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Astrocytes have key regulatory roles in central nervous system (CNS), integrating metabolic, inflammatory and synaptic responses. In this regard, type I interferon (IFN) receptor signaling in astrocytes can regulate synaptic plasticity. Simvastatin is a cholesterol-lowering drug that has shown anti-inflammatory properties, but its effects on astrocytes, a main source of cholesterol for neurons, remain to be elucidated. Herein, we investigated the effects of simvastatin in inflammatory and functional parameters of primary cortical and hypothalamic astrocyte cultures obtained from IFNα/β receptor knockout (IFNα/βR -/- ) mice. Overall, simvastatin decreased extracellular levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which were related to a downregulation in gene expression in hypothalamic, but not in cortical astrocytes. Moreover, there was an increase in anti-inflammatory interleukin-10 (IL-10) in both structures. Effects of simvastatin in inflammatory signaling also involved a downregulation of cyclooxygenase 2 (COX-2) gene expression as well as an upregulation of nuclear factor κB subunit p65 (NFκB p65). The expression of cytoprotective genes sirtuin 1 (SIRT1) and nuclear factor erythroid derived 2 like 2 (Nrf2) was also increased by simvastatin. In addition, simvastatin increased glutamine synthetase (GS) activity and glutathione (GSH) levels only in cortical astrocytes. Our findings provide evidence that astrocytes from different regions are important cellular targets of simvastatin in the CNS, even in the absence of IFNα/βR, which was showed by the modulation of cytokine production and release, as well as the expression of cytoprotective genes and functional parameters., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Preterm birth and hypoxia-ischemia (HI) are major causes of neonatal death and neurological disabilities in newborns. The widely used preclinical HI model combines carotid occlusion with hypoxia exposure; however, the relationship between different hypoxia exposure periods with brain tissue loss, astrocyte reactivity and behavioral impairments following HI is lacking. Present study evaluated HI-induced behavioral and morphological consequences in rats exposed to different periods of hypoxia at postnatal day 3. Wistar rats of both sexes were assigned into four groups: control group, HI-120 min, HI-180 min and HI-210 min. Neurodevelopmental reflexes, exploratory abilities and cognitive function were assessed. At adulthood, tissue damage and reactive astrogliosis were measured. Animals exposed to HI-180 and HI-210 min had delayed neurodevelopmental reflexes compared to control group. Histological assessment showed tissue loss that was restricted to the ipsilateral hemisphere in lower periods of hypoxia exposure (120 and 180 min) but affected both hemispheres when 210 min was used. Reactive astrogliosis was increased only after 210 min of hypoxia. Interestingly, cognitive deficits were induced regardless the duration of hypoxia and there were correlations between behavioral parameters and cortex, hippocampus and corpus callosum volumes. These results show the duration of hypoxia has a close relationship with astrocytic response and tissue damage progression. Furthermore, the long-lasting cognitive memory deficit and its association with brain structures beyond the hippocampus suggests that complex anatomical changes should be involved in functional alterations taking place as hypoxia duration is increased, even when the cognitive impairment limit is achieved.

Neonatal hypoxia-ischemia (HI) is associated to cognitive and motor impairments and until the moment there is no proven treatment. The underlying neuroprotective mechanisms of stem cells are partially understood and include decrease in excitotoxicity, apoptosis and inflammation suppression. This study was conducted in order to test the effects of intracardiac transplantation of human dental pulp stem cells (hDPSCs) for treating HI damage. Seven-day-old Wistar rats were divided into four groups: sham-saline, sham-hDPSCs, HI-saline, and HI-hDPSCs. Motor and cognitive tasks were performed from postnatal day 30. HI-induced cognitive deficits in the novel-object recognition test and in spatial reference memory impairment which were prevented by hDPSCs. No motor impairments were observed in HI animals. Immunofluorescence analysis showed human-positive nuclei in hDPSC-treated animals closely associated with anti-GFAP staining in the lesion scar tissue, suggesting that these cells were able to migrate to the injury site and could be providing support to CNS cells. Our study evidence novel evidence that hDPSC can contribute to the recovery following hypoxia-ischemia and highlight the need of further investigation in order to better understand the exact mechanisms underlying its neuroprotective effects.

