Cannabidiol injected into the bed nucleus of the stria terminalis modulates baroreflex activity through 5-HT(1A) receptors

Cannabidiol (CBD) is a non-psychotomimetic constituent of the Cannabis sativa plant that inhibits behavioral and cardiovascular responses to aversive situations. facilitating 5-HT(1A)-mediated neurotransmission. Previous results from our group suggest that the bed nucleus of the stria terminalis (BNST) may be involved in CBD`s anti-aversive effects. To investigate whether the cardiovascular effects of the CBD could involve a direct drug effect on the BNST, we evaluated the effects of CBD microinjection into this structure on baroreflex activity. We also verified whether these effects were mediated by the activation of 5-HT(1A) receptors. Bilateral microinjection of CBD (60 nmol/100 nL) into the BNST increased the bradycardiac response to arterial pressure increases. However, no changes were observed in tachycardiac responses evoked by arterial pressure decreases. Pretreatment of the BNST with the selective 5-HT(1A) receptor antagonist WAY100635 (0.37 nmol/100 nL) prevented CBD effects on the baroreflex activity. Moreover, microinjection of the 5-HT(1A) receptor agonist 8-OH-DPAT (4 nmol/100 nL) caused effects that were similar to those observed after the microinjection of CBD, which were also blocked by pretreatment with WAY100635. In conclusion, the present studies show that the microinjection of CBD into the BNST has a facilitatory influence on the baroreflex response to blood pressure increases, acting through the activation of 5-HT(1A) receptors. (C) 2010 Elsevier Ltd. All rights reserved.

Exploring cinnamic acid scaffold: development of promising neuroprotective lipophilic antioxidants

New lipophilic hydroxycinnamic acid based derivatives were designed and synthesized and their antioxidant and neuroprotective activities evaluated. The chemical modification introduced in the cinnamic acid scaffold leads to compounds with amplified lipophilicity and in general with increased antioxidant activity when compared to natural models (caffeic and ferulic acids). The compounds did not display cytotoxicity and present a significant neuroprotective effect against 6-OH-DA induced damage to SH-SY5Y cells. Compound 6 stands out as an efficient radical scavenger and iron(II) chelator that ensures drug-like properties. Moreover, neuroprotection against oxidative damage was observed even at low concentration (1 μM). Therefore, compound 6 developed by a biology-oriented approach displays a combination of important features for a further optimization process that will generate a new effective antioxidant with therapeutic application for oxidative-stress-related events, namely neurodegenerative diseases.

Development of polymeric nanoparticles containing neuroprotective compounds of Hypericum perforatum

Tese de Doutoramento em Biologia das Plantas - MAP BIOPLANT

Flavonoids: antioxidants or signalling molecules?

Many studies are accumulating that report the neuroprotective, cardioprotective, and chemopreventive actions of dietary flavonoids. While there has been a major focus on the antioxidant properties, there is an emerging view that flavonoids, and their in vivo metabolites, do not act as conventional hydrogen-donating antioxidants but may exert modulatory actions in cells through actions at protein kinase and lipid kinase signalling pathways. Flavonoids, and more recently their metabolites, have been reported to act at phosphoinositide 3-kinase (PI 3-kinase), Akt/protein kinase B (Akt/PKB), tyrosine kinases, protein kinase C (PKC), and mitogen activated protein kinase (MAP kinase) signalling cascades. Inhibitory or stimulatory actions at these pathways are likely to affect cellular function profoundly by altering the phosphorylation state of target molecules and by modulating gene expression. A clear understanding of the mechanisms of action of flavonoids, either as antioxidants or modulators of cell signalling, and the influence of their metabolism on these properties are key to the evaluation of these potent biomolecules as anticancer agents, cardioprotectants, and inhibitors of neurodegeneration (C) 2004 Elsevier Inc. All rights reserved.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) – Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

Is glutathione an important neuroprotective effector molecule against amyloid beta toxicity?

