Adenosine and neuroprotection in the temporal lobe epilepsy: from A1 receptor activation to A2A receptor blockade

OBJECTIVE: To characterize the effect of the A2A receptor blockage by the SCH58261 in the seizure modulation and neuroprotection of the brain areas vulnerable to injury by pilocarpine. The effect of SCH58261 was also analyzed in combination with the activation of the A1 receptors by R-Pia. METHODS: Eight groups were studied: Pilo, SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, and respective controls. The number of animals in status epilepticus (SE), the latency to the SE onset and the mortality rate were evaluated. The Fluoro Jade B (FJB) method was performed 24 hours and seven days after SE. RESULTS: The pretreatment with SCH58261, R-Pia and R-Pia+SCH58261 reduced the number of animals in SE, increased the latency to the SE and decreased the mortality rate, compared to pilocarpine treatment. The R-Pia and R-Pia+SCH58261 groups exhibited a reduction in the number of FJB stained cells in CA3 and hilus, 24 hours and seven days after SE, and in the piriform cortex only 24 hours after SE, compared to Pilo group. CONCLUSION: The A2A antagonist demonstrated a potent anticonvulsant effect, while the A1 agonist had a crucial role in the seizure modulation and promoted significant neuroprotection.OBJETIVO: Caracterizar o efeito do bloqueio do receptor A2A pelo SCH58261 na modulação da crise e neuroproteção de áreas cerebrais vulneráveis à lesão por pilocarpina. O efeito do SCH58261 foi também analisado em combinação com a ativação dos receptores A1 por R-Pia. MÉTODOS: Oito grupos foram estudados: Pilo, SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, e seus respectivos controles. O número de animais em status epilepticus (SE), a latência para o início do SE e a taxa de mortalidade foram avaliados. O método de Fluoro Jade B (FJB) foi realizado 24 horas e sete dias após SE. RESULTADOS: O pré-tratamento com SCH58261, R-Pia e R-Pia+ SCH58261 reduziu o número de animais em SE, aumentou a latência para o SE e diminuiu a taxa de mortalidade, comparado ao tratamento com pilocarpina. Os grupos R-Pia e R-Pia+SCH58261 apresentaram uma redução no número de células marcadas com FJB em CA3 e hilo, 24 horas e sete dias após SE, e no córtex piriforme apenas 24 horas após SE, comparado ao grupo Pilo. CONCLUSÃO: O antagonista A2A demonstrou um potente efeito anticonvulsivante, enquanto o agonista A1 teve um papel crucial na modulação da crise e promoveu significante neuroproteção.UNIFESP Disciplina de Neurologia Experimental Departamento de Neurologia e NeurocirurgiaUNIFESP, Disciplina de Neurologia Experimental Depto. de Neurologia e NeurocirurgiaSciEL

Epilepsy and neuroprotection: employment of the RPia during status epilepticus induced by pilocarpine