Regular physical activity has shown to improve the quality of life and to prevent age-related memory deficits. Memory processing requires proper regulation of several enzymes such as sodium-potassium adenosine triphosphatase (Na + , K + -ATPase) and acetylcholinesterase (AChE), which have a pivotal role in neuronal transmission. The present study investigated the effects of a treadmill running protocol in young (3 months), mature (6 months) and aged (22 months) Wistar rats, on: (a) cognitive function, as assessed in the Water maze spatial tasks; (b) Na + , K + -ATPase and AChE activities in the hippocampus following cognitive training alone or treadmill running combined with cognitive training. Animals of all ages were assigned to naïve (with no behavioral or exercise training), sedentary (non-exercised, with cognitive training) and exercised (20 min of daily running sessions, 3 times per week for 4 weeks and with cognitive training) groups. Cognition was assessed by reference and working memory tasks run in the Morris Water maze; 24 h after last session of behavioral testing, hippocampi were collected for biochemical analysis. Results demonstrated that: (a) a moderate treadmill running exercise prevented spatial learning and memory deficits in aged rats; (b) training in the Water maze increased both Na + , K + -ATPase and AChE activities in the hippocampus of mature and aged rats; (c) aged exercised rats displayed an even further increase of Na + , K + -ATPase activity in the hippocampus, (d) enzyme activity correlated with memory performance in aged rats. It is suggested that exercise prevents spatial memory deficits in aged rats probably through the activation of Na + , K + -ATPase in the hippocampus.

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 O 2 ) 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.

This study investigated the effects of chronic homocysteine administration on some parameters of inflammation, such as cytokines (TNF-α, IL-1β and IL-6), chemokine CCL(2) (MCP-1), nitrite and prostaglandin E(2) levels, as well as on immunocontent of NF-κB/p65 subunit in hippocampus and/or serum of rats. Since acetylcholinesterase has been associated with inflammation, we also evaluated the effect of homocysteine on this enzyme activity in hippocampus of rats. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 μmol/g body weight) or saline (control) from the 6th to the 28th days-of-age. One or 12 h after the last injection, rats were euthanized and hippocampus and serum were used. Results showed that chronic hyperhomocysteinemia significantly increased pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokine CCL(2) (MCP-1) and prostaglandin E(2) in hippocampus and serum of rats at 1 and 12 h after the last injection of homocysteine. Nitrite levels increased in hippocampus, but decreased in serum at 1 h after chronic hyperhomocysteinemia. Acetylcholinesterase activity and immunocontent of citoplasmic and nuclear NF-κB/p65 subunit were increased in hippocampus of rats subjected to hyperhomocysteinemia at 1 h, but did not alter at 12 h after the last injection of homocysteine. According to our results, chronic hyperhomocysteinemia increases inflammatory parameters, suggesting that this process might be associated, at least in part, with the cerebrovascular and vascular dysfunctions characteristic of some homocystinuric patients.

Folic acid plays an important role in neuroplasticity and acts as a neuroprotective agent, as observed in experimental brain ischemia studies. The aim of this study was to investigate the effects of folic acid on locomotor activity, aversive memory and Na(+),K(+)-ATPase activity in the frontal cortex and striatum in animals subjected to neonatal hypoxia-ischemia (HI). Wistar rats of both sexes at postnatal day 7 underwent HI procedure and were treated with intraperitoneal injections of folic acid (0.011 μmol/g body weight) once a day, until the 30th postnatal day. Starting on the day after, behavioral assessment was run in the open field and in the inhibitory avoidance task. Animals were sacrificed by decapitation 24 h after testing and striatum and frontal cortex were dissected out for Na(+),K(+)-ATPase activity analysis. Results show anxiogenic effect in the open field and an impairment of aversive memory in the inhibitory avoidance test in HI rats; folic acid treatment prevented both behavioral effects. A decreased Na(+),K(+)-ATPase activity in striatum, both ipsilateral and contralateral to ischemia, was identified after HI; a total recovery was observed in animals treated with folic acid. A partial recovery of Na(+),K(+)-ATPase activity was yet seen in frontal cortex of HI animals receiving folic acid supplementation. Presented results support that folic acid treatment prevents memory deficit and anxiety-like behavior, as well as prevents Na(+),K(+)-ATPase inhibition in the striatum and frontal cortex caused by neonatal hypoxia-ischemia.

