Age-dependent effects of resveratrol in hypothalamic astrocyte cultures.

OBJECTIVES: The hypothalamus plays critical roles in maintaining brain homeostasis and increasing evidence has highlighted astrocytes orchestrating several of hypothalamic functions. However, it remains unclear how hypothalamic astrocytes participate in neurochemical mechanisms associated with aging process, as well as whether these cells can be a target for antiaging strategies. In this sense, the aim of this study is to evaluate the age-dependent effects of resveratrol, a well-characterized neuroprotective compound, in primary astrocyte cultures derived from the hypothalamus of newborn, adult, and aged rats. METHODS: Male Wistar rats (2, 90, 180, and 365 days old) were used in this study. Cultured astrocytes from different ages were treated with 10 and 100 µM resveratrol and cellular viability, metabolic activity, astrocyte morphology, release of glial cell line-derived neurotrophic factor (GDNF), transforming growth factor ß (TGF-ß), tumor necrosis factor α (TNF-α), interleukins (IL-1ß, IL-6, and IL-10), as well as the protein levels of Nrf2 and HO-1 were evaluated. RESULTS: In vitro astrocytes derived from neonatal, adults, and aged animals changed metabolic activity and the release of trophic factors (GDNF and TGF-ß), as well as the inflammatory mediators (TNF-α, IL-1ß, IL-6, and IL-10). Resveratrol prevented these alterations. In addition, resveratrol changed the immunocontent of Nrf2 and HO-1. The results indicated that the effects of resveratrol seem to have a dose- and age-associated glioprotective role. CONCLUSION: These findings demonstrate for the first time that resveratrol prevents the age-dependent underlying functional reprogramming of in vitro hypothalamic astrocytes, reinforcing its antiaging activity, and consequently, its glioprotective role.

Effects of long-term resveratrol treatment in hypothalamic astrocyte cultures from aged rats.

Aging is intrinsically related to metabolic changes and characterized by the accumulation of oxidative and inflammatory damage, as well as alterations in gene expression and activity of several signaling pathways, which in turn impact on homeostatic responses of the body. Hypothalamus is a brain region most related to these responses, and increasing evidence has highlighted a critical role of astrocytes in hypothalamic homeostatic functions, particularly during aging process. The purpose of this study was to investigate the in vitro effects of a chronic treatment with resveratrol (1 µM during 15 days, which was replaced once every 3 days), a recognized anti-inflammatory and antioxidant molecule, in primary hypothalamic astrocyte cultures obtained from aged rats (24 months old). We observed that aging process changes metabolic, oxidative, inflammatory, and senescence parameters, as well as glial markers, while long-term resveratrol treatment prevented these effects. In addition, resveratrol upregulated key signaling pathways associated with cellular homeostasis, including adenosine receptors, nuclear factor erythroid-derived 2-like 2 (Nrf2), heme oxygenase 1 (HO-1), sirtuin 1 (SIRT1), proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and phosphoinositide 3-kinase (PI3K). Our data corroborate the glioprotective effect of resveratrol in aged hypothalamic astrocytes, reinforcing the beneficial role of resveratrol in the aging process.

Sintocalmy, a Passiflora incarnata Based Herbal, Attenuates Morphine Withdrawal in Mice.

Chronic opioid use changes brain chemistry in areas related to reward processes, memory, decision-making, and addiction. Both neurons and astrocytes are affected, ultimately leading to dependence. Passiflora incarnata L. (Passifloraceae) is the basis of frequently used herbals to manage anxiety and insomnia, with proven central nervous system depressant effects. Anti-addiction properties of P. incarnata have been reported. The aim of this study was to investigate the effect of a commercial extract of Passiflora incarnata (Sintocalmy®, Aché Laboratory) in the naloxone-induced jumping mice model of morphine withdrawal. In addition, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100B) levels were assessed in the frontal cortex and hippocampus, and DNA damage was verified on blood cells. In order to improve solubilization a Sintocalmy methanol extract (SME) was used. SME is mainly composed by flavonoids isovitexin and vitexin. The effects of SME 50, 100 and 200 mg/kg (i.p.) were evaluated in the naloxone-induced withdrawal syndrome in mice. SME 50 and SME 100 mg/kg decreased naloxone-induced jumping in morphine-dependent mice without reducing locomotor activity. No alterations were found in GFAP levels, however SME 50 mg/kg prevented the S100B increase in the frontal cortex and DNA damage. This study shows anti-addiction effects for a commercial standardized extract of P. incarnata and suggests the relevance of proper clinical assessment.