Cellular thiols are critical moieties in signal transduction, regulation of gene expression, and ultimately are determinants of specific protein activity. Whilst protein bound thiols are the critical effector molecules, low molecular weight thiols, such as glutathione, play a central role in cytoprotection through (1) direct consumption of oxidants, (2) regeneration of protein thiols and (3) export of glutathione containing mixed disulphides. The brain is particularly vulnerable to oxidative stress, as it consumes 20% of oxygen load, contains high concentrations of polyunsaturated fatty acids and iron in certain regions, and expresses low concentrations of enzymic antioxidants. There is substantial evidence for a role for oxidative stress in neurodegenerative disease, where excitotoxic, redox cycling and mitochondrial dysfunction have been postulated to contribute to the enhanced oxidative load. Others have suggested that loss of important trophic factors may underlie neurodegeneration. However, the two are not mutually exclusive; using cell based model systems, low molecular weight antioxidants have been shown to play an important neuroprotective role in vitro, where neurotrophic factors have been suggested to modulate glutathione levels. Glutathione levels are regulated by substrate availability, synthetic enzyme and metabolic enzyme activity, and by the presence of other antioxidants, which according to the redox potential, consume or regenerate GSH from its oxidised partner. Therefore we have investigated the hypothesis that amyloid beta neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels. Using PC12 cells as a model system, amyloid beta 25-35 caused a shift in DCF fluorescence after four hours in culture. This fluorescence shift was attenuated by both desferioxamine and NGF. After four hours, cellular glutathione levels were depleted by as much as 75%, however, 24 hours following oxidant exposure, glutathione concentration was restored to twice the concentration seen in controls. NGF prevented both the loss of viability seen after 24 hours amyloid beta treatment and also protected glutathione levels. NGF decreased the total cellular glutathione concentration but did not affect expression of GCS. In conclusion, loss of glutathione precedes cell death in PC12 cells. However, at sublethal doses the surviving fraction respond to oxidative stress by increasing glutathione levels, where this is achieved, at least in part, at the gene level through upregulation of GCS. Whilst NGF does protect against oxidative toxicity, this is not achieved through upregulation of GCS or glutathione.

Therapeutic azulenyl nitrone antioxidants

The reactions of nitrones with free radicals have been widely studied both in vitro and in vivo. In comparison to classical chain-breaking phenolic antioxidants (such as Vitamin E and butylated hydroxytoluene [BHT]), conventional phenyl-substituted nitrones have much higher oxidation potentials. Azulenyl-substituted nitrones have lower oxidation potentials than conventional nitrones and react efficiently with free radicals in vitro and in vivo. The design and synthesis of novel azulenyl nitrones with yet lower oxidation potentials, prepared from commercially available guaiazulene, has produced several 1,2-trans -bis-azulenyl ethene compounds with enhanced antioxidant activity. A convenient 1H NMR-based assay for assessing the potency of chain-breaking antioxidants has shown these novel nitrones to be more than 300 times more potent in inhibiting the free radical-mediated aerobic peroxidation of cumene than α-phenyl-N-tert-butyl nitrone (PBN) and the experimental stroke drug NXY-059. The low oxidation potential of these novel nitrones and the stability of the corresponding radical cation have been implicated in the explanation of the increased antioxidant potency of these second generation azulenyl nitrones. Based on the results of these in vitro studies, the first of these novel compounds, stilbazulenyl nitrone (STAZN), was investigated in animal models of disease known to involve free radical-mediated pathology. In view of STAZN's marked lipophilicity and anticipated blood brain barrier permeability, neurodegenerative conditions were investigated. All animal experiments were performed at the University of Miami by members of the Ginsberg research group. STAZN was neuroprotective in traumatic brain injury in rats. It also provided exceptional neuroprotection in an animal model of stroke. The concentration of STAZN required for neuroprotection was 300–600 times less than doses of PBN or NXY-059 required for similar effect. Thus, the benefits of greater antioxidant potency sought by lowering the oxidation potential of nitrones appear to have been reaped both in vitro and in vivo. In spite of the challenges and difficulties in understanding free radical-mediated pathology, this work establishes that considerations such as redox potential and lipophilicity can provide a very fruitful rationale for the design of therapeutic azulenyl nitrone antioxidants. ^

Contuse lesion of the rat spinal cord of moderate intensity leads to a higher time-dependent secondary neurodegeneration than severe one - An open-window for experimental neuroprotective interventions

Secondary neurodegeneration takes place in the surrounding tissue of spinal cord trauma and modifies substantially the prognosis, considering the small diameter of its transversal axis. We analyzed neuronal and glial responses in rat spinal cord after different degree of contusion promoted by the NYU Impactor. Rats were submitted to vertebrae laminectomy and received moderate or severe contusions. Control animals were sham operated. After 7 and 30 days post surgery, stereological analysis of Nissl staining cellular profiles showed a time progression of the lesion volume after moderate injury, but not after severe injury. The number of neurons was not altered cranial to injury. However, same degree of diminution was seen in the caudal cord 30 days after both severe and moderate injuries. Microdensitometric image analysis demonstrated a microglial reaction in the white matter 30 days after a moderate contusion and showed a widespread astroglial reaction in the white and gray matters 7 days after both severities. Astroglial activation lasted close to lesion and in areas related to Wallerian degeneration. Data showed a more protracted secondary degeneration in rat spinal cord after mild contusion, which offered an opportunity for neuroprotective approaches. Temporal and regional glial responses corroborated to diverse glial cell function in lesioned spinal cord. (C) 2007 Elsevier Ltd. All rights reserved.