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.

In the present study we investigated the effect of chronic hypermethioninemia on rat performance in the Morris water maze task, as well as on acetylcholinesterase (AChE) activity in rat cerebral cortex. For chronic treatment, rats received subcutaneous injections of methionine (1.34-2.68 micromol/g of body weight), twice a day, from the 6th to the 28th day of age; control rats received the same volume of saline solution. Groups of rats were killed 3 h, 12 h or 30 days after the last injection of methionine to AChE assay and another group was left to recover until the 60th day of life to assess the effect of early methionine administration on reference and working spatial memory of rats. AChE activity was also determined after behavioral task. Results showed that chronic treatment with methionine did not alter reference memory when compared to saline-treated animals. In the working memory task, we observed a significant days effect with significant differences between control and methionine-treated animals. Chronic hypermethioninemia significantly increased AChE activity at 3 h, 12 h or 30 days after the last injection of methionine, as well as before or after behavioral test. The effect of acute hypermethioninemia on AChE was also evaluated. For acute treatment, 29-day-old rats received one single injection of methionine (2.68 micromol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that acute administration of methionine did not alter cerebral cortex AChE activity. Our findings suggest that chronic experimental hypermethioninemia caused cognitive dysfunction and an increase of AChE activity that might be related, at least in part, to the neurological problems presented by hypermethioninemic patients.

The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) alpha and beta and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen--glucose deprivation (OGD). Pretreatment with 17beta-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERalpha protein were significantly reduced by 20% after OGD in both vehicle- and estradiol-treated cultures, whereas ERbeta was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERbeta and, consequently, of the genes influenced by this receptor.

Here we investigated the effects of estradiol replacement in ovariectomized female rats using hippocampal slices exposed to oxygen-glucose deprivation (OGD). OGD induced lactate dehydrogenase (LDH) release to the incubation medium, what was assumed as a parameter of cellular death. In the estradiol-treated group the LDH release was markedly decreased by 23% as compared to the vehicle-treated group. In attempt to study a possible mechanism by which estradiol acts, we investigated some parameters of oxidative stress. In both vehicle-treated and estradiol-treated groups, OGD significantly increased the free radical production by 34% and 16%, respectively, although no significant differences on total antioxidant capacity were observed. Interestingly, estradiol replacement prevented the significant reduction in tryptophan and tyrosine contents caused by OGD observed in vehicle-treated animals. Our results show that estradiol replacement in ovariectomized female rats decreases cellular susceptibility to an ischemic-like injury and suggest a role for the hormone on protein damage prevention.

Here we investigated the neuroprotective effect of 17beta-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17beta-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17beta-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17beta-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3beta. Our results show a clear neuroprotective effect of 17beta-estradiol and suggest that this effect could involve PI3-K pathway.

It has been shown that emotional stress may induce oxidative damage, and considerably change the balance between pro-oxidant and antioxidant factors in the brain. The aim of this study was to verify the effect of repeated restraint stress (RRS; 1 h/day during 40 days) on several parameters of oxidative stress in the hippocampus of adult Wistar rats. We evaluated the lipid peroxide levels (assessed by TBARS levels), the production of free radicals (evaluated by the DCF test), the total radical-trapping potential (TRAP) and the total antioxidant reactivity (TAR) levels, and antioxidant enzyme activities (SOD, GPx and CAT) in hippocampus of rats. The results showed that RRS induced an increase in TBARS levels and in GPx activity, while TAR was reduced. We concluded that RRS induces oxidative stress in the rat hippocampus, and that these alterations may contribute to the deleterious effects observed after prolonged stress.