Glutamatergic Alterations in STZ-Induced Diabetic Rats Are Reversed by Exendin-4.

Diabetes mellitus is a metabolic disorder that results in glucotoxicity and the formation of advanced glycated end products (AGEs), which mediate several systemic adverse effects, particularly in the brain tissue. Alterations in glutamatergic neurotransmission and cognitive impairment have been reported in DM. Exendin-4 (EX-4), an analogue of glucagon-like peptide-1 (GLP-1), appears to have beneficial effects on cognition in rats with chronic hyperglycemia. Herein, we investigated the ability of EX-4 to reverse changes in AGE content and glutamatergic transmission in an animal model of DM looking principally at glutamate uptake and GluN1 subunit content of the N-methyl-D-aspartate (NMDA) receptor. Additionally, we evaluated the effects of EX-4 on in vitro models and the signaling pathway involved in these effects. We found a decrease in glutamate uptake and GluN1 content in the hippocampus of diabetic rats; EX-4 was able to revert these parameters, but had no effect on the other parameters evaluated (glycemia, C-peptide, AGE levels, RAGE, and glyoxalase 1). EX-4 abrogated the decrease in glutamate uptake and GluN1 content caused by methylglyoxal (MG) in hippocampal slices, in addition to leading to an increase in glutamate uptake in astrocyte culture cells and hippocampal slices under basal conditions. The effect of EX-4 on glutamate uptake was mediated by the phosphatidylinositide 3-kinases (PI3K) signaling pathway, which could explain the protective effect of EX-4 in the brain tissue, since PI3K is involved in cell metabolism, inhibition of apoptosis, and reduces inflammatory responses. These results suggest that EX-4 could be used as an adjuvant treatment for brain impairment associated with excitotoxicity.

Grape juice increases the BDNF levels but not alter the S100B levels in hippocampus and frontal cortex from male Wistar Rats.

Several studies have shown that a high consumption of vegetables and fruits is consistently associated with a low risk of oxidative stress-induced diseases, which includes some degenerative diseases such as amyotrophic lateral sclerosis, Alzheimer and Parkinson. Therefore, the objective of this study is to verify the effects of conventional and organic grape juice in the modulation of the neurotrophic factor (BDNF) and astrocytic markers protein (S100B) in hippocampus and frontal cortex of Wistar rats. In this study, 24 male Wistar rats were divided into three groups. To the first one, it was given organic purple grape juice; to the second, conventional grape juice, while the last one received only saline. After 30 days, all rats were sacrificed and hippocampus and frontal cortex were dissected. The animals that received organic and conventional grape juice showed, in frontal cortex, an elevated BNDF levels in relation to saline group. However, S100B levels did not change. These results showed that grape juices are able to modulate important marker in brain tissue, and could be an important factor to prevent brain diseases.

Grape juice increases the BDNF levels but not alter the S100B levels in hippocampus and frontal cortex from male Wistar Rats

ABSTRACT Several studies have shown that a high consumption of vegetables and fruits is consistently associated with a low risk of oxidative stress-induced diseases, which includes some degenerative diseases such as amyotrophic lateral sclerosis, Alzheimer and Parkinson. Therefore, the objective of this study is to verify the effects of conventional and organic grape juice in the modulation of the neurotrophic factor (BDNF) and astrocytic markers protein (S100B) in hippocampus and frontal cortex of Wistar rats. In this study, 24 male Wistar rats were divided into three groups. To the first one, it was given organic purple grape juice; to the second, conventional grape juice, while the last one received only saline. After 30 days, all rats were sacrificed and hippocampus and frontal cortex were dissected. The animals that received organic and conventional grape juice showed, in frontal cortex, an elevated BNDF levels in relation to saline group. However, S100B levels did not change. These results showed that grape juices are able to modulate important marker in brain tissue, and could be an important factor to prevent brain diseases.

