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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.

Background Stroke is a leading cause of death and disability worldwide. A major factor in brain damage following ischemia is excitotoxicity caused by elevated levels of the neurotransmitter glutamate. In the brain, glutamate homeostasis is a primary function of astrocytes. Amburana cearensis has long been used in folk medicine and seed extract obtained with dichloromethane (EDAC) have previously been shown to exhibit cytoprotective activity in vitro. The aim of the present study was to analyse the activity of EDAC in hippocampal brain slices. Methods We prepared a dichloromethane extract (EDAC) from A. cearensis seeds and characterized the chemical constituents by 1H and 13C-NMR. Hippocampal slices from P6-8 or P90 Wistar rats were used for cell viability assay or glutamate uptake test. Hippocampal slices from P10-12 transgenic mice SOX10-EGFP and GFAP-EGFP and immunofluorescence for GS, GLAST and GLT1 were used to study oligodendrocytes and astrocytes. Results Astrocytes play a critical role in glutamate homeostasis and we provide immunohistochemical evidence that in excitotoxicity EDAC increased expression of glutamate transporters and glutamine synthetase, which is essential for detoxifying glutamate. Next, we directly examined astrocytes using transgenic mice in which glial fibrillary acidic protein (GFAP) drives expression of enhanced green fluorescence protein (EGFP) and show that glutamate excitotoxicity caused a decrease in GFAP-EGFP and that EDAC protected against this loss. This was examined further in the oxygen–glucose deprivation (OGD) model of ischemia, where EDAC caused an increase in astrocytic process branching, resulting in an increase in GFAP-EGFP. Using SOX10-EGFP reporter mice, we show that the acute response of oligodendrocytes to OGD in hippocampal slices is a marked loss of their processes and EDAC protected oligodendrocytes against this damage. Conclusion This study provides evidence that EDAC is cytoprotective against ischemia and glutamate excitotoxicity by modulating astrocyte responses and stimulating their glutamate homeostatic mechanisms.

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].

Alzheimer's disease is a neurodegenerative disorder characterized by extracellular deposition of amyloid-β (Aβ) peptide and hyperphosphorylation of Tau protein, which ultimately leads to the formation of intracellular neurofibrillary tangles and cell death. Increasing evidence indicates that genistein, a soy isoflavone, has neuroprotective effects against Aβ-induced toxicity. However, the molecular mechanisms involved in its neuroprotection are not well understood. In this study, we have established a neuronal damage model using retinoic-acid differentiated SH-SY5Y cells treated with different concentrations of Aβ25-35 to investigate the effect of genistein against Aβ-induced cell death and the possible involvement of protein kinase B (PKB, also termed Akt), glycogen synthase kinase 3β (GSK-3β), and Tau as an underlying mechanism to this neuroprotection. Differentiated SH-SY5Y cells were pre-treated for 24 hr with genistein (1 and 10 nM) and exposed to Aβ25-35 (25 μM), and we found that genistein partially inhibited Aβ induced cell death, primarily apoptosis. Furthermore, the protective effect of genistein was associated with the inhibition of Aβ-induced Akt inactivation and Tau hyperphosphorylation. These findings reinforce the neuroprotective effects of genistein against Aβ toxicity and provide evidence that its mechanism may involve regulation of Akt and Tau proteins.

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.

Several environmental factors affect child development, such as the intrauterine environment during the embryonic and fetal development and early postnatal environment provided by maternal behavior. Although mechanistic effects of maternal exercise on offspring health improvement are not yet completely understood, the number of reports published demonstrating the positive influence of maternal exercise have increase. Herein, we addressed issues related to early postnatal environment provided by maternal behavior and early developmental physical landmarks, sensorimotor reflexes, and motor movements ontogeny. In brief, adult female rats underwent involuntary swimming exercise, in a moderated intensity, one week before mating and throughout pregnancy, 30 min a day, 5 days a week. Maternal exercised dams have unchanged gestational outcomes compared to sedentary dams. We found no differences concerning the frequency of pup-directed behavior displayed by dams. However, sedentary dams displayed a poorer pattern of maternal care quality during dark cycle than exercised dams. Physical landmarks and sensorimotor reflexes development of female and male littermates did not differ between maternal groups. Developmental motor parameters such as immobility, lateral head movements, head elevation, pivoting, rearing with forelimb support and crawling frequencies did not differ between groups. Pups born to exercised dams presented higher frequency of walking and rearing on the hind legs. These data suggest that female and male littermates of exercised group present a high frequency of exploratory behavior over sedentary littermates. Taken together, the present findings reinforce that maternal exercise throughout pregnancy represent a window of opportunity to improve offspring’s postnatal health.