OBJECTIVE: The aim of this study was to characterize the neuroprotection of the RPia in rats subjected to status epilepticus (SE) induced by pilocarpine (Pilo). METHODS: We evaluated the mismatch between local cerebral glucose utilisation (LCGU) and local cerebral blood flow (LCBF) 4 hours after SE induction. Neuronal loss was evaluated by Fluoro Jade-B (FJB) 24 hours and 90 days after SE. Four groups were studied: Saline, Pilo, RPia+Saline and RPia+Pilo. RESULTS AND CONCLUSIONS: Significant increases in the LCGU were observed in the almost all brain regions of Pilo and RPia+Pilo groups compared to control. However, significant reduction in the LCGU occurred in the substantia nigra pars reticulata and hippocampal formation of RPia+Pilo group versus Pilo. There was significant increase of the LCBF in all the studied areas, comparing the Pilo and RPia+Pilo groups with the control. The increases of LCBF was more intense in rats from RPia+Pilo compared to Pilo, and located mainly in CA2, CA3, dentate gyrus, entorhinal cortex, thalamic nuclei, mammillary body, red nucleus, zone incerta, pontine nucleus and visual cortex. A great number FJB stained cells was observed in the Pilo group and RPia pretreatment reduced the staining in the hippocampal formation, piriform cortex, basolateral amygdala and substantia nigra pars compacta.OBJETIVO: O objetivo desse estudo foi caracterizar a neuroproteção do RPia em ratos submetidos ao status epilepticus (SE) induzido pela pilocarpina (Pilo). MÉTODOS: Avaliou-se o balanço entre utilização local da glicose cerebral (ULGC) e fluxo sanguíneo cerebral local (FSCL) após 4 horas de SE, e a marcação por Fluoro Jade-B (FJB), 24 horas e 90 dias após SE. Quatro grupos foram avaliados: Salina, Pilo, RPia+Salina e RPia+Pilo. RESULTADOS E CONCLUSÃO: Aumentos significantes na ULGC foram observados na maioria das regiões avaliadas nos grupos Pilo e RPia+Pilo quando comparados ao controle. Entretanto, redução significante na ULGC ocorreu na substância negra pars reticulata e giro denteado do grupo RPia+Pilo versus Pilo. Houve aumento significante do FSCL em todas as áreas estudadas, comparando-se os grupos Pilo e RPia+Pilo com o controle. Foi observado um aumento significante do FSCL durante SE em CA2, CA3, giro denteado, córtex entorrinal, corpo mamilar, núcleos talâmicos, núcleo rubro, zona incerta, núcleo oral da ponte e córtex visual, no grupo pré-tratado com RPia comparado ao tratado somente com Pilo. Grande número de células marcadas com FJB foi observado no grupo Pilo e o pré-tratamento com RPia reduziu essa marcação na formação hipocampal, córtex piriforme, amígdala basolateral e substância negra pars compacta.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)UNIFESP Departamento de Neurologia e NeurocirurgiaUniversité Louis Pasteur INSERM Unité 465UNIFESP Centro de Microscopia EletrônicaUNIFESP, Depto. de Neurologia e NeurocirurgiaUNIFESP, Centro de Microscopia EletrônicaSciEL

The preview benefit in single-feature and conjunction search : constraints of visual marking

Previewing distracters enhances the efficiency of visual search. Watson and Humphreys (1997) proposed that the preview benefit rests on visual marking, a mechanism which actively encodes distracter locations at preview and inhibits them afterwards at search. As Watson and Humphreys did, we used a letter–color search task to study constraints of visual marking in conjunction search and near-efficient single-feature search with single-colored and homogeneous distracter letters. Search performance was measured for fixed target and distracter features (block design) and for randomly changed features across trials (random design). In single-feature search there was a full preview benefit for both block and random designs. In conjunction search a full preview benefit was obtained only for the block design; randomly changing target and distracter features disrupted the preview benefit. However, the preview benefit was restored when the distracters were organized in spatially coherent blocks. These findings imply that the temporal segregation of old and new items is sufficient for visual marking in near-efficient single-feature search, while in conjunction search it is not. We propose a supplanting grouping principle for the preview benefit: When the new items add a new color, conjunction search is initialized and attentional resources are withdrawn from the marking mechanism. Visual marking can be restored by a second grouping principle that joins with temporal asynchrony. This principle can be either spatial or feature based. In the case of the latter, repetition priming is necessary to establish joint grouping by color and temporal asynchrony

The composite effect is face-specific in young but not older adults

In studying holistic face processing across the life-span there are only few attempts to separate face-specific from general aging effects. Here we used the complete design of the composite paradigm (Cheung et al., 2008) with faces and novel non-face control objects (watches) to investigate composite effects in young (18–32 years) and older adults (63–78 years). We included cueing conditions to alert using a narrow or a wide attentional focus when comparing the composite objects, and used brief and relaxed exposure durations for stimulus presentation. Young adults showed large composite effects for faces, but none for watches. In contrast, older adults showed strong composite effects for faces and watches, albeit the effects were larger for faces. Moreover, composite effects for faces were larger for the wide attentional focus in both age groups, while the composite effects for watches of older adults were alike for both cueing conditions. Older adults showed low accuracy at the same levels for both types of stimuli when attended and non-attended halves were incongruent. Increasing presentation times improved performance strongly for congruent but not for incongruent composite objects. These findings suggest that the composite effects of older adults reflect substantial decline in the ability to control irrelevant stimuli, which takes effect both in non-face objects and in faces. In young adults, highly efficient attentional control mostly precludes interference of irrelevant features in novel objects, thus their composite effects reflect holistic integration specific for faces or objects of expertise

