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In this study, anMale Wistar rats received bilateral intracerebroventricular infusions of AβThe Aβ-infused animals showed a significant decrease in ganglioside concentration and relative reduction of GD1b and GQ1b species. Treatment with genistein prevented the decrease in ganglioside levels. Phospholipid and cholesterol contents did not show significant differences.Considering the roles of gangliosides on neuronal function, findings described here can contribute to the knowledge of the potential neuroprotective mechanisms of genistein against Aβ-induced alterations in the frontal cortex of rats and provide a novel view in the multifaceted scenario associated with its beneficial effects.

The Developmental Origins of Health and Disease (DOHaD) states that intrauterine maternal environment influences postnatal life by programming offspring’s metabolism. Intrauterine milieu induced by exercise during pregnancy promotes long-lasting benefits to the offspring’s health and seems to offer some resistance against chronic diseases in adult life. Alzheimer’s disease is a public health concern with limited treatment options. In the present study, we assessed the potential of maternal exercise during pregnancy in long-term programming of young adult male rat offspring’s cerebellar metabolism in conferring neuroprotection against amyloid-β (Aβ) neurotoxicity. Female Wistar rats were submitted to a swimming protocol 1 week prior mating and throughout pregnancy (five sessions/a week lasting 30 min). Aβ oligomers were infused bilaterally in the brain ventricles of 60-day-old male offspring. Fourteen days after surgery, we measured parameters related to redox state, mitochondrial function, and the immunocontent of proteins related to synaptic function. We found that maternal exercise during pregnancy attenuated several parameters in the offspring’s male rat cerebellum, such as the reactive species rise, the increase of inducible nitric oxide synthase immunocontent and tau phosphorylation induced by Aβ oligomers, increased mitochondrial fission indicated by dynamin-related protein 1 (DRP1), and protein oxidation identified by carbonylation. Strikingly, we find that maternal exercise promotes changes in the rat offspring’s cerebellum that are still evident in young adult life. These favorable neurochemical changes in offspring’s cerebellum induced by maternal exercise may contribute to a protective phenotype against Aβ-induced neurotoxicity in young adult male rat offspring., This study was supported by the Pró-Reitoria de Pesquisa/ Universidade Federal do Rio Grande do Sul (PROPESQ/UFRGS). Financial support. CPK is a PhD postgraduate student in Biological Sciences –Biochemistry receiving grants from the Brazilian agency Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq). CM received grants from CNPq [Universal 442406/2014-2 and INCT 465671/2014-4].

Several studies have demonstrated the antitumor effect of doxazosin, an α1-adrenergic blocker, against glioma and breast, bladder and prostate cancers. Doxazosin is also being evaluated as a treatment for posttraumatic stress disorder (PTSD) and alcoholism, and α1-adrenergic blockers have been linked to neuroprotection in neurodegenerative disorders, such as Alzheimer's Disease (AD). Cancer and AD have an inverse relationship in many aspects, with several factors that contribute to apoptosis inhibition and proliferation being increased in cancers but decreased in AD. Neuroblastoma (NB) is a pediatric tumor derived from embryonic neural-crest cells, with an overall cure rate of 40%, despite aggressive treatment. Thus, due to the need of new therapeutic strategies against NB and neurodegenerative disorders and the inverse relationship between these diseases, we investigated whether doxazosin may serve as an antitumor and neuroprotective agent. We analyzed the drug's effects on undifferentiated and retinoic acid-differentiated SH-SY5Y human NB cells and on an in vitro model of organotypic hippocampal cultures exposed to amyloid-β. Doxazosin showed antitumor effect on undifferentiated NB cells by induction of apoptosis, necrosis, cell cycle arrest and decrease of p-EGFRTyr1048 levels. On differentiated cells, doxazosin was less cytotoxic and increased p-EGFRTyr1048, p-AktSer473 and p-GSK-3βSer9 levels. Moreover, the drug was able to protect hippocampal slices from amyloid-β toxicity through prevention of GSK-3β activation and of Tau hyperphosphorylation. Therefore, our results show that doxazosin has antitumor activity against undifferentiated NB and is neuroprotective on an in vitro model of Alzheimer's disease.

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

Cerebral ischemia constitutes the most frequent type of cerebrovascular disease. The reduction of blood supply to the brain initiates the ischemic cascade starting from ionic imbalance to subsequent glutamate excitotoxicity, neuroinflammation and oxidative stress, eventually causing neuronal death. Previously, the authors have demonstrated the in vitro cytoprotective and antioxidant effects of a new arylidene malonate derivative, KM-34, against oxidizing agents like hydrogen peroxide, glutamate or Fe3+/ascorbate. Here, we examined for the first time the neuroprotective effect of KM-34 on ischemia/reperfusion models. In vitro, treatment with 10 and 50 μM KM-34 reduced the cellular death (propidium iodide incorporation) induced by oxygen glucose deprivation (OGD) in rat organotypic hippocampal slices cultures. In vivo, stroke was induced in male Wistar rats through middle cerebral artery occlusion (MCAO), followed by 23 h of reperfusion. KM-34 was orally administered 105 min after MCAO onset. We noticed that 1 mg/kg KM-34 reduced infarct volume and neurological score, and increased the latency to fall in the Hanging Wire test compared to vehicle-treated ischemic animals. While ischemic and sham-operated groups showed similar horizontal locomotor activity, vertical counts decreased after MCAO, suggesting that vertical movements are more sensitive to the ischemic injury. Treatment with KM-34 also alleviated the mitochondrial impairment (ROS generation, swelling and membrane potential dissipation) induced by transient MCAO but not significant alterations were found in oxidative stress parameters. Overall, the study provides preclinical evidences confirming the neuroprotective effects of a novel synthetic molecule and paved the way for future investigations regarding its therapeutic potential against brain ischemia/reperfusion injury.

Achyrocline satureioides, popularly known as "marcela", is a medicinal plant found in South America. This plant is rich in flavonoids, which have been reported to exert numerous biological activities. The aim of this study was to purify, identify and evaluate the mechanisms underlining anticancer activity of A. satureioides flavonoids in glioma cell lines (U87, U251 and C6) as well as their comparative toxicity in normal brain cells (primary astrocytes, neurons and organotypic hippocampal cultures). The main flavonoids present in A. satureioides are luteolin, quercetin, 3-O-methyl-quercetin and achyrobichalcone, the later a very unique metabolite present in this plant. Isolated flavonoids as well as A. satureioides extracts reduced proliferation and clonogenic survival, and induced apoptosis of glioma cell lines. In addition, A. satureioides flavonoids potentiated the cytotoxic effect and apoptosis induction by the glioma chemotherapeutic temozolomide (TMZ). Importantly, A. satureioides flavonoids were less cytotoxic to astrocytes, neuron:astrocytes co-cultures and hippocampal cultures if compared to gliomas. Investigation of 10 cancer-related pathways showed a reduced activation of MYC and the Map kinases ERK and JNK by A. satureioides flavonoid-enriched extract, an effect not observed when individual flavonoids were evaluated. Altogether, the herein presented results show that A. satureioides extract possesses a combination of flavonoids, some unique for this plant, which have synergistic anticancer activity and potential for further studies in vivo.