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.

Doxazosin is an important drug used for treating hypertension and prostatic hyperplasia. An enantiomeric mixture (R)/(S) doxazosin mesylate was complete characterized by NMR and additionally FT-IR spectroscopy techniques. Were performed different NMR experiments, such as APT, HSQC, and HMBC in order to confirm the NMR signals assignments. All the hydrogens and the most of carbons atoms were assigned.

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 (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).

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.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5 % fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5 % FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5 % FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma. Fil: Maier Gaelzer, Mariana. Universidade Federal Do Rio Grande Do Sul. Instituto de Ciências Básicas da Saúde. Departamento de Bioquímica; Brasil Fil: Silva dos Santos, Mariana. Universidade Federal Do Rio Grande Do Sul. Instituto de Ciências Básicas da Saúde. Departamento de Bioquímica; Brasil Fil: Paranhos Coelho, Bárbara. Universidade Federal Do Rio Grande Do Sul. Instituto de Ciências Básicas da Saúde. Departamento de Bioquímica; Brasil Fil: de Quadros, Alice Hoffman. Universidade Federal Do Rio Grande Do Sul. Instituto de Ciências Básicas da Saúde. Departamento de Bioquímica; Brasil Fil: Simão, Fabrício. Harvard Medical School; Estados Unidos Fil: Usach, Vanina. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Guma, Fátima Costa Rodrigues. Universidade Federal Do Rio Grande Do Sul. Instituto de Cs.basicas Da Saude. Dept.de Bioquímica; Brasil Fil: Setton, Clara Patricia. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lenz, Guido. Universidade Federal do Rio Grande do Sul; Brasil Fil: Salbego, Christianne G.. Universidade Federal Do Rio Grande Do Sul. Instituto de Ciências Básicas da Saúde. Departamento de Bioquímica; Brasil

INTRODUCTION: Biochemistry studies chemical transformations in uni or multicellular living beings. It analyzes physiological processes and evaluates their modifications in pathological conditions. It assists in diagnosis and pharmacological intervention of several diseases, characterizing its importance in health area. In Pharmacy graduation course (UFRGS) until 2017 there were two disciplines - Biochemistry I and Biochemistry II - offered by the Biochemistry Department, ICBS. Both were compulsory, present in third and fourth curriculum stages, had eight credits, distributed in theoretical, theoretical-practical and practical contents. OBJECTIVES: The purpose of this work was to report the development, since the second half of 2014 year, of this project which offered extra class assistance to students with difficulty to understand the Biochemistry I contents. MATERIALS AND METHODS: The project was developed weekly by student-monitor, according to the demand of registered students. The reviewed contents were: amino acids, peptides, proteins, enzymes, biological oxidation, chemistry and carbohydrate metabolism. The activities were previously commented by the participating teachers and monitor. The classroom or distance methodologies consisted of: a) discussion of thematic doubts between students and the monitor; b) resolution of pertinent exercises; c) navigation guided in learning objects. DISCUSSION AND RESULTS: One of the goals of the reinforcement project was to reduce the failure rate of students enrolled in the discipline to 25%. We observed that this objective was surpassed in the semester 2015-1, where the percentage of disapproval fell to 8.5%. But in 2015-2 this percentage rose to 31%. However, it is worth mentioning the massive approval of the participating students during the six semesters in which the project was developed (70-85%). CONCLUSION: These data reveal the project efficiency and the participants’ commitment. We intend to continue the reinforcement activities, as an additional tool, for the appropriation of basic Biochemistry knowledge by the future pharmaceutical professional.

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.

Resveratrol, a natural polyphenolic compound, has attracted considerable interest for its anti-inflammatory and neuroprotective properties. However, the biological effects of resveratrol appear strongly limited because it is photosensitive, easily oxidized, and has unfavorable pharmacokinetics. The present study aimed to elucidate the effect of resveratrol on Abeta-triggered neuroinflammation by comparing the effects of free resveratrol (RSV) treatment with those of treatment with resveratrol-loaded lipid-core nanocapsules (RSV-LNC). Organotypic hippocampal cultures were stimulated by Abeta1-42 with or without different concentrations of RSV or RSV-LNC. We found that Abeta triggered a harmful neuroinflammation process in organotypic hippocampal cultures. Pre- and co-treatments with RSV-LNC were able to protect cultures against ROS formation and cell death induced by Abeta, possibly through sustained blocking of TNF-alpha, IL-1beta, and IL-6 release. Furthermore, RSV-LNC was able to increase IL-10 release even in the presence of Abeta and prevent or decrease both glial and JNK activation. On the other hand, both pre- and co-treatment with RSV exhibited a lower ability to prevent or decrease neuroinflammation, ROS formation, and cell death, and failed to increase IL-10 release. Our findings suggest that modulation of neuroinflammation through a combination of resveratrol and a lipid-core nanocapsule-based delivery system might represent a promising approach for preventing or delaying the neurodegenerative process triggered by Abeta. The results open new vistas to the interplay between inflammation and amyloid pathology.

