Emilene B. S. Scherer

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.

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.

Concurrent folate treatment prevents Na+,K+-ATPase activity inhibition and memory impairments caused by chronic hyperhomocysteinemia during rat development.

We investigated the hypothesis that folate administration would prevent hyperhomocysteinemia‐induced memory deficits and Na+,K+‐ATPase activity inhibition. Chronic hyperhomocysteinemia was induced from the 6th to the 28th day of life by subcutaneous injection of homocysteine (0.3–0.6 μmol/g), twice a day; control Wistar rats received the same volume of saline solution (0.9% NaCl). Half of the homocysteine‐ and saline‐treated groups also received intraperitoneal administration of folate (0.011 μmol/g) from the 6th to the 28th day of life. A group of animals was killed 12 h after the last injection, plasma and parietal cortex were collected for biochemical analysis. Another group stayed at Central Animal House until 60th day of life, when the rats were submitted to behavioral testing in water maze or were killed for evaluation of cortical Na+,K+‐ATPase activity. Results showed that hyperhomocysteinemia impaired reference memory for platform location, as assessed by fewer crossings to the platform place and increased latency for the first crossing, when compared to controls. In the working memory task homocysteine‐treated animals also needed more time to find the platform. We also observed that Na+,K+‐ATPase activity was reduced in parietal cortex of hyperhomocysteinemic rats sacrificed 12 h after the last injection of homocysteine (29‐day‐old rats). In contrast, this enzyme was not altered when the rats were sacrificed 31 days after the treatment (60‐day‐old rats). Hyperhomocysteinemic rats treated with folate had all those impairments prevented, an effect probably related to folate antioxidant properties.

Intrastriatal administration of guanidinoacetate inhibits Na+, K+-ATPase and creatine kinase activities in rat striatum

Guanidinoacetate methyltransferase deficiency (GAMT deficiency) is an inherited neurometabolic disorder clinically characterized by epilepsy and mental retardation and biochemically by accumulation of guanidinoacetate (GAA) and depletion of creatine. Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not yet established. In the present study, we investigated the effect of intrastriatal administration of GAA on Na+, K+-ATPase activity, total (tCK), cytosolic (Cy-CK), and mitochondrial (Mi-CK) creatine kinase (CK) activities in rat striatum. We verified that Na+, K+-ATPase, tCK, and Mi-CK activities were significantly inhibited by GAA, in contrast to Cy-CK which was not affected by this guanidino compound. Since these enzyme activities can be affected by reactive species, we also investigated the effect of intrastriatal administration of GAA on thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in rats. We found that this metabolite significantly increased this oxidative stress parameter. Considering the importance of Na+, K+-ATPase and CK activities for brain metabolism homeostasis, our results suggest that the inhibition of these enzymes by increased intracerebral levels of GAA may contribute to the neuropathology observed in patients with GAMT-deficiency.

Evidences that maternal swimming exercise improves antioxidant defenses and induces mitochondrial biogenesis in the brain of young Wistar rats

Physical exercise during pregnancy has been considered beneficial to mother and child. Recent studies showed that maternal swimming improves memory in the offspring, increases hippocampal neurogenesis and levels of neurotrophic factors. The objective of this work was to investigate the effect of maternal swimming during pregnancy on redox status and mitochondrial parameters in brain structures from the offspring. Adult female Wistar rats were submitted to five swimming sessions (30 min/day) prior to mating with adult male Wistar rats, and then trained during the pregnancy (five sessions of 30-min swimming/week). The litter was sacrificed when 7 days old, when cerebellum, parietal cortex, hippocampus, and striatum were dissected. We evaluated the production of reactive species and antioxidant status, measuring the activities of superoxide-dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx), as well as non-enzymatic antioxidants. We also investigated a potential mitochondrial biogenesis regarding mitochondrion mass and membrane potential, through cytometric approaches. Our results showed that maternal swimming exercise promoted an increase in reactive species levels in cerebellum, parietal cortex, and hippocampus, demonstrated by an increase in dichlorofluorescein oxidation. Mitochondrial superoxide was reduced in cerebellum and parietal cortex, while nitrite levels were increased in cerebellum, parietal cortex, hippocampus, and striatum. Antioxidant status was improved in cerebellum, parietal cortex, and hippocampus. SOD activity was increased in parietal cortex, and was not altered in the remaining brain structures. CAT and GPx activities, as well as non-enzymatic antioxidant potential, were increased in cerebellum, parietal cortex, and hippocampus of rats whose mothers were exercised. Finally, we observed an increased mitochondrial mass and membrane potential, suggesting mitochondriogenesis, in cerebellum and parietal cortex of pups subjected to maternal swimming. In conclusion, maternal swimming exercise induced neurometabolic programing in the offspring that could be of benefit to the rats against future cerebral insults.