Stroke is frequently associated with severe neurological decline and mortality, and its incidence is expected to increase due to aging population. The only available pharmacological treatment for cerebral ischemia is thrombolysis, with narrow therapeutic windows. Efforts aimed to identify new therapeutics are crucial. In this study, we look into plausible molecular and cellular targets for JM-20, a new hybrid molecule, against ischemic stroke in vivo. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) following 23 h of reperfusion. Animals treated with 8 mg/kg JM-20 (p.o., 1 h after reperfusion) showed minimal neurological impairment and lower GABA and IL-1β levels in CSF when compared to damaged rats that received vehicle. Immunocontent of pro-survival, phosphorylated Akt protein decreased in the cortex after 24 h as result of the ischemic insult, accompanied by decreased number of NeuN+ cells in the peri-infarct cortex, cornu ammonis 1 (CA1) and dentate gyrus (DG) areas. Widespread reactive astrogliosis in both cortex and hippocampus (CA1, CA3, and DG areas) was observed 24 h post-ischemia. JM-20 prevented the activated Akt reduction, neuronal death, and astrocytes reactivity throughout the brain. Overall, the results reinforce the pharmacological potential of JM-20 as neuroprotective agent and provide important evidences about its molecular and cellular targets in this model of cerebral ischemia.

The translocator protein (TSPO) is an outer mitochondrial membrane protein involved in the transport of cholesterol into the mitochondria, which is the first step for the synthesis of steroid hormones, as well as in the regulation of mitochondrial permeability transition pore opening and apoptosis. Studies have shown that the activation of TSPO may promote neuroprotective actions in experimental models of neurodegeneration and brain injury. In a previous study, our group showed that 4′-chlorodiazepam (4′-CD), a TSPO ligand, was neuroprotective against amyloid-beta (Aβ) in SHSY-5Y neuroblastoma cells. The aim of this study was to evaluate if 4′-CD was also neuroprotective against Aβ in organotypic hippocampal cultures and to identify its mechanisms of action. Aβ decreased the cell viability of organotypic hippocampal cultures, while 4′-CD had a neuroprotective effect when administered at 100 nM and 1000 nM. The neuroprotective effects of 4′-CD against Aβ were associated with an increased expression of superoxide dismutase (SOD). No differences were found in the expression of catalase, glial fibrillary acidic protein, Akt and procaspase-3. In summary, our results show that 4′-CD is neuroprotective against Aβ by a mechanism involving the modulation of SOD protein expression., Bruno D. Arbo received a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Christianne G. Salbego received a CNPq 1C Researcher Productivity Grant. Maria Flávia M. Ribeiro received a CNPq 1D Researcher Productivity Grant.

A redispersible spray-dried formulation containing curcumin-loaded, lipid-core nanocapsules (LNC-C) was developed for oral administration. The neuroprotective activity of curcumin after the spray-drying process was evaluated in vitro. The spray-dried powder (SD-LNC-C) was produced using a drying adjuvant composed of a blend of maltodextrin and L-leucine (90:10 w/w). Acceptable process yield (~ 70%) and drug content (6.5 ± 0.2 mg g-1) were obtained. SD-LNC-C was formed by smooth, spherical-shaped particles, and confocal Raman analysis indicated the distribution of the LNC-C on the surface of the leucine/maltodextrin agglomerates. The surface of the agglomerates was formed by a combination of LNC-C and adjuvants, and laser diffraction showed that SD-LNC-C had adequate aqueous redispersion, with no loss of controlled drug release behaviour of LNC-C. The in vitro curcumin activity against the lipopolysaccharide (LPS)-induced proinflammatory response in organotypic hippocampal slice cultures was evaluated. Both formulations (LNC-C and SD-LNC-C) reduced TNF-α to similar levels. Therefore, neuroprotection of curcumin in vitro may be improved by nanoencapsulation followed by spray-drying, with no loss of this superior performance. Hence, the redispersible spray-dried powder proposed here represents a suitable approach for the development of innovative nanomedicines containing curcumin for the prevention/treatment of neurodegenerative diseases.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular accumulation of amyloid-beta (Aβ) peptide, which induces synaptic dysfunction, alteration of intracellular signaling pathways, hyperphosphorylation of the Tau protein, and cognitive impairment. Genistein, one of the major isoflavones present in soy and soy products, has been shown to modulate some of the pathogenic events associated with the neurodegeneration process. However, its underlying mechanisms remain to be clarified. Therefore, the objectives of the present study were to evaluate the ability of genistein to protect against Aβ1-42-induced cognitive impairment in rats and to elucidate some of the possible mechanisms involved in its neuroprotective effects in the hippocampus. Male Wistar rats received bilateral intracerebroventricular infusions of Aβ1-42 (2 nmol) and genistein 10 mg/kg orally for 10 days. The Aβ-infused animals showed significant impairment of memory, which was accompanied by the following neurochemical alterations in the hippocampus: decreased levels of the synaptic proteins synaptophysin and postsynaptic density protein 95 (PSD-95), hyperphosphorylation of Tau with increased activation of glycogen synthase kinase-3β and c-Jun N-terminal kinase, and inactivation of ERK. Treatment with genistein improved Aβ-induced cognitive impairment by attenuation of synaptotoxicity, hyperphosphorylation of Tau, and inactivation of ERK. Furthermore, treatment with this soy isoflavone did not cause systemic toxicity. These findings provide further evidence of the neuroprotective effect of genistein in an in vivo model of Aβ toxicity and, importantly, extend the current knowledge concerning the mechanisms associated with the neuroprotective effects of this compound in the hippocampus.