Effect of Antioxidants and Corrosion Inhibitor Additives on the Quenching Performance of Soybean Oil

Cooling curve analysis was used to evaluate the effect of corrosion inhibitor additives and antioxidants on the quenching properties of soybean oil. The results showed that addition of corrosion inhibitors provided significant changes in the cooling curve behavior and of the yellow metal corrosion inhibitors evaluated tolyltriazole exhibits the greatest rate acceleration of heat transfer. However, the presence of antioxidants did not exhibit a significant effect on quenching properties of soybean oil. (C)2010 Journal of Mechanical Engineering. All rights reserved.

VEGETABLE OIL STRUCTURE AND ANTIOXIDANTS

Five vegetable oils: canola, soybean, corn, cottonseed and sunflower oils were characterized with respect to their composition by gas chromatography and viscosity. The compositions of the vegetable oils suggest that they exhibit substantially different propensity for oxidation following the order of: canola < corn < cottonseed < sunflower approximate to soybean. Viscosities at 40 degrees C and 100 degrees C and the viscosity index (VI) values were determined for the vegetable oils and two petroleum oil quenchants: Microtemp 157 (a conventional slow oil) and Microtemp 153B (an accelerated or fast oil). The kinematic viscosities of the different vegetable and petroleum oils at 40 degrees C were similar. The VI values for the different vegetable oils were very close and varied between 209-220 and were all much higher than the VI values obtained for Microtemp 157 (96) and Microtemp 153B (121). These data indicate that the viscosity variations of these vegetable oils are substantially less sensitive to temperature variation than are the parafinic oil based Microtemp 157 and Microtemp 153B. Although these data suggest that any of the vegetable oils evaluated could be blended with minimal impact on viscosity, the oxidative stability would surely be substantially impacted. Cooling curve analysis was performed on these vegetable oils at 60 degrees C under non-agitated conditions. These results were compared with cooling curves obtained for Microtemp 157, a conventional, unaccelerated petroleum oil, and Microtemp 153B, an accelerated petroleum oil under the same conditions. The results showed that cooling profiles of the different vegetable oils were similar as expected from the VI values. However, no boiling was observed wit any of the vegetable oils and heat transfer occurs only by convection since there is no full-film boiling and nucleate boiling process as typically observed for petroleum oil quenchants, including those of this study. Therefore, high-temperature cooling is considerable faster for vegetable oils as a class. The cooling properties obtained suggest that vegetable oils would be especially suitable fur quenching low-hardenability steels such as carbon steels.

Wine industry residues extracts as natural antioxidants in raw and cooked chicken meat during frozen storage

The effect of Isabel (IGE) and Niagara (NGE) grape seed and peel extracts on lipid oxidation, instrumental colour, pH and sensory properties of raw and cooked processed chicken meat stored at -18 degrees C for nine months was evaluated. The pH of raw and cooked samples was not affected by the addition of grape extracts. IGE and NGE were effective in inhibiting the lipid oxidation of raw and cooked chicken meat, with results comparable to synthetic antioxidants. The extracts caused alterations in colour, as evidenced by the instrumental (darkening and lower intensity of red and yellow colour) and sensory results of cooked samples. In the sensory evaluation of odour and flavour, IGE produced satisfactory results, which did not differ from synthetic antioxidants. These findings suggest that the ICE and NGE are effective in retarding lipid oxidation of raw and cooked chicken meat during frozen storage. (c) 2011 Elsevier Ltd. All rights reserved.

Natural antioxidants protecting irradiated beef burgers from lipid oxidation

The effect of butylated hydroxytoluene/butylated hydroxyanisole blend (BHT/BHA), and rosemary and oregano extracts, added individually or in combination, on lipid oxidation and fatty acid composition was investigated on irradiated frozen beef burgers. Irradiation treatment was carried out using a (60)CO semi-industrial irradiator at doses of 6, 7 and 8 kGy, and then the treated meat samples were stored at -20 degrees C for 90 days. Lipid oxidation and fatty acid composition of beef samples were evaluated by measurement of TBARS and gas chromatography, respectively. The results of the experiment showed that rosemary extract, applied alone and in combination with either BHT/BHA or oregano extracts was more effective in maintaining a low oxidation level in the samples compared to oregano extract used individually or in combination with BHT/BHA. Results also showed no significant differences (p > 0.05) in fatty acid composition in all analyzed samples, although some changes in terms of decreased PUFA and MUFA, beside of slight increase of SFA content were observed. However, these differences do not correlate positively neither with the irradiation dose nor the type of antioxidant. Thus, there is a potential application of these spices as natural antioxidants in irradiated meats. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of natural antioxidants on the lipid profile of electron beam-irradiated beef burgers