In 2009, the Creation Group of Educational Objects in Biochemistry (GCOEB) started to organize virtually practical-laboratory activities in basic Biochemistry for undergraduate students of Pharmacy Course. This occurred through presentation type files (power point) with detailed photos of technical procedures, reagents, equipment and possible results, all associated with challenging questions. These activities are being gradually transformed into virtual versions of the Flash program, mixing photos, cartoons and proposed interactivities. These simulations aim to help the students in the consolidation of the theoretical and practical biochemical knowledge. We believe that these pedagogic tools support the learning of Biochemistry, increase the scientific curiosity, stimulate the presence in the laboratory and never replace it. ( www.ufrgs.br/gcoeb/ )

The development of novel therapeutic strategies to treat gliomas remains critical as a result of the poor prognoses, inef-. ficient therapies and recurrence associated with these tumors. In this context, biodegradable nanoparticles are emerging as efficient drug delivery systems for the treatment of difficult-to-treat diseases such as brain tumors. In the current study, we evaluated the antiglioma effect of trans-resveratrol-loaded lipid-core nanocapsules (RSV-LNC) based on in vitro (C6 glioma cell line) and in vivo (brain-implanted C6 cells) models of the disease. In vitro, RSV-LNC decreased the viability of C6 glioma cells to a higher extent than resveratrol in solution. Interestingly, RSV-LNC treatment was not cytotoxic to hippocampal organotypic cultures, a model of healthy neural cells, suggesting selectivity for cancer cells. RSV-LNC induced losses in glioma cell viability through induction of apoptotic cell death, as assessed by Annexin-FITC/PI assay, which was preceded by an early arrest in the S and G1 phases of the cell cycle. In brain-implanted C6 tumors, treatment with RSV-LNC (5 mg/kg/day, i.p.) for 10 days promoted a marked decrease in tumor size and also reduced the incidence of some malignant tumor-associated characteristics, such as intratumoral hemorrhaging, intratumoral edema and pseudopalisading, compared to resveratrol in solution. Taken together, the results presented herein suggest that nanoencapsulation of resveratrol improves its antiglioma activity, thus providing a provocative foundation for testing the clinical usefulness of nanoformulations of this natural compound as a new chemotherapeutic strategy for the treatment of gliomas.

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.

INTRODUCTION: The normal blood glucose in rats is 100 mg glucose / 100 ml of blood plasma. After a 24hhour fast, a decrease around 30% of this value occurs. This reduction can reach 45% in a prolonged fast. Several processes controlled by neurohormonal mechanisms prevent a more pronounced decrease in blood glucose of animals subjected to fast. These processes are hepatic glycogenolysis, hepatic gluconeogenesis, decrease of glucose utilization and increase of fatty acids use by muscle tissue, and ketone bodies utilization by the central nervous system. OBJECTIVES: This study presents a learning object, mediated by computer, which simulates the determination of blood glucose (glycemia) obtained from fed rats and from 24-hour fasted rats. MATERIALS AND METHODS: At first, cartoons were planned in order to show the biochemical and methodology fundamentals. The most representative images were selected, edited and inserted into an animation developed with the aid of the Adobe ® Flash 8 software. DISCUSSION AND RESULTS: The animated simulation of a standard glucose curve, followed by virtual evaluation of glucose in blood plasma samples were developed, associated with some questions. This object has been used by students of Biochemistry I (Pharmacy-UFRGS) since second semester of 2009. The navigation features, design and interactivity have been evaluated as excellent by about 80% of them. CONCLUSION: Therefore, this learning object can be considered an adequate teaching resource as well as an innovative support in the construction of theoretical and practical knowledge of Biochemistry. Available at: http://www.ufrgs.br/gcoeb/dosagemglicemia/

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.

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK-3β) and cAMP response element-binding protein (CREB) through phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four-vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK-3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3-K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK-3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3-K, compromised the neuroprotective effect of resveratrol and decreased the level of p-Akt, p-GSK-3β and p-CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt, GSK-3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3-K/Akt signaling pathway, subsequently downregulating expression of GSK-3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.

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.

