treatment with MTX increased CD73 expression and activity in C6 cells, which is in agreement with higher levels of extracellular adenosine. In vivo, MTX-LNC treatment increased CD39 expression on CD3(+)CD8(+) lymphocytes. In addition, MTX-LNC treatment up-regulated CD73 expression in tissue isolated from GBM, a finding that is in agreement with the higher activity of this enzyme. More specifically, the treatment increased CD73 expression on CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes. Treatment with MTX-LNCs decreased the frequencies of T-cytotoxic, T-helper, and Treg lymphocytes in the GME. Although more studies are necessary to better understand the complex cross-talk mediated by supra-physiological concentrations of adenosine in the GME, these studies demonstrate that MTX treatment increases CD73 enzyme expression and AMP hydrolysis, leading to an increase in adenosine production and immunosuppressive capability.

Maternal Hypermethioninemia Affects Neurons Number, Neurotrophins Levels, Energy Metabolism, and Na+,K+-ATPase Expression/Content in Brain of Rat Offspring.

In the current study, we verified the effects of maternal hypermethioninemia on the number of neurons, apoptosis, nerve growth factor, and brain-derived neurotrophic factor levels, energy metabolism parameters (succinate dehydrogenase, complex II, and cytochrome c oxidase), expression and immunocontent of Na+,K+-ATPase, edema formation, inflammatory markers (tumor necrosis factor-alpha and interleukin-6), and mitochondrial hydrogen peroxide levels in the encephalon from the offspring. Pregnant Wistar rats were divided into two groups: the first one received saline (control) and the second group received 2.68 µmol methionine/g body weight by subcutaneous injections twice a day during gestation (approximately 21 days). After parturition, pups were killed at the 21st day of life for removal of encephalon. Neuronal staining (anti-NeuN) revealed a reduction in number of neurons, which was associated to decreased nerve growth factor and brain-derived neurotrophic factor levels. Maternal hypermethioninemia also reduced succinate dehydrogenase and complex II activities and increased expression and immunocontent of Na+,K+-ATPase alpha subunits. These results indicate that maternal hypermethioninemia may be a predisposing factor for damage to the brain during the intrauterine life.

Methylphenidate Causes Behavioral Impairments and Neuron and Astrocyte Loss in the Hippocampus of Juvenile Rats.

Although the use, and misuse, of methylphenidate is increasing in childhood and adolescence, there is little information about the consequences of this psychostimulant chronic use on brain and behavior during development. The aim of the present study was to investigate hippocampus biochemical, histochemical, and behavioral effects of chronic methylphenidate treatment to juvenile rats. Wistar rats received intraperitoneal injections of methylphenidate (2.0 mg/kg) or an equivalent volume of 0.9 % saline solution (controls), once a day, from the 15th to the 45th day of age. Results showed that chronic methylphenidate administration caused loss of astrocytes and neurons in the hippocampus of juvenile rats. BDNF and pTrkB immunocontents and NGF levels were decreased, while TNF-α and IL-6 levels, Iba-1 and caspase 3 cleaved immunocontents (microglia marker and active apoptosis marker, respectively) were increased. ERK and PKCaMII signaling pathways, but not Akt and GSK-3ß, were decreased. SNAP-25 was decreased after methylphenidate treatment, while GAP-43 and synaptophysin were not altered. Both exploratory activity and object recognition memory were impaired by methylphenidate. These findings provide additional evidence that early-life exposure to methylphenidate can have complex effects, as well as provide new basis for understanding of the biochemical and behavioral consequences associated with chronic use of methylphenidate during central nervous system development.

1,25-Dihydroxyvitamin D3 prevents deleterious effects of homocysteine on mitochondrial function and redox status in heart slices.