The purpose of this study was to assess the efficacy of rosemary and oregano extracts in avoiding oxidative changes in beef burgers, and to evaluate the fatty acid profile of these products after electron beam exposition. Extracts, individually or in combination, were added to beef burgers and compared to synthetic antioxidants commonly used in food (butylated hydroxytoluene, butylated hydroxyanisole). The ground beef were submitted to electron beam irradiation at doses of 0, 3.5 and 7 kGy, and stored for 90 days. At regular time intervals, lipid oxidation and fatty acid composition were evaluated through measurement of thiobarbituric acid-reactive substances (TBARS) and gas chromatography, respectively. The results indicate that, although the irradiation process triggers an increase in the lipid oxidation ratio expressed by TBARS values, great changes in the fatty acid profiles were not observed; instead, they continued to present characteristics very similar to that of non-irradiated beef. Thus, as irradiation doses of up to 7 kGy for frozen meat can make foods safe from foodborne pathogens, natural antioxidants derived from spices are able to reduce and avoid lipid changes that may cause a deterioration of the sensory quality of these foods, and these natural extracts offer a good choice for replacing synthetic additives.

Consumer`s evaluation of the effects of gamma irradiation and natural antioxidants on general acceptance of frozen beef burger

The effect of addition of rosemary and oregano extracts on the sensory quality of irradiated beef burger was investigated. Batches of beef burgers were prepared with 400 ppm of rosemary or oregano extract and a group prepared with 200 ppm of synthetic butyl-hydroxytoluene (BHT)/butyl-hydroxy-anisol (BHA) was used as a control. Half of each formulation was irradiated at the maximum dose allowed for frozen meat (7 kGy). Samples were kept under frozen conditions (-20 degrees C) during the whole storage period, including during irradiation. Two analyses were performed after 20 and 90 days to verify the influence of the addition of the different types of antioxidants and the effect of irradiation and storage time on the acceptance of the product. Thirty-three and thirty-four untrained panelists were invited to participate in the first and second test, respectively. A structured hedonic scale ranging from 1 to 9 points was used in both analyses. BHT/BHA formulation obtained the highest score (6.73) and regarding the natural antioxidants, oregano received better acceptance (6.36). Irradiated samples formulated with oregano received a lower score, 6.03 in the first test and 5.06 in the second one, compared to the non-irradiated sample (6.36 and 5.79). In the second test (90 days), the sample formulated with BHT/BHA and which was irradiated received a higher score (6.59) when compared to the non-irradiated one (5.85). In both tests, the irradiated samples formulated with rosemary extract obtained a better score compared to the non-irradiated one, the scores being 5.00-3.82 and 5.00-3.76 in the first and second test, respectively. Our results allowed us to conclude that the natural antioxidants, rosemary and oregano extracts, present a good alternative for replacing synthetic additives in food industries, and that the irradiation process, in some cases, may help to enhance the sensory quality of food. (C) 2008 Elsevier Ltd. All rights reserved.

Neuroprotective Effects of Diazepam, Carbamazepine, Phenytoin and Ketamine after Pilocarpine-induced Status Epilepticus

Cell damage and spatial localization deficits are often reported as long-term consequences of pilocarpine-induced status epilepticus. In this study, we investigated the neuroprotective effects of repeated drug administration after long-lasting status epilepticus. Groups of six to eight Wistar rats received microinjections of pilocarpine (2.4 mg/mu l, 1 mu l) in the right dorsal hippocampus to induce a status epilepticus, which was attenuated by thiopental injection (35 mg/kg, i.p.) 3 hrs after onset. Treatments consisted of i.p. administration of diazepam, ketamine, carbamazepine, or phenytoin at 4, 28, 52, and 76 hr after the onset of status epilepticus. Two days after the treatments, rats were tested in the Morris water maze and 1 week after the cognitive tests, their brains were submitted to histology to perform haematoxylin and eosin staining and glial fibrillary acidic protein (GFAP) immunofluorescence detection. Post-status epilepticus rats exhibited extensive gliosis and cell loss in the hippocampal CA1, CA3 (70% cell loss for both areas) and dentate gyrus (60%). Administration of all drugs reduced cell loss in the hippocampus, with best effects observed in brains slices of diazepam-treated animals, which showed less than 30% of loss in the three areas and decreased GFAP immunolabelling. Treatments improved spatial navigation during training trials and probe trial, with exception of ketamine. Interestingly, in the probe trial, only diazepam-treated animals showed preference for the goal quadrant. Our data point to significant neuroprotective effects of repeated administration of diazepam against status epilepticus-induced cell damage and cognitive disturbances.