Several studies have reported that orally ingested trans-resveratrol is extensively metabolized in the enterocyte before it enters the blood and target organs. Additionally, trans-resveratrol is photosensitive, easily oxidized and presents unfavorable pharmacokinetics. Therefore, it is of great interest to stabilize trans-resveratrol in order to preserve its biological activities and to improve its bioavailability in the brain. Here, trans-resveratrol was loaded into lipid-core nanocapsules and analyzed for particle size, polydispersity and zeta potential. The nanocapsule distribution in brain tissue was evaluated by intraperitoneal (i.p.) and gavage routes in healthy rats. The lipid-core nanocapsules had a mean diameter of 241 nm, a polydispersity index of 0.2, and a zeta potential of -15 mV. No physical changes were observed after 1, 2 and 3 months of storage at 25 degrees C. Lipid-core nanocapsules showed high entrapment of trans-resveratrol and displayed a higher trans-resveratrol concentration in the brain, the liver and the kidney after daily i.p. or gavage administration than that observed for the free trans-resveratrol. Because trans-resveratrol is a potent cyclooxygenase-1 inhibitor, gastrointestinal damage was evaluated. The animals that were administered with trans-resveratrol-loaded lipid-core nanocapsules showed significantly less damage when compared to those administered with free trans-resveratrol. In summary, lipid-core nanocapsules exhibited great trans-resveratrol encapsulation efficiency. trans-Resveratrol-loaded lipid-core nanocapsules increased the concentration of trans-resveratrol in the brain tissue. Gastrointestinal safety was improved when compared with free trans-resveratrol. Thus, trans-resveratrol-loaded lipid-core nanocapsules may be used as an alternative potential therapeutic for several diseases including Alzheimer's disease.

Targeted treatment of diseases of the central nervous system remains problematic due to the complex pathogenesis of these disorders and the difficulty in drug delivery. Here we investigate the neuroprotective effect of indomethacin-loaded nanocapsules (IndOH-NC) in an in vitro model of ischemia. For this purpose we used organotypic hippocampal cultures exposed to oxygen-glucose deprivation (OGD). When the cultures were exposed to 60 min of OGD, 54.5±14.7% of the total area of the hippocampal slices was labeled with propidium iodide. On the other hand, when the cultures were treated with 50 or 100 μM of IndOH-NC the cell death was significantly reduced to 31±7% (P

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.

Cromatografia consiste num método de análise de componentes de uma misturacomplexa baseada em diferentes critérios: adsorbilidade, solubilidade, massa molecular,carga iônica e afinidade. É um importante conteúdo da disciplina Bioquímica. Estetrabalho relata o desenvolvimento e a validação de um software de simulaçãolaboratorial denominado Cromatografia Ascendente em Papel de Aminoácidos usando aferramenta o Adobe® Flash® CS3. A organização e a coleta do material multimídiaocorreram no semestre 2008/2. As imagens mais representativas foram inseridas noobjeto de aprendizagem A primeira aplicação deste objeto foi realizada em 2009/1, naBioquímica I (Farmácia-UFRGS). Com base nesta experiência, uma nova versão foidesenvolvida a qual foi utilizada pelos alunos da mesma disciplina em 2009/2. Apósuma aula expositiva de 50 minutos (teórico-prática), os alunos dos dois semestres foramdivididos em dois grupos. O grupo I respondeu a um questionário sobre conceitosbásicos de cromatografia e depois utilizou o software. O grupo II foi submetido aoprotocolo inverso. Os grupos também avaliaram os aspectos técnicos daanimação/simulação e a atividade realizada. Associando os resultados das duasaplicações (2009/1 e 2009/2), o objeto de aprendizagem em questão pode serconsiderado válido como suporte para o ensino prático de Bioquímica básica.

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.

Glutamate is an excitatory neurotransmitter involved in neuronal plasticity and neurotoxicity. Chronic stress produces several physiological changes on the spinal cord, many of them presenting sex-specific differences, which probably involve glutamatergic system alterations. The aim of the present study was to verify possible effects of exposure to chronic restraint stress and 17beta-estradiol replacement on [3H]-glutamate release and uptake in spinal cord synaptosomes of ovariectomized (OVX) rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided in controls and chronically stressed. Restraint stress or estradiol had no effect on [3H]-glutamate release. The chronic restraint stress promoted a decrease and 17beta-estradiol induced an increase on [3H]-glutamate uptake, but the uptake observed in the restraint stress +17beta-estradiol group was similar to control. Furthermore, 17beta-estradiol treatment caused a significant increase in the immunocontent of the three glutamate transporters present in spinal cord. Restraint stress had no effect on the expression of these transporters, but prevented the 17beta-estradiol effect. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in spinal cord plasticity following repeated stress exposure, and that 17beta-estradiol levels may affect chronic stress effects in this structure.

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.