Alzheimer’s disease (AD) is the main aging-associated neurodegenerative disorder and is characterized by mitochondrial dysfunction, oxidative stress, synaptic failure, and cognitive decline. It has been a challenge to find disease course-modifying treatments. However, several studies demonstrated that regular physical activity and exercise are capable of promoting brain health by improving the cognitive function. Maternal lifestyle, including regular exercise during pregnancy, has also been shown to influence fetal development and disease susceptibility in adulthood through fetal metabolism programming. Here, we investigated the potential neuroprotective role of regular maternal swimming, before and during pregnancy, against amyloid-β neurotoxicity in the adult offspring. Behavioral and neurochemical analyses were performed 14 days after male offspring received a single, bilateral, intracerebroventricular (icv) injection of amyloid-β oligomers (AβOs). AβOs-injected rats of the sedentary maternal group exhibited learning and memory deficits, along with reduced synaptophysin, brain-derived neurotrophic factor (BDNF) levels, and alterations of mitochondrial function. Strikingly, the offspring of the sedentary maternal group had AβOs-induced behavioral alterations that were prevented by maternal exercise. This effect was accompanied by preventing the alteration of synaptophysin levels in the offspring of exercised dams. Additionally, offspring of the maternal exercise group exhibited an augmentation of functional mitochondria, as indicated by increases in mitochondrial mass and membrane potential, α-ketoglutarate dehydrogenase, and cytochrome c oxidase enzymes activities. Moreover, maternal exercise during pregnancy induced long-lasting modulation of fusion and fission proteins, Mfn1 and Drp1, respectively. Overall, our data demonstrates a potential protective effect of exercise during pregnancy against AβOs-induced neurotoxicity in the adult offspring brain, by mitigating the neurodegenerative process triggered by Alzheimer-associated AβOs through programming the brain metabolism., This study was supported by the Pró-Reitoria de Pesquisa/Universidade Federal do Rio Grande do Sul (PROPESQ/UFRGS). CPK is a PhD Postgraduate student in Biological Sciences – Biochemistry receiving grants from the Brazilian agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). CM received grants from CNPq (Universal 442406/2014-2 and INCT 465671/2014-4).

Berberine is an alkaloid derived from herb the Berberis sp. and has long-term use in Oriental medicine. Studies along the years have demonstrated its beneficial effect in various neurodegenerative and neuropsychiatric disorders. The subject of this study was to evaluate whether berberine protects against delayed neuronal cell death in organotypic hippocampal culture (OHC) exposed to oxygen and glucose deprivation (OGD) and the cell signaling mechanism related to its effect. Hippocampal slices were obtained from 6 to 8-days-old male Wistar rat and cultured for 14 days. Following, the cultures were exposed for 1h to OGD and then treated with Berberine (10 and 20μM). After 24h recovery, propidium iodide (PI) uptake was analyzed and a decrease was observed in PI uptake on OGD Ber-treated culture, which means a decrease in cellular death. Western blot analysis showed that proteins Akt, GSK3β, ERK and JNK appear to play a role in berberine-mediated neuroprotection. Furthermore, capase-3 activity of OGD Ber-treated culture was diminished by control level in a fluorimetry assay. These findings suggest that berberine-mediated neuroprotection after ischemia involves Akt/GSK3β/ERK 1/2 survival/apoptotic signaling pathway as well as JNK and caspase-3 activity inhibition.

We have previously demonstrated that quercetin (Quer), a polyphenol widely found in vegetables, decreased glioma cell growth in vitro. Here, we asked whether this compound could affect glioma growth in an in vivo rat glioma model. We found that daily intraperitoneal Quer (50 mg/kg) injections lead to a concentration of 0.15 μg of Quer per gram of brain tissue, which increased the tumor volume in a time dependent manner. We observed a small reduction in lymphocytic infiltration, a marker of good prognosis in gliomas that was accompanied by a small reduction in cell viability of peripheral T-cells. Moreover, after Quer treatment neither body weight alteration nor liver pathology markers were detected. Although in vitro studies and massive literature reports point to the antitumoral properties of Quer, the present results indicate that great caution has to be taken in the design of clinical trials and the indiscriminate use of this polyphenol as dietary supplement.

Alzheimer's disease (AD), a neurodegenerative disorder exhibiting a gradual decline in cognitive function, is characterized by the presence of neuritic plaques composed of neurofibrillary tangles and amyloid-β (Aβ) peptide. Available drugs for AD therapy have small effect sizes and do not alter disease progression. Several studies have been shown that resveratrol is associated with anti-amyloidogenic properties, but therapeutic application of its beneficial effects is limited. Here we compared the neuroprotective effects of free resveratrol treatment with those of resveratrol-loaded lipid-core nanocapsule treatment against intracerebroventricular injection of Aβ1-42 in rats. Animals received a single intracerebroventricular injection of Aβ1-42 (2 nmol), and 1 day after Aβ infusion, they were administered either free resveratrol (RSV) or resveratrol-loaded lipid-core nanocapsules (5 mg/kg, each 12 h, intraperitoneally), for 14 days. Aβ1-42-infused animals showed a significant impairment on learning memory ability, which was paralleled by a significant decrease in hippocampal synaptophysin levels. Furthermore, animals exhibited activated astrocytes and microglial cells, as well as disturbance in c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3β (GSK-3β) activation, beyond destabilization of β-catenin levels. Our results clearly show that by using lipid-core nanocapsules, resveratrol was able to rescue the deleterious effects of Aβ1-42 while treatment with RSV presented only partial beneficial effects. These findings might be explained by the robust increase of resveratrol concentration in the brain tissue achieved by lipid-core nanocapsules. Our data not only confirm the potential of resveratrol in treating AD but also offer an effective way to improve the efficiency of resveratrol through the use of nanodrug delivery systems.

This study examined the effects of two chronic stress regimens upon depressive-like behavior, A(1) and A(2A) adenosine receptor binding and immunocontent. Male rats were subjected to unpredictable chronic mild stress (UCMS) or to chronic restraint stress (CRS) for 40 days. Subsequently, depressive-like behaviors (forced swimming and consumption of sucrose) were evaluated, and A(1) adenosine or A(2A) adenosine receptors were examined in the hippocampus or striatum, respectively. UCMS animals demonstrated depressive-related behaviors (decrease in sucrose consumption and increased immobility in the forced swimming test). This group also presented increased A(1) adenosine receptor binding and immunoreactivity in hippocampus, as well as increased striatal A(2A) adenosine receptor binding in the striatum, without alteration in immunoreactivity. Conversely, the chronic restraint stress group displayed only an increase in A(1) adenosine receptor binding and no alteration in the other parameters evaluated. We suggest that the alteration in adenosine receptors, particularly the upregulation of striatal A(2A) adenosine receptors following UCMS, could be associated with depressive-related behavior.