Because homocysteine (Hcy) is a risk factor for cardiovascular disease, and vitamin D deficiency can contribute to cardiovascular pathologies. In the present study, we tested the hypothesis that Hcy could impair energy metabolism, mitochondrial function, and redox status in heart slices of Wistar rats and that 1,25-dihydroxivitamin D3 (calcitriol) treatment could prevent such effects. Heart slices were first pretreated with 3 different concentrations of calcitriol (50, 100, and 250nmol/L) for 30minutes at 37°C, after which Hcy was added to promote deleterious effects on metabolism. After 1 hour of incubation, the samples were washed, homogenized, and stored at -80°C before analysis. The results showed that Hcy caused changes in energy metabolism (respiratory chain enzymes), mitochondrial function, and cell viability. Homocysteine also induced oxidative stress, increasing lipid peroxidation, reactive oxygen species generation, and protein damage. An imbalance in antioxidant enzymes was also observed. Calcitriol (50nmol/L) reverted the effect of Hcy on the parameters tested, except for the immunocontent of catalase. Both treatments (calcitriol and Hcy) did not alter the vitamin D receptor immunocontent, which combined with the fact that our ex vivo model is acute, suggesting that the beneficial effect of calcitriol occurs directly through antioxidative mechanisms and not via gene expression. In this study, we show that Hcy impairs mitochondrial function and induces changes in the redox status in heart slices, which were reverted by calcitriol. These findings suggest that calcitriol may be a preventive/therapeutic strategy for complications caused by Hcy.

Differential inhibitory effects of methylmalonic acid on respiratory chain complex activities in rat tissues.

Methylmalonic acidemia is an inherited metabolic disorder biochemically characterized by tissue accumulation of methylmalonic acid (MMA) and clinically by progressive neurological deterioration and kidney failure, whose pathophysiology is so far poorly established. Previous studies have shown that MMA inhibits complex II of the respiratory chain in rat cerebral cortex, although no inhibition of complexes I-V was found in bovine heart. Therefore, in the present study we investigated the in vitro effect of 2.5mM MMA on the activity of complexes I-III, II, II-III and IV in striatum, hippocampus, heart, liver and kidney homogenates from young rats. We observed that MMA caused a significant inhibition of complex II activity in striatum and hippocampus (15-20%) at low concentrations of succinate in the medium, but not in the peripheral tissues. We also verified that the inhibitory property of MMA only occurred after exposing brain homogenates for at least 10 min with the acid, suggesting that this inhibition was mediated by indirect mechanisms. Simultaneous preincubation with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) and catalase (CAT) plus superoxide dismutase (SOD) did not prevent MMA-induced inhibition of complex II, suggesting that common reactive oxygen (superoxide, hydrogen peroxide and hydroxyl radical) and nitric (nitric oxide) species were not involved in this effect. In addition, complex II-III (20-35%) was also inhibited by MMA in all tissues tested, and complex I-III only in the kidney (53%) and liver (38%). In contrast, complex IV activity was not changed by MMA in all tissues studied. These results indicate that MMA differentially affects the activity of the respiratory chain pending on the tissues studied, being striatum and hippocampus more vulnerable to its effect. In case our in vitro data are confirmed in vivo in tissues from methylmalonic acidemic patients, it is feasible that that the present findings may be related to the pathophysiology of the tissue damage characteristic of these patients.

Ascorbic acid prevents water maze behavioral deficits caused by early postnatal methylmalonic acid administration in the rat.

Methylmalonic acidemia consists of a group of inherited neurometabolic disorders biochemically characterized by accumulation of methylmalonic acid (MA) and clinically by progressive neurological deterioration whose pathophysiology is not yet fully established. In the present study we investigated the effect of chronic administration (from the 5th to the 28th day of life) of methylmalonic acid (MA) on the performance of adult rats in the Morris water maze task. MA doses ranged from 0.72 to 1.67 micromol/g of body weight as a function of animal age; control rats were treated with the same volume of saline. Chronic postnatal MA treatment had no effect on body weight and in the acquisition of adult rats in the water maze task. However, administration of MA provoked long lasting reversal learning impairment in this task. Motor activity, evaluated by the swim speed in the maze, was not altered by MA administration, indicating no deficit of locomotor activity in rats injected with the metabolite. We also determined the effect of ascorbic acid administered alone or combined with MA on the same behavioral parameters in order to test whether free radicals might be responsible for the behavioral changes observed in MA-treated animals. Ascorbic acid was able to prevent the behavioral alterations provoked by MA. Moreover, the in vitro exposure of hippocampal and striatal preparations to MA revealed that the acid significantly reduced total radical-trapping antioxidant potential (TRAP) and total antioxidant reactivity (TAR) in the striatum, but not in the hippocampus. Furthermore, MA increased the thiobarbituric acid-reactive substances (TBA-RS) measurement in both structures. These data indicate that oxidative stress might be involved in the neuropathology of methylmalonic acidemia and that early MA administration induces long-lasting behavioral deficits, which are possibly caused by oxygen reactive species generation.

