Cristiane Matté

Inhibition of Na+,K+-ATPase Activity in Hippocampus of Rats Subjected to Acute Administration of Homocysteine Is Prevented by Vitamins E and C Treatment

In the present study we evaluated the effect of acute homocysteine (Hcy) administration on Na+,K+-ATPase activity, as well as on some parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP) and on activities of antioxidant enzymes catalase (CAT), superoxide dismutase and glutathione peroxidase in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na+,K+-ATPase and CAT activities, without affecting the activities of superoxide dismutase and glutathione peroxidase. We also verified the effect of chronic pretreatment with vitamins E and C on the reduction of TRAP, Na+,K+-ATPase and CAT activities caused by Hcy. Vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na+,K+-ATPase and CAT activities caused by Hcy. Our results indicate that oxidative stress is probably involved in the pathogenesis of homocystinuria and that reduction of Na+,K+-ATPase activity may be related to the neuronal dysfunction found in homocystinuric patients.

Reduction of Na+,K+-ATPase Activity in Hippocampus of Rats Subjected to Chemically Induced Hyperhomocysteinemia

Hyperhomocysteinemia occurs in homocystinuria, an inherited metabolic disease clinically characterized by thromboembolic episodes and a variable degree of neurological dysfunction whose pathophysiology is poorly known. In this study, we induced elevated levels of homocysteine (Hcy) in blood (500 μM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 μmol/g of body weight) twice a day at 8-hr intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Na+,K+-ATPase and Mg2+-ATPase activities were determined in the hippocampus of treated Hcy- and saline-treated rats. Chronic administration of Hcy significantly decreased (40%) Na+,K+-ATPase activity but did not alter Mg2+-ATPase activity. Considering that Na+,K+-ATPase plays a crucial role in the central nervous system, our results suggest that the brain dysfunction found in homocystinuria may be related to the reduction of brain Na+,K+-ATPase activity.

Reduction of Hippocampal Na+, K+-ATPase Activity in Rats Subjected to an Experimental Model of Depression

The effect of a model of depression using female rats on Na+, K+-ATPase activity in hippocampal synaptic plasma membranes was studied. In addition, the effect of further chronic treatment with fluoxetine on this enzyme activity was verified. Sweet food consumption was measured to evaluate the efficacy of this model in inducing a state of reduced response to rewarding stimili. After 40 days of mild stress, a reduction in sweet food ingestion was observed. Reduction of hippocampal Na+, K+-ATPase activity was also observed. Treatment with fluoxetine increased this enzyme activity and reversed the effect of stress. Chronic fluoxetine decreased the ingestion of sweet food in both groups. This result is in agreement with suggestions that reduction of Na+, K+-ATPase activity is a caracteristic of depressive disorders. Fluoxetine reversed this effect. Therefore it is possible that altered Na+, K+-ATPase activity may be involved in the pathophysiology of depression in patients.

Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid.

We have previously demonstrated that acute hyperhomocysteinemia induces oxidative stress in rat brain. In the present study, we initially investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative damage, namely total radical-trapping antioxidant potential and activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), as well as on DNA damage in parietal cortex and blood of rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of Hcy (0.3-0.6 micromol/g body weight), and/or folic acid (0.011 micromol/g body weight) from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, parietal cortex and total blood was collected. Results showed that chronic homocysteine administration increased DNA damage, evaluated by comet assay, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in parietal cortex and blood/plasma. Folic acid concurrent administration prevented homocysteine effects, possibly by its antioxidant and DNA stability maintenance properties. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in disorders that accumulate homocysteine.

Pretreatment with vitamins E and C prevent the impairment of memory caused by homocysteine administration in rats

Homocystinuria is a metabolic disorder caused by deficiency of cystathione β-synthase activity leading to tissue accumulation of homocysteine (Hcy); affected patients present neurological dysfunction. Considering that Hcy induces free radical formation and that memory is impaired by oxidative stress, in the present study we investigated the effect of an acute administration of Hcy on retrieval of step-down inhibitory avoidance in adult rats. The action of vitamins E and C on the effects produced by Hcy was also tested. Adult Wistar rats were pretreated for 1 week with daily i.p. administration of saline (control group) and vitamins E and C (vitamin E 40 mg/kg and vitamin C 100 mg/kg). Hcy (11 mmol/kg) or an equivalent volume of 0.9% saline were administered 1 h before training, 1 h before testing, or immediately after training sessions. Memory was significantly impaired in Hcy-treated group, whereas the rats chronically treated with vitamins E and C had this effect prevented. Present data strongly indicate that Hcy administration impairs memory, an effect probably mediated by oxidative stress since treatment with vitamins E and C prevented amnesia. Assuming the possibility that this might occur in the human condition, reported results may be relevant to explain, at least in part, neurologic dysfunction associated with homocystinuria.

