Contextual fear conditioning in maternal separated rats: the amygdala as a site for alterations.

The first 2 weeks of life are a critical period for neural development in rats. Repeated long-term separation from the dam is considered to be one of the most potent stressors to which rat pups can be exposed, and permanently modifies neurobiological and behavioral parameters. Prolonged periods of maternal separation (MS) usually increase stress reactivity during adulthood, and enhance anxiety-like behavior. The aim of this study was to verify the effects of maternal separation during the neonatal period on memory as well as on biochemical parameters (Na(+), K(+)-ATPase and antioxidant enzymes activities) in the amygdala of adult rats. Females and male Wistar rats were subjected to repeated maternal separation (incubator at 32 °C, 3 h/day) during postnatal days 1-10. At 60 days of age, the subjects were exposed to a Contextual fear conditioning task. One week after the behavioral task, animals were sacrificed and the amygdala was dissected for evaluation of Na(+), K(+)-ATPase and antioxidant enzymes activities. Student-t test showed significant MS effect, causing an increase of freezing time in the three exposures to the aversive context in both sexes. Considering biochemical parameters Student-t test showed significant MS effect causing an increase of Na(+), K(+)-ATPase activity in both sexes. On the other hand, no differences were found among the groups on the antioxidant enzymes activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] in male rats, but in females, we found a significant MS effect, causing an increase of CAT activity and no differences were found among the groups on SOD and GPx activities. Our results suggest a role of early rearing environment in programming fear learning and memory in adulthood. An early stress experience such as maternal separation may increase activity in the amygdala (as pointed by the increased activity of Na(+), K(+)-ATPase), affecting behaviors related to fear in adulthood, and this effect could be task-specific.

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.

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.

Acute hyperhomocysteinemia alters the coagulation system and oxidative status in the blood of rats.

In the present study, we investigated the effect of the acute administration of homocysteine (Hcy) on parameters of the coagulation system, as well as fibrinogen and nitrite levels in the blood of rats. In addition, we evaluated the effect of acute hyperhomocysteinemia on thiobarbituric acid-reactive substances in plasma and on antioxidant enzymes activities (superoxide dismutase, catalase, and gluthatione peroxidase) in the erythrocytes of rats. Wistar rats, aged 29 days, received a single subcutaneous dorsal injection of saline (control) or Hcy (0.6 µmol/g body weight). Fifteen minutes, 1 h, 6 h or 12 h after the injection, the rats were euthanized and the blood, plasma, and erythrocytes were collected. Results showed that Hcy significantly increased platelet count in the blood and plasma fibrinogen levels of rats at 15 min and 1 h, but not at 6 h and 12 h, when compared with the control group. Prothrombin time, activated partial thromboplastin time, and nitrite levels significantly decreased in plasma at 15 min and 1 h, but not at 6 h and 12 h after Hcy administration. In addition, hyperhomocysteinemia increased thiobarbituric acid-reactive, an index of lipid peroxidation, in plasma at 15 min and 1 h; decreased the superoxide dismutase and gluthatione peroxidase activity, and increased the catalase activity at 15 min in erythrocytes of rats, suggesting that acute Hcy administration may alter the oxidative status in the blood of rats. Our findings suggest that hypercoagulability and oxidative stress can occur after acute hyperhomocysteinemia, possibly in association, at least in part, with the vascular dysfunction and thromboembolic complications observed in homocystinuric patients.

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.

Antioxidants prevent memory deficits provoked by chronic variable stress in rats.

Learning and memory deficits occur in depression and other stress related disorders. Although the pathogenesis of cognitive impairment after stress has not been fully elucidated, factors such as oxidative stress and neurotrophins are thought to play possible roles. Here we investigated the effect of treatment with vitamin E (40 mg/kg) and vitamin C (100 mg/kg) on the effects elicited by chronic variable stress on rat performance in Morris water maze. Brain-derived neurotrophic factor (BDNF) immunocontent was also evaluated in hippocampus of rats. Sixty-day old Wistar rats were submitted to different stressors for 40 days (stressed group). Half of stressed group received administration of vitamins once a day, during the period of stress. Chronically stressed rats presented a marked decrease in reference memory in the water maze task as well as a reduced efficiency to find the platform in the working memory task. Rats treated with vitamins E and C had part of the above effects prevented, suggesting the participation of oxidative stress in such effects. The BDNF levels were not altered in hippocampus of stressed group when compared to controls. Our findings lend support to a novel therapeutic strategy, associated with these vitamins, to the cognitive dysfunction observed in depression and other stress related diseases.

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.

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.

Experimental Lung Injury Promotes Changes in Oxidative/Nitrative Status and Inflammatory Markers in Cerebral Cortex of Rats.

In the present study, we investigate the effect of lung injury on parameters of oxidative/nitrative stress [reactive oxygen species production, nitrite levels, thiobarbituric acid-reactive substances (TBARS), carbonyl content, sulfhydryl content, activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), total radical-trapping antioxidant potential, glutathione content, and glucose-6-phosphate dehydrogenase], as well as on inflammation mediators [immunocontent of nuclear factor-kappaB (NF-κB) total (p65), NF-κB phosphorylated (pp65) subunit (cytosolic and nuclear), TNF-α, IL-1ß, IL-6, and IL-10] in the cerebral cortex. Cytokine levels in serum were also evaluated. Adult Wistar rats were submitted to lung injury induced by intratracheal instillation of lipopolysaccharide in a dose of 100 µg/100 g body weight. Sham group (control) received isotonic saline instillation. Twelve hours after the injury, rats were decapitated and blood samples were collected and the cerebral cortex dissected out. Results showed an increase in reactive oxygen species production, TBARS, and nitrite and carbonyl levels in the cerebral cortex of rats submitted to lung injury. Antioxidant enzymatic defenses were altered, superoxide dismutase and glutathione peroxidase activities decreased, and catalase activity increased. Non-enzymatic antioxidant capacity, glutathione content, and glucose-6-phosphate dehydrogenase were decreased. Inflammatory parameters were also altered in the cerebral cortex of rats subjected to lung injury; it was observed an increase in the immunocontent of NF-κB/p65 (nuclear fraction) and NF-κB/pp65 (cytosolic and nuclear faction), as well as an increase in TNF-α, IL-1ß, IL-6, and IL-10 levels. The levels of IL-10 also increased in the serum. Our findings show that the lung injury alters oxidative/nitrative status and induces inflammation in the cerebral cortex of rats, which might be associated with cognitive impairments present in patients with lung injury.

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

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.

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 (H(2)DCF) 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.