MPP+-Lesioned Mice: an Experimental Model of Motor, Emotional, Memory/Learning, and Striatal Neurochemical Dysfunctions.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces motor and nonmotor dysfunctions resembling Parkinson's disease (PD); however, studies investigating the effects of 1-methyl-4-phenylpyridinium (MPP+), an active oxidative product of MPTP, are scarce. This study investigated the behavioral and striatal neurochemical changes (related to oxidative damage, glial markers, and neurotrophic factors) 24 h after intracerebroventricular administration of MPP+ (1.8-18 µg/mouse) in C57BL6 mice. MPP+ administration at high dose (18 µg/mouse) altered motor parameters, since it increased the latency to leave the first quadrant and reduced crossing, rearing, and grooming responses in the open-field test and decreased rotarod latency time. MPP+ administration at low dose (1.8 µg/mouse) caused specific nonmotor dysfunctions as it produced a depressive-like effect in the forced swim test and tail suspension test, loss of motivational and self-care behavior in the splash test, anxiety-like effect in the elevated plus maze test, and short-term memory deficit in the step-down inhibitory avoidance task, without altering ambulation. MPP+ at doses of 1.8-18 µg/mouse increased tyrosine hydroxylase (TH) immunocontent and at 18 µg/mouse increased α-synuclein and decreased parkin immunocontent. The astrocytic calcium-binding protein S100B and glial fibrillary acidic protein (GFAP)/S100B ratio was decreased following MPP+ administration (18 µg/mouse). At this highest dose, MPP+ increased the ionized calcium-binding adapter molecule 1 (Iba-1) immunocontent, suggesting microglial activation. Also, MPP+ at a dose of 18 µg/mouse increased thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels and increased glutathione peroxidase (GPx) and hemeoxygenase-1 (HO-1) immunocontent, suggesting a significant role for oxidative stress in the MPP+-induced striatal damage. MPP+ (18 µg/mouse) also increased striatal fibroblast growth factor 2 (FGF-2) and brain-derived neurotrophic factor (BDNF) levels. Moreover, MPP+ decreased tropomyosin receptor kinase B (TrkB) immunocontent. Finally, MPP+ (1.8-18 µg/mouse) increased serum corticosterone levels and did not alter acetylcholinesterase (AChE) activity in the striatum but increased it in cerebral cortex and hippocampus. Collectively, these results indicate that MPP+ administration at low doses may be used as a model of emotional and memory/learning behavioral deficit related to PD and that MPP+ administration at high dose could be useful for analysis of striatal dysfunctions associated with motor deficits in PD.

Methylglyoxal can mediate behavioral and neurochemical alterations in rat brain.

Diabetes is associated with loss of cognitive function and increased risk for Alzheimer's disease (AD). Advanced glycation end products (AGEs) are elevated in diabetes and AD and have been suggested to act as mediators of the cognitive decline observed in these pathologies. Methylglyoxal (MG) is an extremely reactive carbonyl compound that propagates glycation reactions and is, therefore, able to generate AGEs. Herein, we evaluated persistent behavioral and biochemical parameters to explore the hypothesis that elevated exogenous MG concentrations, induced by intracerebroventricular (ICV) infusion, lead to cognitive decline in Wistar rats. A high and sustained administration of MG (3µmol/µL; subdivided into 6days) was found to decrease the recognition index of rats, as evaluated by the object-recognition test. However, MG was unable to impair learning-memory processes, as shown by the habituation in the open field (OF) and Y-maze tasks. Moreover, a single high dose of MG induced persistent alterations in anxiety-related behavior, diminishing the anxiety-like parameters evaluated in the OF test. Importantly, MG did not alter locomotion behavior in the different tasks performed. Our biochemical findings support the hypothesis that MG induces persistent alterations in the hippocampus, but not in the cortex, related to glyoxalase 1 activity, AGEs content and glutamate uptake. Glial fibrillary acidic protein and S100B content, as well as S100B secretion (astroglial-related parameters of brain injury), were not altered by ICV MG administration. Taken together, our data suggest that MG interferes directly in brain function and that the time and the levels of exogenous MG determine the different features that can be seen in diabetic patients.