Glutamatergic mechanisms are thought to be involved in stress-induced changes of brain function, especially in the hippocampus. We hypothesized that alterations caused by the hormonal changes associated with chronic and acute stress may affect glutamate uptake and release from hippocampal synaptosomes in Wistar rats. It was found that [3H]glutamate uptake and release by hippocampal nerve endings, when measured 24 h after 1 h of acute restraint, presented no significant difference. The exposure to repeated restraint stress for 40 days increased neuronal presynaptic [3H]glutamate uptake as well as basal and K+-stimulated glutamate release when measured 24 h after the last stress session. Chronic treatment also caused a significant decrease in [3H]glutamate binding to hippocampal membranes. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in nervous system plasticity following repeated stress exposure.

Ketogenic diets have been used in the treatment of refractory childhood epilepsy for almost 80 years; however, we know little about the underlying biochemical basis of their action. In this study, we evaluate oxidative stress in different brain regions from Wistar rats fed a ketogenic diet. Cerebral cortex appears to have not been affected by this diet, and cerebellum presented a decrease in antioxidant capacity measured by a luminol oxidation assay without changes in antioxidant enzyme activities--glutathione peroxidase, catalase, and superoxide dismutase. In the hippocampus, however, we observed an increase in antioxidant activity accompanied by an increase of glutathione peroxidase (about 4 times) and no changes in lipoperoxidation levels. We suggest that the higher activity of this enzyme induced by ketogenic diet in hippocampus might contribute to protect this structure from neurodegenerative sequelae of convulsive disorders.

Neonatal Hypoxia-Ischemia (HI) triggers a cascade of biochemical events that result in neuronal injury, but the mechanisms underlying these processes are not completely understood, and information regarding the effect of HI on the synthesis of brain glycoconjugates is lacking. The present work evaluates the effects of neonatal HI on hippocampal ganglioside synthesis. Seven-day-old rat pups were exposed to HI for 2.5 h according to the modified Levine model and samples from hyppocampus were obtained at 30 min as well as at 1, 2 and 4 days later. The activity for synthesis of gangliosides was evaluated by determining the incorporation of N-acetyl [3H]neuraminc acid ([3H]NeuAc) into the endogenous gangliosides of Golgi membranes and by determining the activity of Sial-T2 (GD3 synthase) and GalNAc-T (GM2 synthase), the two enzymes acting on sialyllactosylceramide (GM3) at the branching point of synthesis of a- and b-ganglioside pathway. Northern blot experiments were also conducted to determine transcription levels of the mRNAs specific for these transferases. Neonatal HI caused a relative increase of in vitro [3H]NeuAc incorporation into endogenous lactosylceramide, which was most noticeable at 30 min and I day post-event and disappeared by day 2 and 4. The transient accumulation of [3H]GM3 correlated with decreases in the activities of GD3- and GM2 synthase measured at 30 min and at 1 day after the HI insult. No significant variations in the expression of the genes for these enzymes were observed. Results suggest that transient accumulation of GM3 may be due to post-translational events negatively modulating both GD3- and GM2 synthase activities.

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na+,K+-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na+,K+-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na+,K+-ATPase activity afforded by preconditioning be related to cellular neuroprotection.

Synapsins are phosphoproteins related to the anchorage of synaptic vesicles to the actin skeleton. Hypoxia-ischemia causes an increased calcium influx into neurons through ionic channels gated by activation of glutamate receptors. In this work seven-day-old Wistar rats were submitted to hypoxia-ischemia and sacrificed after 21 hours, 7, 30, or 90 days. Synaptosomal fractions were obtained by Percoll gradients and incubated with 32P (10 microCi/g). Proteins were analysed by SDS-PAGE and radioactivity incorporated into synapsin 1 was counted by liquid scintillation. Twenty-one hours after hypoxia-ischemia we observed a reduction on the in vitro phosphorylation of synapsin 1, mainly due to hypoxia, rather than to ischemia; this effect was reversed at day 7 after the insult. There was another decrease in phosphorylation 30 days after the event interpreted as a late effect of hypoxia-ischemia. No changes were observed at day 90. Our results suggest that decreased phosphorylation of synapsin 1 could be related to neuronal death that follows hypoxia-ischemia.

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [14Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of 32P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and the proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.