Maira J. da Cunha

Methylphenidate affects memory, brain-derived neurotrophic factor immunocontent and brain acetylcholinesterase activity in the rat

Methylphenidate, a psychostimulant that affects both dopaminergic and noradrenergic systems, is one of the most frequently prescribed treatments for attention-deficit hyperactivity disorder. The present study investigated the effects of chronic administration of methylphenidate to juvenile rats on spatial memory, brain-derived neurotrophic factor immunocontent and acetylcholinesterase activity in hippocampus and prefrontal cortex. Rats received intraperitoneal injections of methylphenidate (2.0mg/kg) once a day, from the 15th to the 45th day of age or an equivalent volume of 0.9% saline solution (controls). Twenty-four hours after the last injection, animals were subjected to testing in the Morris water maze. After that, animals were sacrificed and hippocampus and prefrontal cortex were dissected out for determination of brain-derived neurotrophic factor immunocontent and acetylcholinesterase activity. Chronic administration of methylphenidate provoked cognitive impairments on spatial reference and working memory tasks. A reduction on brain-derived neurotrophic factor immunocontent and increased acetylcholinesterase activity in prefrontal cortex, but not in hippocampus, of rats treated with methylphenidate were also observed. These results suggest that the deficit in spatial memory may be associated to decreased brain-derived neurotrophic factor immunocontent and increased acetylcholinesterase in prefrontal cortex of juvenile rats subjected to methylphenidate administration.

Development of an animal model for chronic mild hyperhomocysteinemia and its response to oxidative damage.

The purpose of this study was to develop a chronic chemically induced model of mild hyperhomocysteinemia in adult rats. We produced levels of Hcy in the blood (30 μM), comparable to those considered a risk factor for the development of neurological and cardiovascular diseases, by injecting homocysteine subcutaneously (0.03 μmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Using this model, we evaluated the effect of chronic administration of homocysteine on redox status in the blood and cerebral cortex of adult rats. Reactive oxygen species and thiobarbituric acid reactive substances were significantly increased in the plasma and cerebral cortex, while nitrite levels were reduced in the cerebral cortex, but not in the plasma, of rats subjected to chronic mild hyperhomocysteinemia. Homocysteine was also seen to disrupt enzymatic and non‐enzymatic antioxidant defenses in the blood and cerebral cortex of rats. Since experimental animal models are useful for understanding the pathophysiology of human diseases, the present model of mild hyperhomocysteinemia may be useful for the investigation of additional mechanisms involved in tissue alterations caused by homocysteine.

Role of antioxidants on Na(+),K (+)-ATPase activity and gene expression in cerebral cortex of hyperprolinemic rats.

Considering that Na+,K+-ATPase is an embedded-membrane enzyme and that experimental chronic hyperprolinemia decreases the activity of this enzyme in brain synaptic plasma membranes, the present study investigated the effect of chronic proline administration on thiobarbituric acid-reactive substances, as well as the influence of antioxidant vitamins E plus C on the effects mediated by proline on Na+,K+-ATPase activity in cerebral cortex of rats. The expression of Na+,K+-ATPase catalytic subunits was also evaluated. Results showed that proline increased thiobarbituric acid-reactive substances, suggesting an increase of lipid peroxidation. Furthermore, concomitant administration of vitamins E plus C significantly prevented the increase of lipid peroxidation, as well as the inhibition of Na+,K+-ATPase activity caused by proline. We did not observe any change in levels of Na+,K+-ATPase mRNA transcripts after chronic exposure to proline and vitamins E plus C. These findings provide insights into the mechanisms through which proline exerts its effects on brain function and suggest that treatment with antioxidants may be beneficial to treat neurological dysfunctions present in hyperprolinemic patients.

Physical exercise reverses glutamate uptake and oxidative stress effects of chronic homocysteine administration in the rat

The influence of physical exercise on the effects elicited by homocysteine on glutamate uptake and some parameters of oxidative stress, namely thiobarbituric acid‐reactive substances, 2′,7′‐dichlorofluorescein (H2DCF) oxidation, as well as enzymatic antioxidant activities, superoxide dismutase, catalase and glutathione peroxidase in rat cerebral cortex were investigated. Wistar rats received subcutaneous administration of homocysteine or saline (control) from the 6th to 29th day of life. The physical exercise was performed from the 30th to 60th day of life; 12 h after the last exercise session animals were sacrificed and the cerebral cortex was dissected out. It is shown that homocysteine reduces glutamate uptake increases thiobarbituric acid‐reactive substances and disrupts enzymatic antioxidant defenses in cerebral cortex. Physical activity reversed the homocysteine effects on glutamate uptake and on antioxidant enzymes activities; although the increase in thiobarbituric acid‐reactive substances was only partially reversed by exercise. These findings allow us to suggest that physical exercise may have a protective role against homocysteine‐induced oxidative imbalance and brain damage to the glutamatergic system.