Repeated forced swimming impairs prepulse inhibition and alters brain-derived neurotrophic factor and astroglial parameters in rats.

Glutamate perturbations and altered neurotrophin levels have been strongly associated with the neurobiology of neuropsychiatric disorders. Environmental stress is a risk factor for mood disorders, disrupting glutamatergic activity in astrocytes in addition to cognitive behaviours. Despite the negative impact of stress-induced neuropsychiatric disorders on public health, the molecular mechanisms underlying the response of the brain to stress has yet to be fully elucidated. Exposure to repeated swimming has proven useful for evaluating the loss of cognitive function after pharmacological and behavioural interventions, but its effect on glutamate function has yet to be fully explored. In the present study, rats previously exposed to repeated forced swimming were evaluated using the novel object recognition test, object location test and prepulse inhibition (PPI) test. In addition, quantification of brain-derived neurotrophic factor (BDNF) mRNA expression and protein levels, glutamate uptake, glutathione, S100B, GluN1 subunit of N-methyl-D-aspartate receptor and calmodulin were evaluated in the frontal cortex and hippocampus after various swimming time points. We found that swimming stress selectively impaired PPI but did not affect memory recognition. Swimming stress altered the frontal cortical and hippocampal BDNF expression and the activity of hippocampal astrocytes by reducing hippocampal glutamate uptake and enhancing glutathione content in a time-dependent manner. In conclusion, these data support the assumption that astrocytes may regulate the activity of brain structures related to cognition in a manner that alters complex behaviours. Moreover, they provide new insight regarding the dynamics immediately after an aversive experience, such as after behavioural despair induction, and suggest that forced swimming can be employed to study altered glutamatergic activity and PPI disruption in rodents.

Lipoic acid increases glutamate uptake, glutamine synthetase activity and glutathione content in C6 astrocyte cell line.

Alpha lipoic acid (LA) is a sulfhydryl compound, used as dietary supplement and to treat a variety of conditions associated to oxidative stress. Glial cells are key modulators of neuroprotection. We show here that LA modulates specific glial parameters in C6 astrocyte cell line, such as glutamate uptake, glutamine synthetase (GS) activity and glutathione content, commonly associated with the protective role of glial cells. LA (10 and 50µM) after 24h of treatment significantly decreased the formation of reactive oxygen species (ROS) and nitric oxide (NO) levels, and increased glutamate uptake (up to 20%), GS activity (25%) and GSH content (up to 40%). LA increase glutamate uptake probably by decreasing oxidizing conditions and/or by mechanism dependent of protein kinase C (PKC). In contrast, high concentrations of LA (1000µM) decreased these glial functions. Moreover, this concentration increased ROS production and NO levels. In summary, these findings show that low doses of LA were able to modulate glial functions and it appears to have remarkable therapeutic potential in neurological diseases involving oxidative stress by improving glutamatergic metabolism.

Green tea (-)epigallocatechin-3-gallate reverses oxidative stress and reduces acetylcholinesterase activity in a streptozotocin-induced model of dementia.

Alzheimer's disease (AD) is the most prevalent form of dementia. Intracerebroventricular (ICV) infusion of streptozotocin (STZ) provides a relevant animal model of chronic brain dysfunction that is characterized by long-term and progressive deficits in learning, memory, and cognitive behavior, along with a permanent and ongoing cerebral energy deficit. Numerous studies on green tea epigallocatechin gallate (EGCG) demonstrate its beneficial effects on cognition and memory. As such, this study evaluated, for the first time, the effects of sub-chronic EGCG treatment in rats that were submitted to ICV infusion of STZ (3mg/kg). Male Wistar rats were divided into sham, STZ, sham+EGCG and STZ+EGCG groups. EGCG was administered at a dose of 10mg/kg/day for 4 weeks per gavage. Learning and memory was evaluated using Morris' Water Maze. Oxidative stress markers and involvement of the nitric oxide (NO) system, acetylcholinesterase activity (AChE) and glucose uptake were evaluated as well as glial parameters including S100B content and secretion and GFAP content. Our results show that EGCG was not able to modify glucose uptake and glutathione content, although cognitive deficit, S100B content and secretion, AChE activity, glutathione peroxidase activity, NO metabolites, and reactive oxygen species content were completely reversed by EGCG administration, confirming the neuroprotective potential of this compound. These findings contribute to the understanding of diseases accompanied by cognitive deficits and the STZ-model of dementia.