Considerable evidence has been accumulated to suggests that blocking the inflammatory reaction promotes neuroprotection and shows therapeutic potential for clinical treatment of ischemic brain injury. Consequently, anti-inflammatory therapies are being explored for prevention and treatment of these diseases. Induction of brain tolerance against ischemia by pretreatment with resveratrol has been found to influence expression of different molecules. It remains unclear, however, whether and how resveratrol preconditioning changes expression of inflammatory mediators after subsequent global cerebral ischemia/reperfusion (I/R). Therefore, we investigated the effect of resveratrol pretreatment on NF-κB inflammatory cascade, COX-2, iNOS and JNK levels in experimental I/R. Adult male rats were subjected to 10 min of four-vessel occlusion and sacrificed at selected post-ischemic time points. Resveratrol (30 mg/kg) pretreatment was injected intraperitoneally 7 days prior to I/R induction. We found that resveratrol treatment before insult remarkably reduced astroglial and microglial activation at 7 days after I/R. It greatly attenuated I/R-induced NF-κB and JNK activation with decreased COX-2 and iNOS production. In conclusion, the neuroprotection of resveratrol preconditioning may be due in part to the suppression of the inflammatory response via regulation of NF-κB, COX-2 and iNOS induced by I/R. JNK was also suggested to play a protective role through in neuroprotection of resveratrol, which may also be contributing to reduction in neuroinflammation. The study adds to a growing literature that resveratrol can have important anti-inflammatory actions in the brain.

Increased oxidative stress and energy metabolism deficit have been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In this study, we investigated the oxidative mechanisms underlying the neuroprotective effects of resveratrol, a potent polyphenol antioxidant found in grapes, on structural and biochemical abnormalities in rats subjected to global cerebral ischemia. Experimental model of transient global cerebral ischemia was induced in Wistar rats by the four vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl and fluoro jade C stained indicated extensive neuronal death at 7 days after I/R. These findings were preceded by a rapid increase in the generation of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation, as well as by a decrease in Na + K + -ATPase activity and disrupted antioxidant defenses (enzymatic and non-enzymatic) in hippocampus and cortex. Administrating resveratrol 7 days prior to ischemia by intraperitoneal injections (30 mg/kg) significantly attenuated neuronal death in both studied structures, as well as decreased the generation of ROS, lipid peroxidation and NO content. Furthermore, resveratrol brought antioxidant and Na + K + -ATPase activity in cortex and hippocampus back to normal levels. These results support that resveratrol could be used as a preventive, or therapeutic, agent in global cerebral ischemia and suggest that scavenging of ROS contributes, at least in part, to resveratrol-induced neuroprotection.

The effect of Aβ25–35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C14] galactose and results showed that Aβ25–35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25–35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 μM was able to prevent the toxicity triggered by the fibrillar Aβ25–35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24 h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer’s disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer’s disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer’s treatment.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.

The role of omega-3 polyunsaturated fatty acids (3PUFAs) on brain function is increasingly demonstrated. Here, the effect of dietary deprivation of essential 3PUFAs on some parameters related to neuroprotection was investigated. Rats were fed with two different diets: omega-3 diet and omega-3-deprived diet. To assess the influence of 3PUFAs on brain responses to ischemic insult, hippocampal slices were subjected to an oxygen and glucose deprivation (OGD) model of in vitro ischemia. The omega-3-deprived group showed higher cell damage and stronger decrease in the [ 3 H]glutamate uptake after OGD. Moreover, omega-3 deprivation influenced antiapoptotic cell response after OGD, affecting GSK-3beta and ERK1/2, but not Akt, phosphorylation. Taken together, these results suggest that 3PUFAs are important for cell protection after ischemia and also seem to play an important role in the activation of antiapoptotic signaling pathways.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSC) seems to be a new alternative for the treatment of neurological diseases, including stroke. In order to investigate the response of hippocampal tissue to factors secreted by MSC and if these factors are neuroprotective in a model of oxygen and glucose deprivation (OGD), we used organotypic hippocampal cultures exposed to conditioned medium from bone marrow-derived MSC. Our results suggest that the conditioned medium obtained from these cells aggravates lesion caused by OGD. In addition, the presence of the conditioned medium alone was toxic mainly to cells in the CA1, CA2 and CA3 areas of the hippocampal organotypic culture even in basal conditions. GABA stimulation and NMDA and AMPA receptors antagonists were able to reduce propidium iodide staining, suggesting that the cell death induced by the toxic factors secreted by MSC could involve these receptors.

Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent.

Gliomas are the most common and devastating tumors of the central nervous system. Several studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) are promising anticancer agents. Biodegradable nanoparticulate systems have received considerable attention as potential drug delivery vehicles. The aim of this study was to evaluate the effects of indomethacin-loaded nanocapsules and indomethacin ethyl ester-loaded nanocapsules on glioma cell lines. In addition, the effect of these formulations on normal neural tissue was also evaluated. In order to investigate this, glioma cell lines (U138-MG and C6) and hippocampal organotypic cultures were used. The main finding of the present study is that indomethacin-loaded nanocapsules formulation was more potent than a solution of indomethacin in decreasing the viability and cell proliferation of glioma lines. Indomethacin and indomethacin ethyl ester associated together in the same nanocapsule formulation caused a synergic effect decreasing glioma cell proliferation. In addition, when the glioma cells were exposed to 25 microM of indomethacin-loaded nanocapsules or indomethacin ethyl ester-loaded nanocapsules, a necrotic cell death was observed. Interestingly, 5 microM of indomethacin-loaded nanocapsules was able to cause an antiproliferative effect without promoting necrosis in glioma cells. Another important finding was that the cytotoxic effect induced by 25 microM or 50 microM of indomethacin-loaded nanocapsules or indomethacin ethyl ester-loaded nanocapsules, in glioma cells was not observed in the organotypic cultures, indicating selective cytotoxicity of those formulations for tumoral cells. Further investigations using in vivo glioma model should be helpful to confirm the distinct effects of indomethacin-loaded nanocapsules and indomethacin ethyl ester-loaded nanocapsules, in normal versus tumoral cells.

We have recently demonstrated that high intensity training exercise exacerbates brain damage, while a moderate intensity (2 weeks of 20 min/day of treadmill training) reduces the injury caused by in vitro ischemia, oxygen and glucose deprivation (OGD), to hippocampal slices from Wistar rats. In the present paper, the effect of different running programs on severity of ischemic OGD lesion was examined, by the evaluation of three protocols designed to simulate exercise conditions common to humans: one or three 20-min sessions per week, during 12 weeks (moderate intensity), and two 20-min daily sessions for 3 weeks. OGD caused an increase of lactate dehydrogenase (LDH) release into the incubation media, a marker of tissue necrosis, and a decline of cell viability, as assessed by the decrease of mitochondrial dehydrogenase activity (MTT method). Moderate exercise, three times a week during 12-week treadmill training, decreased LDH release after OGD, while one weekly session and 3 weeks of two daily sessions did not affect OGD-induced LDH released. No exercise protocol evaluated altered MTT reduction. Our data support the hypothesis that moderate intensity exercise reduces hippocampal susceptibility to in vitro ischemia.