The face inversion effect in opponent-stimulus rivalry

BACKGROUND: The face inversion effect is regarded as a hallmark of face-specific processing, and can be observed in a large variety of visual tasks. Face inversion effects are also reported in binocular rivalry. However, it is unclear whether these effects are face-specific, and distinct from the general tendency of visual awareness to privilege upright objects. We studied continuous rivalry across more than 600 dominance epochs for each observer, having faces and houses rival against their inverted counterparts, and letting faces rival against houses in both upright and inverted orientation. We found strong inversion effects for faces and houses in both the frequency of dominance epochs and their duration. Inversion effects for faces, however, were substantially larger, reaching a 70:30 distribution of dominance times for upright versus inverted faces, while a 60:40 distribution was obtained for upright versus inverted houses. Inversion effects for faces reached a Cohen's d of 0.85, compared to a value of 0.33 for houses. Dominance times for rivalry of faces against houses had a 60:40 distribution in favor of faces, independent of the orientation of the objects. These results confirm the general tendency of visual awareness to prefer upright objects, and demonstrate the outstanding role of faces. Since effect size measures clearly distinguish face stimuli in opponent-stimulus rivalry, the method is highly recommended for testing the effects of face manipulations against non-face reference objects

Analysis of small molecules using MALDI mass spectrometry

Im Rahmen der vorliegenden Arbeit wurde eine allgemein anwendbare Methode zur Identifizierung und Quantifizierung kleiner Moleküle mittels MALDI-TOF-MS entwickelt. Dabei wurden zahlreiche Analyten, wie unterschiedliche Arzneistoffe, Neurotransmitter und Lebensmittelinhaltsstoffe in verschiedenen Probenmatrizes analysiert. Bei den verwendeten Matrizes wurden mit a-Cyano-4-Hydroxy-Zimtsäure (CHCA) die besten Ergebnisse erzielt. Es zeigte sich jedoch, dass die Probenpräparation wichtiger war als die Wahl der Matrix, da auch mit anderen Matrizes bei optimierter Probenpräparation sensitive Messungen im niedrigen Massenbereich möglich waren. Insbesondere eine schnelle Trocknung des Probenspots, und damit verbunden die Bildung kleiner Kristalle, ist für die Analytik kleiner Moleküle hilfreich. Bei gleichzeitiger Verwendung geringer Matrixkonzentrationen und geringer Laserintensität konnte so der störende Matrixhintergrund minimiert werden. Eine noch stärkere Suppression der Matrixsignale bei gleichzeitiger Anreicherung der Analyten auf dem Probenspot konnte durch eine Dünnschichtpräparation (TLP) mit CHCA und Nitrozellulose erreicht werden. Allerdings war die TLP für eine automatische Quantifizierung kleiner Moleküle nur bedingt geeignet. Die für kleine Moleküle so wichtige Quantifizierung war durch Verwendung einer optimierten schnell trocknenden Dried Droplet Präparation (DDP) möglich. Die Kombination dieser optimierten DDP mit ausreichend aufsummierten Einzelschussspektren bei gleichzeitiger Verwendung eines internen Standards (IS) ermöglichte eine valide Quantifizierung mit guter Präzision, Linearität und Richtigkeit. Dabei erfüllten die quantifizierten Analyten die Vorgaben der FDA für Präzision und Richtigkeit. Diese Quantifizierungsmethode wurde an Mischungen unterschiedlicher Arzneistoffe, sowohl in Standardlösungen als auch in humanem Plasma, erfolgreich durchgeführt. Dabei genügte ein einziger interner Standard, um alle Arzneistoffe der Mischung