Evaluation of the effect of chronic administration of drugs on rat behavior in the water maze task.

Tissue accumulation of intermediates of the metabolism occurs in various inherited neurodegenerative disorders, including methylmalonic acidemia (MA). Animal cognition is usually tested by measuring learning/memory of rats in behavioral tasks. A procedure in which rats are chronically injected with the metabolites accumulating in the neurometabolic disorder methylmalonic acidemia from the 5th to the 28th day of life is described. The animals were allowed to recover for approximately 30 days, after which they were submitted to the Morris water maze task. This behavioral task consisted of two steps. The first one is called the acquisition phase, where rats were trained for 5 consecutive days performing four trials per day to find the submerged platform. On each trial, the rat was placed in the water in one of four start locations (N, S, W and E). The animal was then allowed to search for the platform for 60 s. Once the rat located the platform, it was permitted to remain on it for 10 s. The acquisition phase was followed by the probe trial 24 h later, in which the platform is not present. The time spent in the quadrant of the former platform position and the correct annulus crossings were obtained as a measure for spatial memory. The next step was the reversal learning (reversal phase) performed 2 weeks later. Animals were trained for 4 days (four trials per day) to find the hidden platform, which had now been moved to a position diagonally opposite (reversed) from its location in the acquisition phase. On the next day, all animals were submitted to a second probe trial, similar to the first one. We observed that rats chronically injected with methylmalonic acid (MA), although presenting no alterations in the acquisition phase, showed a long lasting reversal learning impairment. Moreover, motor activity, evaluated by the swim speed in the maze, was not altered by MA administration. These results are consistent with perseverative behavior.

Ascorbic acid prevents cognitive deficits caused by chronic administration of propionic acid to rats in the water maze.

Propionic acidemia is an inherited neurometabolic disorder characterized by progressive neurological deterioration with psychomotor delay/mental retardation, convulsions and coma, and whose pathophysiology is poorly unknown. In the present study, we investigated the effect of chronic administration (from the 5th to the 28th days of life) of propionic acid (PA), the major metabolite accumulating in tissues of patients affected by propionic acidemia, on the cognitive performance of adult rats in the Morris water maze task. PA doses ranged from 1.44 to 1.92 micromol/g body weight as a function of animal age. Control rats were treated with saline in the same volumes. Chronic postnatal days (5-28) PA treatment had no effect on body weight. However, it impaired spatial performance in the water maze. We also determined the effect of ascorbic acid (AA) administered, alone or combined with PA, on the same behavioral parameters in order to test whether free radicals could be responsible for the behavioral alterations observed in PA-treated animals. AA was able to prevent the behavioral alterations provoked by PA, implying that oxidative stress may be involved in these effects. Furthermore, we also investigated the total radical-trapping antioxidant potential (TRAP) in the hippocampus of the animals. We observed that TRAP was significantly reduced in the brain of propionic acidemic rats and that co-administration of AA prevented this effect. The results provide evidence that early PA treatment induces long-lasting behavioral deficits, which are possibly caused by oxygen reactive species generation, and suggest that oxidative stress may be involved in the neuropathology of propionic acidemia.

Resveratrol induces pro-oxidant effects and time-dependent resistance to cytotoxicity in activated hepatic stellate cells.