Acute and chronic hypermethioninemia alter Na+,K+-ATPase activity in rat hippocampus: prevention by antioxidants

In the current study we initially investigated the influence of antioxidants (vitamins E plus C) on the effect mediated by acute and chronic administration of methionine (Met) on Na+,K+‐ATPase activity in rat hippocampus. We also verified whether the alterations on the enzyme after administration of Met and/or antioxidants were associated with changes in relative expression of Na+,K+‐ATPase catalytic subunits (isoforms α1, α2 and α3). For acute treatment, young rats received a single subcutaneous injection of Met or saline (control) and were sacrificed 12 h later. In another set of experiments, rats were pretreated for 1 week with daily intraperitoneal administration of vitamins E (40 mg/kg) and C (100 mg/kg) or saline. After that, rats received a single injection of Met or saline and were killed 12 h later. For chronic treatment, Met was administered to rats from the 6th to the 28th day of life; controls and treated rats were sacrificed 12 h after the last injection. In parallel to chronic treatment, rats received a daily intraperitoneal injection of vitamins E and C from the 6th to the 28th day of life and were killed 12 h after the last injection. Results showed that administration of antioxidants partially prevented the inhibition of enzyme activity caused by acute and chronic hypermethioninemia. Besides, we demonstrated that transcription of catalytic subunits of Na+,K+‐ATPase was not altered by chronic and acute exposure to Met and/or vitamins E plus C. These data strongly suggest the oxidative damage as one possible mechanism involved in the reduction of Na+,K+‐ATPase activity caused by hypermethioninemia and if confirmed in human beings, we might propose the use of antioxidants as an adjuvant therapy in hypermethioninemic patients.

Coumestrol treatment prevents Na+, K+ -ATPase inhibition and affords histological neuroprotection to male rats receiving cerebral global ischemia.

Abstract Objective: In this study, we investigated the possible mechanisms underlying the neuroprotective effects of coumestrol, a potent isoflavonoid with antioxidant activities and binding affinities for both estrogen receptors (ER) ER-alpha and ER-beta that are comparable to those of 17beta-estradiol, in a model of global ischemia in male subjects. Methods: Wistar rats underwent global ischemia (10 minutes) or sham surgery and received a single intracerebroventricular (icv) infusion of 20 μg of coumestrol or vehicle 1 hour before ischemia or 0, 3, 6, or 24 hours after reperfusion. Results: The data analysis revealed an extensive neuronal death in the CA1 hippocampal subfield at 7 days, and a significant decrease in the Na+, K+-ATPase activity at 1 and 24 hours after ischemia, and both injuries were attenuated by coumestrol administration. Conclusions: Coumestrol treatment was effective in preventing neuronal loss in all times of administration as well as able to rescue the Na+, K+-ATPase activity, suggesting its potential benefits for either prevention or therapeutics use against cerebral ischemia in males.

Cytoskeleton of cortical astrocytes as a target to proline through oxidative stress mechanisms

Hyperprolinemia is an inherited disorder of proline (Pro) metabolism and patients affected by this disease may present neurological manifestations. However, the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering the pivotal role of cytoskeletal remodeling in several neurodegenerative pathologies and the potential links between cytoskeleton, reactive oxygen species production and cell death, the aim of the present work was to study the effects of Pro on astrocyte and neuron cytoskeletal remodeling and the possible oxidative stress involvement. Pro induced a shift of actin cytoskeleton in stress fibers together with increased RhoA immunocontent and ERK1/2 phosphorylation/activation in cortical astrocytes. Unlike astrocytes, results evidenced little susceptibility of neuron cytoskeleton remodeling, since Pro-treated neurons presented unaltered neuritogenesis. We observed increased hydrogen peroxide production characterizing oxidative stress together with decreased superoxide dismutase (SOD) and catalase (CAT) activities in cortical astrocytes after Pro treatment, while glutathione peroxidase (GSHPx) activity remained unaltered. However, coincubation with Pro and Trolox/melatonin prevented decreased SOD and CAT activities in Pro-treated astrocytes. Accordingly, these antioxidants were able to prevent the remodeling of the actin cytoskeleton, RhoA increased levels and ERK1/2 phosphorylation in response to high Pro exposure. Taken together, these findings indicated that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Pro and such effects could be mediated, at least in part, by redox imbalance, RhoA and ERK1/2 signaling pathways. The vulnerability of astrocyte cytoskeleton may have important implications for understanding the effects of Pro in the neurotoxicity linked to inborn errors of Pro metabolism.