Alterations of PI3K and Akt signaling pathways in the hippocampus and hypothalamus of Wistar rats treated with highly palatable food.

BACKGROUND/OBJECTIVES: Highly palatable food (HPF), which is enriched in simple sugars and saturated fat, contributes to obesity and insulin resistance in humans. These metabolic changes are associated with serious complications of the central nervous system, including an elevated risk of cognitive dysfunction. We, herein, treated rats with HPF and then examined the insulin-signaling pathway, in particular, the levels of phosphatidylinositol-3 kinase (PI3K), Akt, and insulin receptor substrate-1 (IRS-1) in the hippocampus and hypothalamus. METHODS: Adult Wistar rats fed with HPF (heated or not during preparation) for 4 months and then measured the levels of PI3K, Akt, and IRS-1 in the hippocampus and hypothalamus, by western blotting and quantitative real-time polymerase chain reaction. RESULTS: We observed changes in body weight, glucose intolerance, and lipidemia, confirming that peripheral metabolic alterations were induced using this model. Hippocampal PI3K and hypothalamic Akt were affected in rats that are submitted to chronic exposure to an HPF diet. Moreover, heated HPF caused differentiated alterations in the regulatory subunit of PI3K in the hippocampus. DISCUSSION: Our data suggest that this diet alters insulin signaling differentially in each brain region, and that hippocampal changes induced by this diet could contribute to the understanding of cognitive impairments that are dependent on the hippocampus.

Investigation of the in vitro and ex vivo acetylcholinesterase and antioxidant activities of traditionally used Lycopodium species from South America on alkaloid extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: The study was aimed at evaluating medicinal and therapeutic potentials of two Lycopodiaceae species, Lycopodium clavatum (L.) and Lycopodium thyoides (Humb. & Bonpl. ex Willd), both used in South American folk medicine for central nervous system conditions. Alkaloid extracts were evaluated for chemical characterization, acetylcholinesterase and antioxidant activities. MATERIALS AND METHODS: The alkaloid extracts obtained by alkaline extraction were determined for each species by GC/MS examination. The evaluation of the anticholinesterase and the antioxidant activities of the extracts were tested by determining in vitro and ex vivo models. Effects on acetylcholinesterase (AChE) were tested in vitro using rat brain homogenates and ex vivo after a single administration (25, 10 and 1mg/kg i.p.) of the alkaloid extracts in mice. The in vitro antioxidant effects were tested for the 2-deoxyribose degradation, nitric oxide (NO) interaction, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity and total reactive antioxidant potential (TRAP). After an acute administration (25 and 10mg/kg i.p.) of the extracts in middle-aged (12 months) mice, the antioxidant effects were estimated through the thiobarbituric acid reactive substances test (TBARS), and the antioxidant enzymes activities for catalase (CAT) and superoxide dismutase (SOD) were measured. RESULTS: AChE activity was inhibited in vitro by the alkaloid-enriched extracts of both Lycopodium species in a dose and time-dependent manner in rat cortex, striatum and hippocampus. A significant inhibition was also observed in areas of the brain after acute administration of extracts, as well as decreased lipid peroxidation and increased CAT activity in the cortex, hippocampus and cerebellum. A moderate antioxidant activity was observed in vitro for the extracts. Chemically, the main alkaloids found for the two species were lycopodine and acetyldihidrolycopodine. CONCLUSION: This study showed that the biological properties of the folk medicinal plants Lycopodium clavatum and Lycopodium thyoides include AChE inhibitory activity and antioxidant effects, two possible mechanisms of action in Alzheimer's related processes.

Differential effects of insulin on peripheral diabetes-related changes in mitochondrial bioenergetics: involvement of advanced glycosylated end products.