Resveratrol prevents oxidative stress and inhibition of Na+K+-ATPase activity induced by transient global cerebral ischemia in rats

Increased oxidative stress and energy metabolism deficit have been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In this study, we investigated the oxidative mechanisms underlying the neuroprotective effects of resveratrol, a potent polyphenol antioxidant found in grapes, on structural and biochemical abnormalities in rats subjected to global cerebral ischemia. Experimental model of transient global cerebral ischemia was induced in Wistar rats by the four vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl and fluoro jade C stained indicated extensive neuronal death at 7 days after I/R. These findings were preceded by a rapid increase in the generation of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation, as well as by a decrease in Na(+)K(+)-ATPase activity and disrupted antioxidant defenses (enzymatic and non-enzymatic) in hippocampus and cortex. Administrating resveratrol 7 days prior to ischemia by intraperitoneal injections (30 mg/kg) significantly attenuated neuronal death in both studied structures, as well as decreased the generation of ROS, lipid peroxidation and NO content. Furthermore, resveratrol brought antioxidant and Na(+)K(+)-ATPase activity in cortex and hippocampus back to normal levels. These results support that resveratrol could be used as a preventive, or therapeutic, agent in global cerebral ischemia and suggest that scavenging of ROS contributes, at least in part, to resveratrol-induced neuroprotection.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats.

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical‐trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid‐reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia.

Vitamins E and C pretreatment prevents ovariectomy-induced memory deficits in water maze.

We investigated whether the pretreatment with vitamins E (alpha-tocopherol) and C (ascorbic acid) would act on ovariectomy-induced memory deficits in Morris water maze tasks. Adult female Wistar rats were divided into three groups: (1) naive (control), (2) sham (submitted to surgery without removal of ovaries) and (3) ovariectomized. Thirty days after surgery, they were trained in the Morris water maze in order to verify ovariectomy effects both on reference and working memory tasks. Results show that ovariectomized rats presented impairment in spatial navigation in the acquisition phase, as well as in the time spent in target quadrant and in the latency to cross over the location of the platform in test session, when compared to naive and sham groups (controls), in the reference memory task. Ovariectomy did not affect performance in the working memory task. Confirming our hypothesis, ovariectomized rats pretreated for 30 days with vitamins E and C had those impairments prevented. We conclude that ovariectomy significantly impairs spatial reference learning/memory and that pretreatment with vitamins E and C prevents such effect. Assuming this experimental memory impairment might mimic, at least in part, the cognitive deficit sometimes present in the human condition of lack of reproductive hormones, our findings lend support to a novel therapeutic strategy, based on vitamins E and C, to cognitive impairments in post-menopausal women.

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.

In vivo and in vitro effects of homocysteine on Na+,K+-ATPase activity in parietal, prefrontal and cingulate cortex of young rats

In the present study we determined the effect of chronic administration of homocysteine on Na+,K+‐ATPase activity in synaptic membranes from parietal, prefrontal and cingulate cortex of young rats. We also studied the in vitro effect of homocysteine on this enzyme activity and on some oxidative stress parameters, namely thiobarbituric acid‐reactive substances (TBA‐RS) and total radical‐trapping antioxidant potential (TRAP) in the same cerebral structures. For the in vivo studies, we induced elevated levels of homocysteine in blood (500 μM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3–0.6 μmol/g of body weight) twice a day at 8 h intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Rats were killed 12 h after the last injection. Chronic administration of homocysteine significantly decreased (50%) Na+,K+‐ATPase activity in parietal, increased (36%) in prefrontal and did not alter in cingulate cortex of young rats. In vitro homocysteine decreased Na+,K+‐ATPase activity and TRAP and increased TBA‐RS in all cerebral structures studied. It is proposed that the alteration of Na+,K+‐ATPase and induction of oxidative stress by homocysteine in cerebral cortex may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.