Lithium and valproate (VPA) are the most commonly prescribed mood-stabilizing drugs. Recently, several studies have reported their neuroprotective properties in several models of neural toxicity and, in some pathological conditions, large amounts of intracellular ATP can be released from damaged cells. In the present study, we investigate the potential neuroprotective effect of lithium and VPA against ATP-induced cell death in hippocampal slices of adult rats. Acute (in vitro) and chronic (in vivo) treatment at therapeutic doses with lithium or VPA significantly prevent the ATP-induced cell death. Lithium and VPA also exerted a synergic effect in the prevention of ATP-induced cell death. Moreover, hippocampal slices prepared from rats chronically treated with lithium or VPA presented a significant reduction in cell death in the presence of cytotoxic extracellular ATP. Although further investigations are necessary, our results show the neuroprotective effect of lithium and VPA against neuronal death induced by extracellular ATP, probably through a different pathway, and suggest novel uses of these drugs in neurogenerative diseases.

We evaluated the effects of two levels of daily forced exercise intensity (moderate and high) in the treadmill over cell susceptibility to oxygen and glucose deprivation (OGD) in hippocampal slices from Wistar rats. Moderate exercise decreased lactate dehydrogenase (LDH) release after OGD, while a significant increase in LDH release was observed in the high intensity group submitted to OGD. Our data corroborate the hypothesis that higher training intensity exacerbates brain damage, while a moderate intensity reduces the injury caused by in vitro ischemia.

Here we investigated the neuroprotective effect of resveratrol in an in vitro model of ischemia. We used organotypic hippocampal cultures exposed to oxygen-glucose deprivation (OGD). In OGD-vehicle exposed cultures, about 46% of the hippocampus was labeled with PI, indicating a robust percentage of cell death. When cultures were treated with resveratrol 10, 25 and 50 microM, the cell death was reduced to 22, 20 and 13% respectively. To elucidate a possible mechanism by which resveratrol exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase (PI3-k) pathway using LY294002 (5 microM) and mitogen-activated protein kinase (MAPK) using PD98059 (20 microM). The resveratrol (50 microM) neuroprotection was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that resveratrol 50 microM induced the phosphorylation/activation of Akt and extracellular signal-regulated kinase-1 and -2 (ERK1/2) and the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that PI3-k/Akt pathway are involved in the neuroprotective effect of resveratrol.

Recent epidemiological and dietary intervention studies in animals and humans have suggested that diet-derived flavonoids, in particular quercetin, may play a beneficial role by preventing or inhibiting tumorigenesis. The aim of this study was to evaluate whether quercetin may act differently on cancer and normal neuronal tissue. In order to investigate this, the U138MG human glioma cell line and hippocampal organotypic cultures were used. The study showed that quercetin induced in glioma cell cultures results in (a) a decrease in cell proliferation and viability, (b) necrotic and apoptotic cell death, (c) arrest in the G2 checkpoint of the cell cycle, and (d) a decrease of the mitotic index. Furthermore, we demonstrated that while quercetin promotes cancer regression it was able to protect the hippocampal organotypic cultures from ischemic damage. To sum up, our results suggest that quercetin induced growth inhibition and cell death in the U138MG human glioma cell line, while exerting a cytoprotective effect in normal cell cultures.

In the present study we evaluate the effects of homocysteine on cellular damage using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation), an in vitro model of hypoxic–ischemic events. For chronic treatment, we induced elevated levels of homocysteine in blood (500 mM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by subcutaneous injections of homocysteine (0.3–0.6 mmol/g of body weight), twice a day with 8 h intervals, from the 6th to the 28th postpartum day and controls received saline. Rats were sacrificed 1, 3 or 12 h after the last injection. For acute treatment, 29-day-old rats received one single injection of homocysteine (0.6 mmol homocysteine/g body weight) or saline and were sacrificed 1 h later. In another set of experiments rats were pretreated with Vitamins E (40 mg/kg) and C (100 mg/kg) or folic acid (5 mg/kg) during 1 week; 12 h after the last administration they received a single injection of homocysteine or saline and were sacrificed 1 h later. Results showed that both chronic (1 h after homocysteine administration) and acute hyperhomocysteinemia increased the cellular damage measured by LDH released to de incubation medium, suggesting an increase of tissue damage caused by OGD. Pretreatment with folic acid completely prevented the damage caused by acute hyperhomocysteinemia, whereas Vitamin E just partially prevented such effect. These findings may be relevant to explain, at least in part, the higher susceptibility of hyperhomocysteinemic patients to be susceptible to ischemic events and point to a possible preventive treatment. # 2006 ISDN. Published by Elsevier Ltd. All rights reserved.

Several studies have shown that high corticosteroid hormone levels increase neuronal vulnerability. Here we evaluate the consequences of in vivo acute or repeated restraint stress on cellular viability in rat hippocampal slices suffering an in vitro model of ischemia. Cellular injury was quantified by measuring lactate dehydrogenase (LDH) and neuron-specific enolase released into the medium. Acute stress did not affect cellular death when oxygen and glucose deprivation (OGD) was applied both immediately or 24h after restraint. The exposure to OGD, followed by reoxygenation, resulted in increased LDH in the medium. Repeated stress potentiated the effect of OGD both, on LDH and neuron-specific enolase released to the medium. There was no effect of repeated stress on the release of S100B, an astrocytic protein. Additionally, no effect of repeated stress was observed on glutamate uptake by the tissue. These results suggest that repeated stress increases the vulnerability of hippocampal cells to an in vitro model of ischemia, potentiating cellular damage, and that the cells damaged by the exposure to repeated stress+OGD are mostly neurons. The uptake of glutamate was not observed to participate in the mechanisms responsible for rendering the neurons more susceptible to ischemic damage after repeated stress.