Large scale clinical trials have demonstrated that an intensive antihyperglycemic treatment in diabetes mellitus (DM) in individuals reduces the incidence of micro- and macrovascular complications, e.g. nephropathy, retinopathy, DM-accelerated atherosclerosis, myocardial infarction, or limb amputations. Here, we investigated the effect of short- and long-term insulin administration on mitochondrial function in peripheral tissues of streptozotocin (STZ)-induced hyperglycemic rats. In addition, the in vitro effect of methylglyoxal (MG), advanced glycation end products (AGEs) and human diabetic plasma on mitochondrial activity was investigated in skeletal muscle and liver mitochondria and in rat skin primary fibroblasts. Hyperglycemic STZ rats showed tissue-specific patterns of energy deficiency, evidenced by reduced activities of complexes I, II and/or IV after 30 days of hyperglycemia in heart, skeletal muscle and liver; moreover, cardiac tissue was found to be the most sensitive to the diabetic condition, since energy metabolism was impaired after 10 days of the hyperglycemia. Insulin-induced tight glycemic control was effective in protecting against the hyperglycemia-induced inhibition of mitochondrial enzyme activities. Furthermore, the long-term hormone replacement (30 days) also increased these activities in kidney from STZ-treated animals, where the hyperglycemic state did not modify the electron transport activity. Results from in vitro experiments indicate that mitochondrial impairment could result from oxidative stress-induced accumulation of MG and/or AGEs. Further investigations demonstrated that human plasma AGE accumulation elicits reduced mitochondrial function in skin fibroblast. These data suggest that persistent hyperglycemia results in tissue-specific patterns of energy deficiency and that early and continuous insulin therapy is necessary to maintain proper mitochondrial metabolism.

In vitro S100B secretion is reduced by apomorphine: effects of antipsychotics and antioxidants.

Astrocytes express dopamine receptors and respond to dopamine stimulation. However, the role of astrocytes in psychiatric disorders and the effects of antipsychotics on astroglial cells have only been investigated recently. S100B is a glial-derived protein, commonly used as a marker of astroglial activation in psychiatric disorders, particularly schizophrenia. We investigated S100B secretion in three different rat brain preparations (fresh hippocampal slices, C6 glioma cells and primary astrocyte cultures) exposed to apomorphine and antipsychotics (haloperidol and risperidone), aiming to evaluate, ex vivo and in vitro, whether dopamine activation and dopaminergic antagonists modulate astroglial activation, as measured by changes in the extracellular levels of S100B. The serum S100B elevation observed in schizophrenic patients is not reflected by the in vitro decrease of S100B secretion that we observed in hippocampal slices, cortical astrocytes and C6 glioma cells treated with apomorphine, which mimics dopaminergic hyperactivation. This decrease in S100B secretion can be explained by a stimulation of D2 receptors negatively coupled to adenyl cyclase. Antipsychotic medications and antioxidant supplementation were able to prevent the decline in S100B secretion. Findings reinforce the benefits of antioxidant therapy in psychiatric disorders. Based on our results, in hippocampal slices exposed to apomorphine, it may be suggested that antipsychotics could help to normalize S100B secretion by astrocytes.

Ascorbate uptake is decreased in the hippocampus of ageing rats.

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.

Genoprotective effects of the green tea-derived polyphenol/epicatechin gallate in C6 astroglial cells.

In vitro and in vivo studies have recently reported significant chemopreventive effects of green tea-derived polyphenols in different diseases. However, it remains unclear how such effects could be triggered. In order to elucidate the effects of epicatechin gallate (ECG) in C6 cells, both by itself and against H2O2-induced genotoxicity, measurements of DNA strand breaks and chromosome loss were performed. DNA damage was measured by comet and micronucleus assays. The present study shows for the first time how ECG, the major green tea-derived polyphenol, is able to exert dose-dependent genoprotective effects in an H2O2-induced toxicity model of C6 astroglial cells. We demonstrate that doses of ECG in a range from 0.1 to 1 µM were able to completely prevent H2O2-induced genotoxicity in vitro. In contrast, considerably higher concentrations of ECG (10 µM) were able to reverse previous positive effects in a dose- and time-dependent manner. The same results were confirmed by both comet (F(3,9) = 336,148; P < .001) and micronucleus (F(3,9) = 23,228; P < .001) methods. Together, our data show ECG as a dose-dependent genoprotective compound in C6 astroglial cells. This indicates that small doses of polyphenols included in our diet could have beneficial effects on neural cells, contributing to prevention of oxidative stress-associated brain pathologies. In addition, our data highlight the importance of strictly modulating doses and/or consumption of antioxidant-fortified foods or additional supplements containing such beneficial molecules.