Resveratrol (RSV) is known for its antioxidant properties; however, this compound has been proposed to have cytotoxic and pro-oxidant effects depending on its concentration and time of exposure. We previously reported the cell cycle arrest effect of low doses of RSV in GRX cells, an activated hepatic stellate cell model. Here, we evaluated the effects of RSV treatment (0.1-50 µM) for 24 and 120 h on GRX viability and oxidative status. Only treatment with 50 µM of RSV reduced the amount of live cells. However, even low doses of RSV induced an increased reactive species production at both treatment times. While being diminished within 24 h, RSV induced an increase in the SOD activity in 120 h. The cellular damage was substantially increased at 24 h in the 50 µM RSV-treated group, as indicated by the high lipoperoxidation, which may be related to the significant cell death and low proliferation. Paradoxically, this cellular damage and lipoperoxidation were considerably reduced in this group after 120 h of treatment while the surviving cells proliferated. In conclusion, RSV induced a dose-dependent pro-oxidant effect in GRX cells. The highest RSV dose induced oxidative-related damage, drastically reducing cell viability; but this cytotoxicity seems to be attenuated during 120 h of treatment.

The effect of unpredictable chronic mild stress on depressive-like behavior and on hippocampal A1 and striatal A2A adenosine receptors.

This study examined the effects of two chronic stress regimens upon depressive-like behavior, A(1) and A(2A) adenosine receptor binding and immunocontent. Male rats were subjected to unpredictable chronic mild stress (UCMS) or to chronic restraint stress (CRS) for 40 days. Subsequently, depressive-like behaviors (forced swimming and consumption of sucrose) were evaluated, and A(1) adenosine or A(2A) adenosine receptors were examined in the hippocampus or striatum, respectively. UCMS animals demonstrated depressive-related behaviors (decrease in sucrose consumption and increased immobility in the forced swimming test). This group also presented increased A(1) adenosine receptor binding and immunoreactivity in hippocampus, as well as increased striatal A(2A) adenosine receptor binding in the striatum, without alteration in immunoreactivity. Conversely, the chronic restraint stress group displayed only an increase in A(1) adenosine receptor binding and no alteration in the other parameters evaluated. We suggest that the alteration in adenosine receptors, particularly the upregulation of striatal A(2A) adenosine receptors following UCMS, could be associated with depressive-related behavior.

Acute renal failure potentiates brain energy dysfunction elicited by methylmalonic acid.

The influence of acute renal failure induced by gentamicin administration on the effects of MMA on mitochondrial respiratory chain complexes, citrate synthase, succinate dehydrogenase and creatine kinase activities in cerebral cortex and kidney of young rats were investigated. Animals received one intraperitoneal injection of saline or gentamicin (70 mg/kg). One hour after, the animals received three consecutive subcutaneous injections of MMA (1.67 µmol/g) or saline (11 h interval between injections) and 60 min after the last injection the animals were killed. Acute MMA administration decreased creatine kinase activity in both tissues and increased complexes I-III activity in cerebral cortex. Creatine kinase activity was also inhibited by gentamicin administration. Simultaneous administration of MMA and gentamicin increased the activities of citrate synthase in cerebral cortex and kidney and complexes II-III in cerebral cortex. The other enzyme activities in cerebral cortex and kidney of animals receiving MMA plus gentamicin did not significantly differ from those observed in animals receiving only MMA. Our present data is line with the hypothesis that MMA acts as a toxin in brain and kidney of rats and suggest that renal injury potentiates the toxicity of MMA on the Krebs cycle and respiratory chain in brain and peripheral tissues.

Effect of chronic administration of tamoxifen and/or estradiol on feeding behavior, palatable food and metabolic parameters in ovariectomized rats.

Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in the treatment of breast cancer; however many women complain of weight gain during TAM treatment. The anorectic effects of estradiol (E) and TAM are well known, although the effects of E on the consumption of palatable food are controversial and there is no information regarding the effects of TAM on palatable food consumption. The aim of this study was to investigate the effects of chronic treatment with estradiol and/or tamoxifen on feeding behavior in ovariectomized rats exposed to standard chow and palatable foods (Froot Loops® or chocolate). Additionally, parameters such as body weight, uterine weight, lipid profile and plasma glucose were also measured. Wistar rats were ovariectomized (OVX) and subsequently injected (ip.) for 40 days with: E, TAM, E+TAM or vehicle (OVX and SHAM - controls). Behavioral tests were initiated 25 days after the start of treatment. Froot Loops® consumption was evaluated in a novel environment for 3 min. Standard chow intake was evaluated for two days and chocolate intake for 7 days in the home cage in a free choice model (chocolate or standard chow). Rats injected with E, TAM and E+TAM groups showed a reduction in body weight and standard chow intake, compared with control groups. With regard to palatable food intake, the E, TAM and E+TAM groups demonstrated increased consumption of Froot Loops®, compared with the SHAM and OVX groups. In contrast, all groups increased their consumption of chocolate, compared with standard chow; however the E group consumed more chocolate than the OVX, TAM and E+TAM groups. Despite these differences in chocolate consumption, all groups showed the same caloric intake during the chocolate exposure period; however the TAM and E+TAM groups presented decreased body weight. Treatment with estradiol and tamoxifen showed a favorable lipid profile with low levels of TC, LDL, LDL/HDL ratio and lower levels of plasma glucose. The E group presented high levels of TG and HDL, when compared with the TAM and E+TAM groups. Taken together, results suggest that TAM acted in an estrogen-like manner on the majority of parameters analyzed. However, tamoxifen acts in a different manner depending on the type of palatable food and the exposure. In addition, the TAM group demonstrated weight loss, compared with other groups independently of the type of food presented (palatable food or standard chow), showing a low caloric efficiency.

Neonatal hypoxia-ischemia induces sex-related changes in rat brain mitochondria.

The effects of neonatal hypoxia-ischemia (HI) on energy metabolism in male and female rats were investigated, testing the hypothesis that HI-induced brain mitochondrial dysfunction could present in a dimorphic pattern. Impairment in electron transport chain complex activities at 2 and 18 h after HI was observed in cortex and hippocampus in rats of both sexes, with females presenting an overall activity higher than that of males. Females also showed loss of mitochondrial mass and membrane potential 18 h after HI, while males were only slightly affected. These findings suggest a dimorphism in mitochondrial dysfunction and provide information that may lead to new neuroprotection strategies.

Amyloid-ß induced toxicity involves ganglioside expression and is sensitive to GM1 neuroprotective action.

The effect of Aß25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C(14)] galactose and results showed that Aß25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aß25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 µM was able to prevent the toxicity triggered by the fibrillar Aß25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aß-induced alterations on GSK3ß dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aß-induced dephosphorylation (activation) of GSK3ß, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer's disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer's disease experimental models and suggest a protective role for GM1 in Aß-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer's treatment.

Early biochemical effects after unilateral hypoxia-ischemia in the immature rat brain.

Perinatal hypoxia-ischemia (HI) gives rise to inadequate substrate supply to the brain tissue, resulting in damage to neural cells. Previous studies at different time points of development, and with different animal species, suggest that the HI insult causes oxidative damage and changes Na+, K+-ATPase activity, which is known to be very susceptible to free radical-related lipid peroxidation. The aim of the present study was to establish the onset of the oxidative damage response in neonatal Wistar rats subjected to brain HI, evaluating parameters of oxidative stress, namely nitric oxide production, lipoperoxidation by thiobarbituric acid reactive substances (TBA-RS) production and malondialdehyde (MDA) levels, reactive species production by DCFH oxidation, antioxidant enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase as well as Na+, K+-ATPase activity in hippocampus and cerebral cortex. Rat pups were subjected to right common carotid ligation followed by exposure to a hypoxic atmosphere (8% oxygen and 92% nitrogen) for 90 min. Animals were sacrificed by decapitation 0, 1 and 2 h after HI and both hippocampus and cerebral cortex from the right hemisphere (ipsilateral to the carotid occlusion) were dissected out for further experimentation. Results show an early decrease of Na+, K+-ATPase activity (at 0 and 1 h), as well as a late increase in MDA levels (2 h) and superoxide dismutase activity (1 and 2 h after HI) in the hippocampus. There was a late increase in both MDA levels and DCFH oxidation (1 and 2 h) and an increase in superoxide dismutase activity (2 h after HI) in cortex; however Na+, K+-ATPase activity remained unchanged. We suggest that neonatal HI induces oxidative damage to both hippocampus and cortex, in addition to a decrease in Na+, K+-ATPase activity in hippocampus early after the insult. These events might contribute to the later morphological damage in the brain and indicate that it would be essential to pursue neuroprotective strategies, aimed to counteract oxidative stress, as early as possible after the HI insult.