Evidence that Hyperprolinemia Alters Glutamatergic Homeostasis in Rat Brain: Neuroprotector Effect of Guanosine

This study investigated the effects of acute and chronic hyperprolinemia on glutamate uptake, as well as some mechanisms underlying the proline effects on glutamatergic system in rat cerebral cortex. The protective role of guanosine on effects mediated by proline was also evaluated. Results showed that acute and chronic hyperprolinemia reduced glutamate uptake, Na+, K+-ATPase activity, ATP levels and increased lipoperoxidation. GLAST and GLT-1 immunocontent were increased in acute, but not in chronic hyperprolinemic rats. Our data suggest that the effects of proline on glutamate uptake may be mediated by lipid peroxidation and disruption of Na+, K+-ATPase activity, but not by decreasing in glutamate transporters. This probably induces excitotoxicity and subsequent energy deficit. Guanosine was effective to prevent most of the effects promoted by proline, reinforcing its modulator role in counteracting the glutamate toxicity. However, further studies are needed to assess the modulatory effects of guanosine on experimental hyperprolinemia.

Physical Exercise Reverses Cognitive Impairment in Rats Subjected to Experimental Hyperprolinemia

This study investigated whether physical exercise would reverse proline-induced performance deficits in water maze tasks, as well as its effects on brain-derived neurotrophic factor (BDNF) immunocontent and brain acetylcholinesterase (AChE) activity in Wistar rats. Proline administration followed partial time (6th–29th day of life) or full time (6th–60th day of life) protocols. Treadmill exercise was performed from 30th to 60th day of life, when behavioral testing was started. After that, animals were sacrificed for BDNF and AChE determination. Results show that proline impairs cognitive performance, decreases BDNF in cerebral cortex and hippocampus and increases AChE activity in hippocampus. All reported effects were prevented by exercise. These results suggest that cognitive, spatial learning/memory, deficits caused by hyperprolinemia may be associated, at least in part, to the decrease in BDNF levels and to the increase in AChE activity, as well as support the role of physical exercise as a potential neuroprotective strategy.

The effect of exercise on the oxidative stress induced by experimental lung injury

AIM The effects of physical exercise on oxidative stress parameters and immunocontent of NF-кβ/p65 in lung of rats submitted to lung injury, as well as its possible protective effect on the changes in the alveolar-capillary barrier (total cell count, lactate dehydrogenase and total protein) in the bronchoalveolar lavage fluid (BALF) and the inflammatory infiltration in the pulmonary parenchyma were evaluated. MAIN METHODS Wistar rats were submitted to two months of physical exercise and after this period, lung injury was induced by intratracheal instillation of lipopolysaccharide (dose of 100 μg/100 g body weight). Twelve hours after injury, the animals were sacrificed and lung and BALF were collected. KEY FINDINGS Results showed an increase in reactive species production, lipid peroxidation, oxidative damage to protein, as well as in nitrite levels and NF-кβ/p65 immunocontent in lung of rats submitted to lung injury. Physical exercise was able to totally prevent the increase in reactive species, nitrite levels and NF-кβ/p65 immunocontent, but partially prevented the damage to protein. Superoxide dismutase and catalase were not changed in lung injury group, but the activities of these enzymes were increased in lung injury plus exercise group. Non-enzymatic antioxidant capacity, glutathione content and glutathione peroxidase were decreased and exercise totally prevented such effects. Rats subjected to lung injury presented an increase in total cell, lactate dehydrogenase and total protein; exercise partially prevented the increase in lactate dehydrogenase. SIGNIFICANCE These findings suggest that physical exercise may prevent, at least partially, the oxidative damage caused by experimental lung injury, suggesting that exercise may have an important role as protector in this condition.