Administration of Huperzia quadrifariata Extract, a Cholinesterase Inhibitory Alkaloid Mixture, has Neuroprotective Effects in a Rat Model of Cerebral Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) is an etiologic component of several neurologic pathologies associated to cognitive impairment. The mechanisms involved in HI-induced tissue damage start immediately after HI and extend for days. Acetylcholine is an important neurotransmitter in the central nervous system and exerts a protector effect on tissue damage by modulating inflammation, and cholinesterase inhibitors have shown neuroprotective properties and their action are often attributed to inhibition of the immune response. The administration of Huperzia quadrifariata alkaloid extract (HqAE), with potent and selective cholinesterase inhibitor properties, will reduce the HI induced behavioral deficits and tissue damage. A total of 84 newborn Wistar rat pups at post natal day 7 (PND7) were subjected to right carotid occlusion followed by 1 h of hypoxia (8% of O2) and i.p. injections of saline, vehicle or HqAE (10 mg/kg). Morris Water Maze and inhibitory avoidance tests were used to assess the cognitive function. Flow cytometry was performed at PND11. Histological analysis was performed at PND45. HqAE treatment was able to prevent the HI induced cognitive deficits in both tests and, at PND45, histological analysis showed that HqAE treatment reduced hippocampus tissue damage. Flow cytometry of the injured hippocampus revealed that the treatment was able to reduce cellular death and the number of infiltrating T cells. Altogether, these results show the therapeutic potential of the Huperzia quadrifariata alkaloid extract to prevent cognitive deficits and histological damage caused by neonatal hypoxia-ischemia, probably by reducing cellular death and T cell mobilization.

Metabolic and feeding behavior alterations provoked by prenatal exposure to aspartame.

The use of artificial sweeteners has increased together with the epidemic growth of obesity. In addition to their widespread use in sodas, artificial sweeteners are added to nearly 6000 other products sold in the US, including baby foods, frozen dinners and even yogurts. It has been suggested that the use of nonnutritive sweeteners can lead to body weight gain and an altered metabolic profile. However, very few studies have evaluated the effects of maternal consumption of artificial non-caloric sweeteners on body weight, feeding behavior or the metabolism of offspring in adult life. In this study, we found that animals exposed to aspartame during the prenatal period presented a higher consumption of sweet foods during adulthood and a greater susceptibility to alterations in metabolic parameters, such as increased glucose, LDL and triglycerides. These effects were observed in both males and females, although they were more pronounced in males. Despite the preliminary nature of this study, and the need for further confirmation of these effects, our data suggest that the consumption of sweeteners during gestation may have deleterious long-term effects and should be used with caution.

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.

Acute renal failure potentiates methylmalonate-induced oxidative stress in brain and kidney of rats.

Tissue methylmalonic acid (MMA) accumulation is the biochemical hallmark of methylmalonic acidemia. The disease is clinically characterized by progressive neurological deterioration and kidney failure, whose pathophysiology is still unclear. In the present work we investigated the effects of acute MMA administration on various parameters of oxidative stress in cerebral cortex and kidney of young rats, as well as the influence of acute renal failure on MMA-elicited effects on these parameters. Acute renal failure was induced by gentamicin, an aminoglycoside antibiotic whose utilization over prolonged periods causes nephrotoxicity. The administration of gentamicin alone increased carbonyl content and inhibited superoxide dismutase (SOD) activity in cerebral cortex, as well as increased thiobarbituric acid-reactive substances (TBA-RS) and sulfhydryl levels and diminished glutathione peroxidase activity in kidney. On the other hand, MMA administration increased TBA-RS levels in cerebral cortex and decreased SOD activity in kidney. Furthermore, the simultaneous administration of MMA and gentamicin to the rats provoked an augment in TBA-RS levels and superoxide generation in cerebral cortex and in TBA-RS, carbonyl and sulfhydryl levels in kidney, while diminished SOD activity in both studied tissues. Finally, nitrate/nitrite content, reduced glutathione levels, 2',7'-dihydrodichlorofluorescein oxidation and catalase activity were not affected by this animal treatment in either tissue. In conclusion, our present data are in line with the hypothesis that MMA acts as a toxin in brain and kidney of rats and suggest that renal injury potentiates the toxicity of MMA on oxidative stress parameters in brain and peripheral tissues.