Glioblastomas are the most common form of primary tumors of the central nervous system (CNS) and despite treatment, patients with these tumors have a very poor prognosis. ATP and other nucleotides and nucleosides are very important signaling molecule in physiological and pathological conditions in the CNS. ATP is degraded very slowly by gliomas when compared to astrocytes, potentially resulting in the accumulation of extracellular ATP around gliomas. Cell lysis caused by excitotoxic death or by tumor resection may liberate intracellular ATP, a known mitotic factor for glioma cells. The aim of this study is to examine the effects on cytotoxicity induced by extracellular ATP in U138-MG human glioma cell line and C6 rat glioma cell line compared to hippocampal organotypic cell cultures. The cytotoxicity of ATP (0.1, 0.5, 5 mM) was measured using propidium iodide and LDH assays. Caspases assay was performed to identify apoptotic cell death. Results showed that the glioma cells present resistance to death induced by ATP when compared with a normal tissue. High ATP concentrations (5 mM) induced cell death after 24 h in organotypic cell cultures but not in glioma cell lines. Our data indicate that ATP released in these situations can induce cell death of the normal tissue surrounding the tumor, potentially opening space to the fast growth and invasion of the tumor.

Aging is an important risk factor for stroke. We evaluated the effects of aging on cell susceptibility to oxygen and glucose deprivation (OGD) in slices of the hippocampus from Wistar rats aged 2, 11 and 24 months. Lactate dehydrogenase (LDH) released to the incubation media and free radical content were markedly increased in the 24-month group submitted to OGD. These results confirm that hippocampal tissue from old animals is more susceptible to ischemia-reoxygenation injury.

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.

Treated glioblastoma multiforme (GBM) patients only survive 6 to 14months after diagnosis; therefore, the development of novel therapeutic strategies to treat gliomas remains critically necessary. Considering that phenolic compounds, like quercetin, have the potential to be used in the chemotreatment of gliomas and that some flavonoids exhibit the ability to cross the BBB, in the present study, we investigated the antitumor effect of flavonoids (including chalcones, flavones, flavanones and flavonols). Initially their activities were tested in C6 glioma cells screened using the MTT method, resulting in the selection of chalcone 2 whose feasibility was confirmed by a Trypan Blue exclusion assay in the low μM range on C6 glioma cells. Cell cycle and apoptotic death analyses on C6 glioma cells were also performed, and chalcone 2 increased the apoptosis of the cells but did not alter the cell cycle progression. In addition, treatments with these two compounds were not cytotoxic to hippocampal organotypic cultures, a model of healthy neural cells. Furthermore, the results indicated that 2 induced apoptosis by inhibition of NF-κB and activation of active caspase-3 in glioma cells, suggesting that it is a potential prototype to develop new treatments for GBM in the future.

We described the first synthesis of fatty acid 3,4-dihydropyrimidinones (DHPM-fatty acids) using the Biginelli multicomponent reaction. Antiproliferative activity on two glioma cell lines (C6 rat and U-138-MG human) was also reported. The novel DHPM-fatty acids reduced glioma cell viability relative to temozolomide. Hybrid oxo-monastrol-palmitic acid was the most potent, reducing U-138-MG human cell viability by ca. 50% at 10 μM. In addition, the DHPM-fatty acids showed a large safety range to neural cells, represented by the organotypic hippocampal culture. These results suggest that the increased lipophilicity of DHPM-fatty acids offer a promising approach to overcoming resistance to chemotherapy and may play an important role in the development of new antitumor drugs.

Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus. CA1, a hippocampal vulnerable area, showed an increase in HSP27 phosphorylation, parallel with immunocontent. In dentate gyrus, a resistant area, the increase in HSP phosphorylation was lower than immunocontent. After preconditioned ischemia (2+10 min), when CA1 neurons are protected to a lethal, 10 min insult, we observed an increase in HSP immunocontent and a decrease in phosphorylation in both regions of the hippocampus, suggesting that, when there is no neuronal death, HSP27 in a vulnerable area responds similarly to the resistant area.When dephosphorylated, HSP27 acts as a chaperone, protecting other proteins from denaturation. As it is markedly expressed in astrocytes, we suggest that HSP27 could be protecting hippocampal astrocytes, which could then be helping neurons to resist to the insult, maintaining tissue normal homeostasis.

We previously showed that JM-20, a novel 1,5-benzodiazepine fused to a dihydropyridine moiety, possessed an anxiolytic profile similar to diazepam and strong neuroprotective activity in different cell models relevant to cerebral ischemia. Here, we investigated whether JM-20 protects against ischemic neuronal damage in vitro and in vivo. The effects of JM-20 were evaluated on hippocampal slices subjected to oxygen and glucose deprivation (OGD). For in vivo studies, Wistar rats were subjected 90 min of middle cerebral artery occlusion (MCAo) and oral administration of JM-20 at 2, 4 and 8 mg/kg 1 h following reperfusion. Twenty-four hours after cerebral blood flow restoration, neurological deficits were scored, and the infarct volume, histopathological changes in cortex, number of hippocampal and striatal neurons, and glutamate/aspartate concentrations in the cerebrospinal fluid were measured. Susceptibility to brain mitochondrial swelling, membrane potential dissipation, H2O2 generation, cytochrome c release, Ca2+ accumulation, and morphological changes in the organelles were assessed 24 h post-ischemia. In vitro, JM-20 (1 and 10 μM) administered during reperfusion significantly reduced cell death in hippocampal slices subjected to OGD. In vivo, JM-20 treatment (4 and 8 mg/kg) significantly decreased neurological deficit scores, edema formation, total infarct volumes and histological alterations in different brain regions. JM-20 treatment also protected brain mitochondria from ischemic damage, most likely by preventing Ca2+ accumulation in organelles. Moreover, an 8-mg/kg JM-20 dose reduced glutamate and aspartate concentrations in cerebrospinal fluid and the deleterious effects of MCAo even when delivered 8 h after blood flow restoration. These results suggest that in rats, JM-20 is a robust neuroprotective agent against ischemia/reperfusion injury with a wide therapeutic window. Our findings support the further examination of potential clinical JM-20 use to treat acute ischemic stroke.