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.

Chemically induced model of hypermethioninemia in rats

In the present study, we developed a chronic chemically induced model of hypermethioninemia in rats. We induced elevated concentrations of methionine in the blood by injecting subcutaneously methionine (1.34-2.68 micromol/g of body weight) to developing animals of various ages. Brain methionine concentrations were approximately 1.25 micromol/g wet tissue ( approximately 1.0mM). We then injected the same doses of methionine to young rats twice a day at 8h intervals from the 6(th) to the 28(th) postpartum day. Controls received saline in the same volumes. The body, brain and hippocampus of rats were weighed after treatment and showed that hypermethioninemic animals had no differences in these parameters, when compared to the control group, suggesting that methionine did not cause malnutrition in the rats. Considering that experimental animal models are useful to understand the pathophysiology of human disease, the present model of hypermethioninemia may contribute to the investigation of the mechanisms of brain damage caused by high tissue methionine levels.

Running exercise effects on spatial and avoidance tasks in ovariectomized rats

Since previous studies have shown that ovariectomy impairs memory and cognition, we investigated whether physical exercise would affect ovariectomy-induced memory deficits in inhibitory avoidance and Morris water maze tasks. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries), exercise, ovariectomy (Ovx) and Ovx plus exercise. Thirty days after ovariectomy or sham surgery, animals were submitted to 1 month of treadmill exercise training for 20 min, three times per week. Rats were than tested in inhibitory avoidance and Morris water maze tasks in order to verify ovariectomy effects on aversive and spatial memory performance. Results show that ovariectomized rats were impaired in aversive memory and spatial navigation, both in reference and working memory protocols. Confirming the working hypothesis, ovariectomized rats submitted to exercise had those impairments prevented. These findings support that physical exercise might constitute an important strategy to minimize cognitive deficits found in post-menopausal women.

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.

Homocysteine induces energy imbalance in rat skeletal muscle: is creatine a protector?

Homocystinuria is a neurometabolic disease caused by a severe deficiency of cystathionine beta‐synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction. In this study, we investigated the effect of chronic hyperhomocysteinemia on the cell viability of the mitochondrion, as well as on some parameters of energy metabolism, such as glucose oxidation and activities of pyruvate kinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, respiratory chain complexes and creatine kinase in gastrocnemius rat skeletal muscle. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injections of homocysteine (0.3–0.6 µmol/g body weight) and/or creatine (50 mg/kg body weight) from the 6th to the 28th days of age. The animals were decapitated 12 h after the last injection. Homocysteine decreased the cell viability of the mitochondrion and the activities of pyruvate kinase and creatine kinase. Succinate dehydrogenase was increased other evaluated parameters were not changed by this amino acid. Creatine, when combined with homocysteine, prevented or caused a synergistic effect on some changes provoked by this amino acid. Creatine per se or creatine plus homocysteine altered glucose oxidation. These findings provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function, more studies are needed to elucidate them. Although creatine prevents some alterations caused by homocysteine, it should be used with caution, mainly in healthy individuals because it could change the homeostasis of normal physiological functions. Copyright