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.

Sulfite Impairs Bioenergetics and Redox Status in Neonatal Rat Brain: Insights into the Early Neuropathophysiology of Isolated Sulfite Oxidase and Molybdenum Cofactor Deficiencies.

Isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies are genetic diseases biochemically characterized by the toxic accumulation of sulfite in the tissues of patients, including the brain. Neurological dysfunction and brain abnormalities are commonly observed soon after birth, and some patients also have neuropathological alterations in the prenatal period (in utero). Thus, we investigated the effects of sulfite on redox and mitochondrial homeostasis, as well as signaling proteins in the cerebral cortex of rat pups. One-day-old Wistar rats received an intracerebroventricular administration of sulfite (0.5 µmol/g) or vehicle and were euthanized 30 min after injection. Sulfite administration decreased glutathione levels and glutathione S-transferase activity, and increased heme oxygenase-1 content in vivo in the cerebral cortex. Sulfite also reduced the activities of succinate dehydrogenase, creatine kinase, and respiratory chain complexes II and II-III. Furthermore, sulfite increased the cortical content of ERK1/2 and p38. These findings suggest that redox imbalance and bioenergetic impairment induced by sulfite in the brain are pathomechanisms that may contribute to the neuropathology of newborns with ISOD and MoCD. Sulfite disturbs antioxidant defenses, bioenergetics, and signaling pathways in the cerebral cortex of neonatal rats. CII: complex II; CII-III: complex II-III; CK: creatine kinase; GST: glutathione S-transferase; HO-1: heme oxygenase-1; SDH: succinate dehydrogenase; SO32-: sulfite.

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.

Leptin stimulates the release of pro-inflammatory cytokines in hypothalamic astrocyte cultures from adult and aged rats.

Leptin is an adipose tissue-derived hormone that acts on the hypothalamus in order to maintain energy homeostasis. However, leptin can also induce an inflammatory response. Increasing evidence has highlighted a critical role of astrocytes in the effects of leptin on the hypothalamus. In addition, astrocytes participate in neuroinflammation by producing and releasing a wide range of inflammatory mediators. In this study, we aimed to investigate the age-dependent effect of leptin on pro- and anti-inflammatory cytokines released by the hypothalamic astrocyte cultures obtained from newborn, adult, and aged Wistar rats. In hypothalamic astrocytes from newborn rats, leptin did not change the release of pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). On the other contrary, leptin increased the release of both TNF-α and IL-1ß in astrocyte cultures from adult and aged animals. Regarding the anti-inflammatory cytokine interleukin 10 (IL-10), we did not observe any change in response to leptin. In conclusion, our data suggests a pro-inflammatory action of leptin on the hypothalamus during aging. This in turn may be related to the triggering of metabolic disorders, as both of these conditions are associated with neuroinflammation.

Age-Dependent Neurochemical Remodeling of Hypothalamic Astrocytes.

The hypothalamus is a crucial integrative center in the central nervous system, responsible for the regulation of homeostatic activities, including systemic energy balance. Increasing evidence has highlighted a critical role of astrocytes in orchestrating hypothalamic functions; they participate in the modulation of synaptic transmission, metabolic and trophic support to neurons, immune defense, and nutrient sensing. In this context, disturbance of systemic energy homeostasis, which is a common feature of obesity and the aging process, involves inflammatory responses. This may be related to dysfunction of hypothalamic astrocytes. In this regard, the aim of this study was to evaluate the neurochemical properties of hypothalamic astrocyte cultures from newborn, adult, and aged Wistar rats. Age-dependent changes in the regulation of glutamatergic homeostasis, glutathione biosynthesis, amino acid profile, glucose metabolism, trophic support, and inflammatory response were observed. Additionally, signaling pathways including nuclear factor erythroid-derived 2-like 2/heme oxygenase-1 p38 mitogen-activated protein kinase, nuclear factor kappa B, phosphatidylinositide 3-kinase/Akt, and leptin receptor expression may represent putative mechanisms associated with the cellular alterations. In summary, our findings indicate that as age increases, hypothalamic astrocytes remodel and exhibit changes in their neurochemical properties. This process may play a role in the onset and/or progression of metabolic disorders.

Effect of a trans fatty acid-enriched diet on mitochondrial, inflammatory, and oxidative stress parameters in the cortex and hippocampus of Wistar rats.

PURPOSE: Previously showed that dietary trans fatty acids (TFAs) may cause systemic inflammation and affect the central nervous system (CNS) in Wistar rats by increased levels of cytokines in the cerebrospinal fluid (CSF) and serum (Longhi et al. Eur J Nutr 56(3):1003-1016, 1). Here, we aimed to clarifying the impact of diets with different TFA concentrations on cerebral tissue, focusing on hippocampus and cortex and behavioral performance. METHODS: Wistar rats were fed either a normolipidic or a hyperlipidic diet for 90 days; diets had the same ingredients except for fat compositions, concentrations, and calories. We used lard in the cis fatty acid (CFA) group and PHSO in the TFA group. The intervention groups were as follows: (1) low lard (LL), (2) high lard (HL), (3) low partially hydrogenated soybean oil (LPHSO), and (4) high partially hydrogenated soybean oil (HPHSO). Mitochondrial parameters, tumor necrosis factor alpha (TNF-α), 2'7'-dichlorofluorescein (DCFH) levels in brain tissue, and open field task were analyzed. RESULTS: A worse brain tissue response was associated with oxidative stress in cortex and hippocampus as well as impaired inflammatory and mitochondrial parameters at both PHSO concentrations and there were alterations in the behavioral performance. In many analyses, there were no significant differences between the LPHSO and HPHSO diets. CONCLUSIONS: Partially hydrogenated soybean oil impaired cortical mitochondrial parameters and altered inflammatory and oxidative stress responses, and the hyperlipidic treatment caused locomotor and exploratory effects, but no differences on weight gain in all treatments. These findings suggest that quality is more important than the quantity of fat consumed in terms of CFA and TFA diets.

Effect of a trans fatty acid-enriched diet on biochemical and inflammatory parameters in Wistar rats.

PURPOSE: Recent data regarding trans fatty acids (TFAs) have implicated these lipids as particularly deleterious to human health, causing systemic inflammation, endothelial dysfunction and possibly inflammation in the central nervous system (CNS). We aimed to clarify the impact of partially hydrogenated soybean oil (PHSO) with different TFA concentrations on cerebrospinal fluid (CSF), serum and hepatic parameters in adult Wistar rats. METHODS: Wistar rats (n = 15/group) were fed either a normolipidic diet or a hyperlipidic diet for 90 days. The normolipidic and hyperlipidic diets had the same ingredients except for fat compositions, concentrations and calories. We used lard in the cis fatty acid group and PHSO in the trans fatty acid group. The intervention groups were as follows: (1) low lard (LL), (2) high lard (HL), (3) low partially hydrogenated soybean oil (LPHSO) and (4) high partially hydrogenated soybean oil (HPHSO). Body weight, lipid profiles and the inflammatory responses in the CSF, serum and liver tissue were analyzed. RESULTS: Surprisingly, with the PHSO diet we observed a worse metabolic response that was associated with oxidative stress in hepatic tissue as well as impaired serum and CSF fluid parameters at both PHSO concentrations. In many analyses, there were no significant differences between the LPHSO and HPHSO diets. CONCLUSIONS: Dietary supplementation with PHSO impaired inflammatory parameters in CSF and blood, induced insulin resistance, altered lipid profiles and caused hepatic damage. Overall, these findings suggest that fat composition is more important than the quantity of fat consumed in terms of cis and trans fatty acid diets.

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.

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.

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.

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.