Fernanda R. Machado

Homocysteine alters glutamate uptake and Na+,K+-ATPase activity and oxidative status in rats hippocampus: protection by vitamin C

In the present study we investigate the effect of homocysteine on glutamate uptake, Na+,K+-ATPase, enzymatic antioxidant defenses, as well as reactive species levels in hippocampus of rats. The influence of vitamin C, a classic antioxidant, on the effects elicited by homocysteine was also tested. Results showed that chronic hyperhomocysteinemia decreased glutamate uptake and the activities of Na+,K+-ATPase, catalase and superoxide dismutase in hippocampus of rats. Reactive species levels were increased by chronic homocysteine administration. Concomitant administration of vitamin C significantly prevented these alterations caused by homocysteine. According to our results, it seems possible to suggest that the reduction in glutamate uptake and Na+,K+-ATPase activity may be mediated by oxidative stress, since vitamin C prevented these effects. We suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diet in homocystinuria.

Methylphenidate induces lipid and protein damage in prefrontal cortex, but not in cerebellum, striatum and hippocampus of juvenile rats

The use of psychostimulant methylphenidate has increased in recent years for the treatment of attention-deficit hyperactivity disorder in children and adolescents. However, the behavioral and neurochemical changes promoted by its use are not yet fully understood, particularly when used for a prolonged period during stages of brain development. Thus, the aim of this study was to determine some parameters of oxidative stress in encephalic structures of juvenile rats subjected to chronic methylphenidate treatment. Wistar rats received intraperitoneal injections of methylphenidate (2.0 mg/kg) once a day, from the 15th to the 45th day of age or an equivalent volume of 0.9% saline solution (controls). Two hours after the last injection, animals were euthanized and the encephalic structures obtained for determination of oxidative stress parameters. Results showed that methylphenidate administration increased the activities of superoxide dismutase and catalase, but did not alter the levels of reactive species, thiobarbituric acid reactive substances levels and sulfhydryl group in cerebellum of rats. In striatum and hippocampus, the methylphenidate-treated rats presented a decrease in the levels of reactive species and thiobarbituric acid reactive substances, but did not present changes in the sulfhydryl groups levels. In prefrontal cortex, methylphenidate promoted an increase in reactive species formation, SOD/CAT ratio, and increased the lipid peroxidation and protein damage. These findings suggest that the encephalic structures respond differently to methylphenidate treatment, at least, when administered chronically to young rats. Notably, the prefrontal cortex of juvenile rats showed greater sensitivity to oxidative effects promoted by methylphenidate in relation to other encephalic structures analyzed.

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.

Experimental hyperprolinemia induces mild oxidative stress, metabolic changes, and tissue adaptation in rat liver

The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid‐reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation. J. Cell. Biochem. 113: 174–183, 2012.

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.

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.

Hyperhomocysteinemia decreases NA+,K+- atpase activity and glutamate uptake in hippocampus of rats: Prevention of vitamin C

CB1 cannabinoid receptors exert a crucial neuromodulatory role upon engagement by the retrograde agonists anandamide and 2arachidonoylglycerol in different brain areas. In addition, owing to their ability to activate pro-survival signaling mechanisms, CB1 receptors are protective against neuronal degeneration and control neural cell generation and survival. Recent studies have unraveled a previously unknown role of CB1 receptor signalling during cortical development. CB1 receptors regulate pyramidal neuronal specification, together with a role in inhibitory GABAergic interneuron migration and morphogenesis. Thus, CB1 deficient mice at early postnatal stages show aberrant corticofugal connectivity. In the present study we aim to investigate the signaling mechanism by which CB1 receptors participate in pyramidal cell specification and to analyze in detail the different projecting neuronal subtypes regulated by endocannabinoid signaling. Immunofluorescence studies showed reduced cell number of apical and basal progenitors resident in the ventricular zone and subventricular zone of CB1-deficient mouse embryos as compared to wild-type littermates. In agreement, CB1 receptor knockout or electroporated siRNA mediated-CB1 knockdown in wild-type mice, decreased the expression of the pyramidal progenitor markers paired box gene 6 (pax6) and T-box-brain 2 (tbr2, eomes). At more advanced stages and using conditional CB1-deficient mice in the pyramidal lineage (using Nex/Math2-Cre recombinase expression) we analyzed the development of deep and upper cortical neurons with selective layer markers. CB1 receptor knock-out mice show, among others, altered expression of the Tbr2, Tbr1, CTIP2 and SATB2 transcription regulators, and this results in deregulated generation of deep and upper layer cortical neurons. A predominant role of CB1 receptor deficiency in the generation or commitment of layer V neurons is observed, indicating the involvement of CB1 receptors in corticofugal projecting neuronal generation. In summary, our results identify CB1 receptor signalling as critical regulator of the molecular transcription regulatory network involved in the specification of cortical projecting neuron subpopulations.