Accumulating evidence indicates that curcumin potently protects against beta-amyloid (Abeta) due to its oxygen free radicals scavenging and anti-inflammatory properties. However, cellular mechanisms that may underlie the neuroprotective effect of curcumin in Abeta-induced toxicity are not fully understood yet. The present study was undertaken to investigate the mechanisms involved in neuroprotective effects of curcumin, particularly involving Wnt/beta-catenin and PI3K pathways.Organotypic hippocampal slice cultures were treated with curcumin and exposed to Abeta1-42 for 48 hours. Synaptic dysfunction, cell death, ROS formation, neuroinflammation and beta-catenin, Akt, and GSK-3beta phosphorylation were measured to determine the effects of curcumin against Abeta toxicity.Curcumin significantly attenuated Abeta-induced cell death, loss of synaptophysin, and ROS generation. Furthermore, curcumin was able to decrease IL-6 release and increase IL-10 release, and prevented glial activation. The phosphorylation of beta-catenin was avoided and the levels of free beta-catenin were increased by curcumin to promote cell survival upon treatment with Abeta. Curcumin, in the presence of Abeta, activated Akt which in turn phosphorylates GSK-3beta, and resulted in the inhibition of GSK-3beta. The presence of LY294002, an inhibitor of PI3K pathway, blocked the pro-survival effect of curcumin.These results reinforce the neuroprotective effects of curcumin on Abeta toxicity and add some evidence that its mechanism may involve beta-catenin and PI3K signaling pathway in organotypic hippocampal slice culture.

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-β peptide (Aβ). Aβ-induced toxicity is believed to involve alterations on as Na(+),K(+)-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aβ1-42 infusion and GM1 treatment on recognition memory and on Na(+),K(+)-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aβ1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aβ-induced cognitive deficit, corroborating its neuroprotective function. Aβ impaired Na(+),K(+)-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aβ-induced alteration on Na(+),K(+)-ATPase, though with no impact on AChE activity. Aβ caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aβ-triggered cognitive deficit involves region-specific alterations on Na(+),K(+)-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na(+),K(+)-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment.

Alzheimer's disease (AD), a neurodegenerative disorder exhibiting progressive loss of memory and cognitive functions, is characterized by the presence of neuritic plaques composed of neurofibrillary tangles and β-amyloid (Aβ) peptide. Drug delivery to the brain still remains highly challenging for the treatment of AD. Several studies have been shown that curcumin is associated with anti-amyloidogenic properties, but therapeutic application of its beneficial effects is limited. Here we investigated possible mechanisms involved in curcumin protection against Aβ(1-42)-induced cognitive impairment and, due to its poor bioavailability, we developed curcumin-loaded lipid-core nanocapsules in an attempt to improve the neuroprotective effect of this polyphenol. Animals received a single intracerebroventricular injection of Aβ(1-42) and they were administered either free curcumin or curcumin-loaded lipid-core nanocapsules (Cur-LNC) intraperitoneally for 10days. Aβ(1-42)-infused animals showed a significant impairment on learning-memory ability, which was paralleled by a significant decrease in hippocampal synaptophysin levels. Furthermore, animals exhibited activated astrocytes and microglial cells, as well as disturbance in BDNF expression and Akt/GSK-3β signaling pathway, beyond tau hyperphosphorylation. Our findings demonstrate that administration of curcumin was effective in preventing behavioral impairments, neuroinflammation, tau hyperphosphorylation as well as cell signaling disturbances triggered by Aβ in vivo. Of high interest, Cur-LNC in a dose 20-fold lower presented similar neuroprotective results compared to the effective dose of free curcumin. Considered overall, the data suggest that curcumin is a potential therapeutic agent for neurocognition and nanoencapsulation of curcumin in LNC might constitute a promising therapeutic alternative in the treatment of neurodegenerative diseases such as AD.

Age-dependent changes in the activity of protein phosphorylating systems stimulated by phorbol dibutyrate (M r range 40–80 kDa) were studied in micro-slices from rat brain labelled with [ 32 P]orthophosphate. In adult animals phorbol stimulated the phosphorylation of the known substrates of protein kinase C (B-50/GAP-43 and MARCKS) and 3 unknown polypeptides: a basic molecule of 74 kDa (pp74B) and acidic molecules of 60 kDa (pp60A) and 42 kDa (pp42C). In neonatal animals the labelling of MARCKS was relatively very high as previously reported. Labelling of pp60A was present at birth, but increased during development; labelling of pp74B was only detected after days 8–10. The activity of the system labelling pp42C was very high during the first 2 weeks postnatal and then declined rapidly. The labelling of pp74B was considerably higher in slices from the cerebral cortex compared with the hippocampus; no regional differences in the labelling of pp60A were observed. Compared to B-50/GAP-43 and MARCKS, stimulation of phosphorylation of pp74B and pp60A required a higher concentration of phorbol. The substrates of all the phorbol-responsive systems, with the exception of pp74B, were soluble in 40% acetic acid. The possible identity of pp60A, pp74B and pp42C is discussed. Copyright © 1996 Elsevier Science Ltd.

Objective: The present study was undertaken to evaluate whether resveratrol (RSV) modulates membrane lipid composition, as well as on ganglioside profile in ischemia/reperfusion injury. Methods: Global cerebral ischemia was induced by four-vessel occlusion for 10 minutes. RSV (30 mg/kg) or vehicle was intraperitoneally administered to rats 7 days prior to ischemia. Brain structures were homogenized with chloroform/methanol for ganglioside, phospholipids, and cholesterol levels. Results: RSV significantly prevented the reduction in the total content of gangliosides, phospholipids, and cholesterol in hippocampi and cerebral cortex induced by global cerebral ischemia. Although ischemia/ reperfusion decreased ganglioside content, the ganglioside profiles were apparently not modified. Conclusions: Our experiments suggest that lipid metabolism is important for development of ischemic damage and indicate that RSV treatment 7 days prior to ischemia may prevent membrane lipid loss.

The coumarins 5-methoxy-6,7-methylenedioxycoumarin 1 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7-methylenedioxycoumarin 3 isolated from Pterocaulon species showed significant cytotoxicity against two glioma cells lines. Compound 1 presented IC(50) values of 34.6 μM and 31.6 μM against human (U138-MG) and rat (C6) glioma cells, respectively, and this compound was at least two times more potent than compounds 2 and 3. This result could be explained by the planar conformation adopted by 1 through a non-classical hydrogen bond between a hydrogen of the methoxy and the oxygen of the methylenedioxy groups. Another important finding was that the cytotoxic effect induced by 1 in glioma cells was not observed in organotypic cultures, indicating a selective cytotoxicity for tumor cells.