Long-term effects of environmental stimulation following hypoxia-ischemia on the oxidative state and BDNF levels in rat hippocampus and frontal cortex.

Environmental enrichment recovers memory deficits without affecting atrophy of the hippocampus adult rats submitted to neonatal hypoxia-ischemia (HI). The present study was designed to investigate whether the modulation of brain oxidative status and/or BDNF content, as assessed in adulthood, are involved with the functional neuroprotection caused by environmental enrichment in animals receiving neonatal HI. Male Wistar rats, in the 7th postnatal day, were submitted to the Levine-Rice model of neonatal hypoxia-ischemia, comprising permanent occlusion of the right common carotid artery and a 90 min period of hypoxia (8% O(2)-92% N(2)). Starting 2 weeks after the HI event, animals were stimulated by the enriched environment (1 h/day for 9 weeks). Rats were sacrificed approximately 24 h after the end of enrichment period and some oxidative stress parameters, specifically the free radical levels, macromolecules damage and superoxide dismutase activity, in hippocampus and frontal cortex samples were determined. BDNF levels were also measured in the same encephalic structures. Indexes of macromolecules damage, TBARS levels and total cellular thiols, as well as free radical levels were unchanged in both studied structures. An increased SOD activity in the right hippocampus of HI group maintained in standard environment was found, this effect was reversed in HI enriched group. Moreover, BDNF levels were increased only in the hippocampus of non-stimulated HI group. These results suggest that the environmental enrichment protocol bearing cognitive protection is not associated to increases in BDNF expression nor SOD activity in hippocampus of the rats, as assessed in adulthood, submitted to neonatal hypoxia-ischemia.

Effect of the atypical neuroleptic risperidone on morphology and S100B secretion in C6 astroglial lineage cells.

We investigated the effect of the atypical neuroleptic risperidone on morphology and S100B secretion in C6 glioma cells, considering the putative involvement of astroglial cells in neuropsychiatric disorders. In the presence of high experimental doses of risperidone, C6 cells become stellate, with process-bearing cells and partial retraction of the cell body followed by detachment from the adhesion surface with practically no cell death. These results indicate that risperidone is able to interfere with C6 cell adhesion without toxic effects. RhoA activator LPA prevented the effects of risperidone on cell morphology. From 6 h risperidone induced a statistically significant increment of about 80% in S100B secretion. These data contribute to the proposal that glial cells are targets of risperidone, which could be involved in the therapeutic response of risperidone to improve autism symptoms.

Beta-hydroxy-butyrate alters the extracellular content of S100B in astrocyte cultures.

Astrocytes have a variety of roles in maintaining neural tissue physiology, including energetic support, uptake and metabolism of glutamate and secretion of neurotrophic factors. Glutamate toxicity has been implicated in neurodegenerative disorders associated with conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. Glucose is the main energetic substrate for brain cells but, in some circumstances, the ketone bodies are used as a supplementary source and have been suggested to be neuroprotective agents against seizure disorders. Here, we investigate some possible biochemical changes in astrocyte cultures induced by beta-hydroxy-butyrate, the predominant blood ketone body. Its effect upon S100B secretion, astrocyte morphology and glutamate uptake was particularly investigated. S100B, a calcium-binding protein expressed and secreted by astrocytes, has neurotrophic activity and a possible role in epileptogenesis. Cell morphology was investigated by phase-contrast microscopy and immunocytochemistry for actin, GFAP and S100B. Our data show that beta-hydroxy-butyrate induces dramatic changes in astrocyte morphology and, independent of this, causes changes in the extracellular content of S100B. We observed an increment in S100B 1 h after beta-hydroxy-butyrate addition and a decrease 24 h later. No changes were observed in glutamate uptake. These astrocytic modifications may be associated with reduced neuronal excitability observed in the ketogenic condition.