Isolation stress exposure and consumption of palatable diet during the prepubertal period leads to cellular changes in the hippocampus.

Social isolation is one of the most potent stressors in the prepubertal period and may influence disease susceptibility or resilience in adulthood. The glucocorticoid response and, consequently, the adaptive response to stress involve important changes in mitochondrial functions and apoptotic signaling. Previous studies have shown that consumption of a palatable diet reduces some stress effects. Therefore, the aim of the present study was to investigate whether isolation stress in early life can lead to cellular alterations in the hippocampus. For this, we evaluated oxidative stress parameters, DNA breakage index, mitochondrial mass and potential, respiratory chain enzyme activities, apoptosis, and necrosis in the hippocampus of juvenile male rats submitted or not to isolation stress during the pre-puberty period. We also verified whether consumption of a palatable diet during this period can modify stress effects. Results show that stress led to an oxidative imbalance, DNA breaks, increased the mitochondrial potential and early apoptosis, and decreased the number of live and necrotic cells. In addition, the palatable diet increased glutathione peroxidase activity, high mitochondrial potential and complex I-III activity in the hippocampus of juvenile rats. The administration of a palatable diet during the isolation period prevented the stress effects that caused the reduction in live cells and increased apoptosis. In conclusion, the stress experienced during the pre-pubertal period induced a hippocampal oxidative imbalance, DNA damage, mitochondrial dysfunction, and increased apoptosis, while consumption of a palatable diet attenuated some of these effects of exposure, such as the reduction in live cells and increased apoptosis, besides favoring an increase in antioxidant enzymes activities.

Isolation stress during the prepubertal period in rats induces long-lasting neurochemical changes in the prefrontal cortex.

Social isolation during postnatal development leads to behavioral and neurochemical changes, and a particular susceptibility of the prefrontal cortex to interventions during this period has been suggested. In addition, some studies showed that consumption of a palatable diet reduces some of the stress effects. Therefore, our aim is to investigate the effect of isolation stress in early life on some parameters of oxidative stress and energy metabolism (Na(+),K(+)-ATPase activity, respiratory chain enzymes activities and mitochondrial mass and potential) in prefrontal cortex of juvenile and adult male rats. We also verified if the consumption of a palatable diet during the prepubertal period would reduce stress effects. The results showed that, in juvenile animals, isolation stress increased superoxide dismutase and Complex IV activities and these effects were still observed in the adulthood. An interaction between stress and diet was observed in catalase activity in juveniles, while only the stress effect was detected in adults, reducing catalase activity. Access to a palatable diet increased Na(+),K(+)-ATPase activity in juveniles, an effect that was reversed after removing this diet. On the other hand, isolation stress induced a decreased activity of this enzyme in adulthood. No effects were observed on glutathione peroxidase, total thiols and free radicals production, as well as on mitochondrial mass and potential. In conclusion, isolation stress in the prepubertal period leads to long-lasting changes on antioxidant enzymes and energetic metabolism in the prefrontal cortex of male rats, and a palatable diet was not able to reverse these stress-induced effects.

GD1a modulates GM-CSF-induced cell proliferation.

Gangliosides have been extensively described to be involved in the proliferation and differentiation of various cell types, such including hematopoietic cells. Our previous studies on murine models of stroma-mediated myelopoiesis have shown that gangliosides are required for optimal capacity of stromal cells to support proliferation of myeloid precursor cells, being shed to the supernatant and selectively incorporated into myeloid cell membranes. Here we describe the effect of gangliosides on the specific granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced proliferation. For that, we used the monocytic FDC-P1 cell line, which is dependent upon GM-CSF for survival and proliferation. Cells were cultured in the presence of GM-CSF and exogenous gangliosides (GM3, GD1a or GM1) or in the absence of endogenous ganglioside synthesis by the use of a ceramide-synthase inhibitor, D-PDMP. We observed that exogenous addition of GD1a enhanced the GM-CSF-induced proliferation of the FDC-P1 cells. Also, we detected an increase in the expression of the α isoform of the GM-CSF receptor (GMRα) as well as of the transcription factor C/EBPα. On the contrary, inhibition of glucosylceramide synthesis was accompanied by a decrease in cell proliferation, which was restored upon the addition of exogenous GD1a. We also show a co-localization of GD1a and GMR by immunocytochemistry. Taken together, our results suggest for the first time that ganglioside GD1a play a role on the modulation of GM-CSF-mediated proliferative response, which might be of great interest not only in hematopoiesis, but also in other immunological processes, Alzheimer disease, alveolar proteinosis and wherever GM-CSF exerts its effects.