This study was undertaken to evaluate the effects of chronic variate stress and lithium treatment on glutamatergic activity and neuronal vulnerability of rat hippocampus. Male Wistar rats were simultaneously treated with lithium and submitted to a chronic variate stress protocol during 40 days, and afterwards the hippocampal glutamatergic uptake and release, measured in slices and synaptosomes, were evaluated. We observed an increased synaptosomal [(3)H]glutamate uptake and an increase in [(3)H]glutamate stimulated release in hippocampus of lithium-treated rats. Chronic stress increased basal [(3)H]glutamate release by synaptosomes, and decreased [(3)H]glutamate uptake in hippocampal slices. When evaluating cellular vulnerability, both stress and lithium increased cellular death after oxygen and glucose deprivation (OGD). We suggest that the manipulation of glutamatergic activity induced by stress may be in part responsible for the neuroendangerment observed after stress exposure, and that, in spite of the described neuroprotective effects of lithium, it increased the neuronal vulnerability after OGD.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid-beta (Abeta) peptide in brain regions that are important for memory and cognition. The buildup of Abeta aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Abeta-induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 microm of Abeta(25-35) in the absence or in the presence of melatonin (25, 50, or 100 microm). In addition, the authors have investigated the involvement of GSK-3beta, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Abeta-induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Abeta(25-35). In addition, melatonin significantly reduced the activation of GSK-3beta, the phosphorylation of tau protein, the glial activation and the Abeta-induced increase of TNF-alpha and IL-6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Abeta-induced phosphorylation of tau protein, and preventing GSK-3beta activation and neuroinflammation.

In the present study we evaluated the effect of acute and chronic homocysteine administrations on glutamate uptake in parietal cortex of rats. The immunocontent of glial glutamate transporter (GLAST) and sodium-dependent glutamate/aspartate transporter (GLT-1) in the same cerebral structure was also investigated. For acute treatment, neonate or young rats received a single injection of homocysteine or saline (control) and were sacrificed 1, 8, 12 h, 7 or 30 days later. For chronic treatment, homocysteine was administered to rats twice a day at 8 h interval from their 6th to their 28th days old; controls and treated rats were sacrificed 12 h, 1, 7 or 30 days after the last injection. Results show that acute hyperhomocysteinemia caused a reduction on glutamate uptake in parietal cortex of neonate and young rats, and that 12 h after homocysteine administration the glutamate uptake returned to normal levels in young rats, but not in neonate. Chronic hyperhomocysteinemia reduced glutamate uptake, and GLAST and GLT-1 immunocontent. According to our results, it seems reasonable to postulate that the reduction on glutamate uptake in cerebral cortex of rats caused by homocysteine may be mediated by the reduction of GLAST and GLT-1 immunocontent, leading to increased extracellular glutamate concentrations, promoting excitotoxicity.

Brain ischemia results in cellular degeneration and loss of brain function. Oxcarbazepine (OXC), one of the newer antiepileptic drugs, has been demonstrating its efficacy on wide spectrum neurological disorders. In this paper, we investigated the neuroprotective profile of OXC in an in vitro model of ischemia, which consists in the exposure of organotypic hippocampal slice cultures to oxygen and glucose deprivation.OXC (30 microM) was added to the medium before and/or during and/or after the oxygen and glucose deprivation induction. Cell death was quantified by propidium iodide uptake measurement. Immunoblotting was used to detect the phosphorylation of Akt.Our results showed a decrease in propidium iodide incorporation when OXC was added before oxygen and glucose deprivation, suggesting a neuroprotective effect. This effect was prevented when cultures were previously treated with LY294002, an inhibitor of phosphoinositide-3-kinase (PI3K) pathway. We also analysed the effect of OXC on Akt phosphorylation. Immunoblotting revealed that OXC did not induce any change in phosphorylation/activation of Akt.Our results reinforce the neuroprotective effect of OXC and add some evidence that its mechanism may involve the PI3K pathway, suggesting that such effect could be upstream Akt. This indicates that with respect to OXC neuroprotective, Akt may not play a crucial role in determining cell survival.

Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities, which may arise from its free radical scavenging properties. Given that the pathogenesis of brain ischemia/reperfusion has been associated with an excessive generation of oxygen free radicals, the aim of this study was to evaluate the neuroproperties of boldine using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, to mimic an ischemic condition. The OGD ischemic condition significantly impaired cellular viability, increased lactate dehydrogenase (LDH) leakage and increased free radical generation. In non-OGD slices, incubation with 100microM boldine significantly increased LDH released into incubation media and decreased mitochondrial activity, suggesting an increase of tissue damage caused by boldine. However, slices incubated with 10microM boldine during and after OGD exposure had significantly increased cellular viability with no effect on cell damage. Total reactive antioxidant potential (TRAP) levels measured for this alkaloid showed an antioxidant potential three times higher than Trolox, which acts as a peroxyl radical scavenger. Moreover, boldine prevented the increase in lipoperoxidation levels induced by ischemia, but higher concentrations potentiated this parameter. These results confirm the potent antioxidant properties of this alkaloid, and add evidence to support the need for further investigations in order to confirm the potential pro-oxidant effects of boldine at higher doses.

The effect of undernutrition during suckling on phosphoryl serine levels was investigated in rats. Young rats were undernourished by suckling their lactating mothers fed an 8% (by weight) protein diet. Mothers of normal rats were fed a 25% protein diet. After weaning (21 d), normal and undernourished rats were fed a 25% protein diet until 90-120 d of age. Previously we observed that undernutrition during suckling decreases the levels of phosphoryl serine in nuclear proteins from the whole brain of adult rats. In this paper we report more specifically on the effect of undernutrition during this period on the levels of phosphoryl serine in nuclear and synaptosomal proteins from the cerebral cortex of young and nutritionally rehabilitated adult rats. In young, undernourished rats the levels of phosphoryl serine in nuclear proteins were significantly lower (28%) than those in control animals. This alteration persisted in nutritionally rehabilitated adult rats. Serine and protein levels in cerebral cortex were the same in all groups. No alteration in the levels of phosphoryl serine in the synaptosomal fraction was observed.

Stroke syndromes are a major cause of disability in middle and later life resulting in severe neuronal degeneration and loss of brain functions. In situations with energy failure, glutamate transport is impaired and high levels of this amino acid accumulate on the synaptic cleft. Our group has showed that guanosine exerts neuroprotection against neurotoxicity situations. The aim of this work is draw a post-ischemic profile of glutamate uptake and cell damage using an oxygen and glucose deprivation model (OGD) in hippocampal slices from young (P10) and adult (P60) rats, analyzing guanosine effect. OGD decreases glutamate uptake in both ages and recovery times, although decrease in cell viability was only observed 1 and 3 h after OGD in young and adult animals, respectively. Guanosine partially protected cell damage from 1 h in P10 and at 3 h in P60 rats and avoided glutamate uptake decrease from P10 rats at 3 h. The impairment of glutamate transporters since immediately after the insult observed here is probably due to an energetic failure; loss of cell viability was only observed from 1 h after OGD. The mechanism by which guanosine acts in the 'ischemic' model used here is still unknown, but evidence leads to its antiapoptotic effect.