The effects of hypercaloric diets on glucose homeostasis in the rat: influence of saturated and monounsaturated dietary lipids.

Consumption of energy-dense/high-fat diets is strongly and positively associated with overweight and obesity, which are associated with increase in the prevalence of certain chronic diseases. We evaluated the effect of hypercaloric/fat or normocaloric diets on some biochemical parameters in rats. Seventy-two rats were divided into four groups that were fed for 16 weeks with diets: normocaloric [9.12% soy oil, normocaloric soy oil (NSO)], hypercaloric olive oil [43.8% olive oil, hypercaloric olive oil (HOO)], hypercaloric saturated fat [43.8% saturated fat, hypercaloric saturated fat (HSF)] and normocaloric saturated fat [43.8% saturated fat, normocaloric saturated fat (NSF)]. HSF rats consumed more calories daily than the others and gained more retroperitoneal fat, although HSF and HOO rats had higher body weight. In liver, glycogen synthesis and concentration were higher in rats HSF and NSF. In plasma, total cholesterol (TC) levels were higher in HSF rats than in the others, and triacylglycerol (TAG) levels were lower in HOO and higher in HSF rats in relation to the others. In liver, TC and TAG were elevated in HSF, NSF and HOO rats. Paraoxonase 1 activity, which is related to high-density lipoprotein cholesterol and has anti-atherogenic role was lower in rats HSF. In HOO rats, glucose tolerance test was altered, but insulin tolerance test was normal. These results suggest that consumption of energy-dense/high-fat diets, both saturated or monounsaturated, causes damaging effects. However, more studies are necessary to understand the mechanisms by which these diets cause the metabolic alterations observed.

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid.

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.

Ganglioside alterations in the central nervous system of rats chronically injected with methylmalonic and propionic acids.

Neurological dysfunction and structural cerebral abnormalities are commonly found in patients with methylmalonic and propionic acidemia. However, the mechanisms underlying the neuropathology of these disorders are poorly understood. We have previously demonstrated that methylmalonic and propionic acids induce a significant reduction of ganglioside N-acetylneuraminic acid in the brain of rats subjected to chronic administration of these metabolites. In the present study, we investigated the in vivo effects of chronic administration of methylmalonic (MMA) and propionic (PA) acids (from the 6th to the 28th day of life) on the distribution and composition of gangliosides in the cerebellum and cerebral cortex of rats. Control rats were treated with the same volumes of saline. It was first verified that MMA and PA treatment did not modify body, cerebellum, or cortical weight, nor the ganglioside concentration in the cerebral cortex of the animals. In contrast, a significant reduction in total ganglioside content in the cerebellum of approximately 20-30% and 50% of control levels occurred in rats injected with MMA and PA, respectively. Moreover, chronic MMA and PA administration did not interfere with the ganglioside pattern in the cerebral cortex, whereas the distribution of individual gangliosides was altered in the cerebellum of MMA- and PA-treated animals. Rats injected with MMA demonstrated a marked decrease in GM1 and GD3, whereas chronic PA treatment provoked a significant reduction of all ganglioside species, with the exception of an increase in GM2. Since gangliosides are closely related to the dendritic surface and other neural membranes, indirectly reflecting synaptogenesis, these ganglioside abnormalities may be associated with the brain damage found in methylmalonic and propionic acidemias.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats.

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 umol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 umol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 æmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 æmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children