schnell und sensitiv zu bestimmen (Bestimmungsgrenze 1-5 ng/ml). Weiterhin war es möglich, den Wirkstoff einer pharmazeutischen Tablette ohne aufwändige Probenvorbereitung schnell und genau zu bestimmen. Dass MALDI über eine hohe Salztoleranz verfügt, wurde bei der Bestimmung von Acetylcholin (ACh) und Cholin (Ch) in Mikrodialysaten bestätigt. Trotz des hohen Salzgehalts der Proben (~150 mM) war eine direkte Messung ohne vorherige Aufarbeitung möglich. Dabei lag die Nachweis- und Bestimmungsgrenze für ACh bei 0,3 bzw. 1 fmol/µl und für Ch bei 20 bzw. 100 fmol/µl. Nach den Standardlösungen wurden in-vivo Mikrodialysate aus dem rechten Striatum verschiedener CD1-Mäuse quantifiziert. Durch Verbindung der Dialysesonde mit einem MALDI-Spotter konnte außerdem die zeitliche Auflösung der Dialyse deutlich verbessert werden. Ein weiterer Anwendungsbereich stellte die Bestimmung von Melamin in Milch und Milchpulver dar. Nach einfacher Probenvorbereitung war es möglich, Melamin mit einer Bestimmungsgrenze von 0,25 ppm in Milchpulver bzw. 0,625 ppm in Milch direkt und schnell zu quantifizieren. Damit wurden die geforderten Grenzwerte von 1 ppm für Babynahrung bzw. 2,5 ppm für übrige Lebensmittel leicht erreicht. Als letzte Methode wurden verschiedene Inhaltstoffe in Energy Drinks bestimmt. Bei Verwendung von Theophyllin als IS konnte so neben Koffein auch Niacin und Pyridoxin erfolgreich und ohne Probenvorbereitung schnell quantifiziert werden. Zusammenfassend zeigt diese Arbeit, dass MALDI auch für die Analytik kleiner Moleküle gut geeignet ist. Neben der Identifizierung der Analyten war auch die Quantifizierung mit guter Linearität, Reproduzierbarkeit und Richtigkeit möglich. Dabei konnten mit der Standardmatrix CHCA verschiedenste niedermolekulare Analyten in unterschiedlichsten Probenmatrizes bestimmt werden. Im direkten Vergleich war die Sensitivität der vorgestellten MALDI-Methoden mindestens vergleichbar, wenn nicht gar besser als bestehende LC-MS-Methoden. Da auf den Einsatz einer LC verzichtet werden kann, ermöglich MALDI einen sehr viel höheren Probendurchsatz. Zudem war keine spezielle Matrix, besondere Geräte oder zeitaufwändige Probenvorbereitung und Präparation notwendig.A generally applicable method for the identification and quantification of small molecules by MALDI-TOF-MS was developed. A large number of analytes, such as different drugs, neurotransmitters and food ingredients in different sample matrices were analyzed. The best results have been achieved with the MALDI matrix a-cyano-4-hydroxy-cinnamic acid (CHCA). It appears, however, that the sample preparation is more important than the choice of matrix. Also, with the use of other matrices, a sensitive analysis in the low mass range was possible. A rapid drying of the sample spots and the associated formation of small crystals is useful for the analysis of small molecules. The use of low matrix concentrations and low laser intensity could minimize the disturbing matrix background. An even greater suppression of matrix signals and an enrichment of the analytes on the sample spot was achieved by a thin layer preparation (TLP) with CHCA and nitrocellulose. Quantification of small molecules was carried out with an optimized dried droplet preparation method (DDP). The combination of the optimized DDP and the use of an internal standard (IS) allowed for quantification with good precision, linearity and accuracy. The quantified analytes met the requirements of the FDA for precision and accuracy. The quantification method was carried out successfully for mixtures of different drugs in standard solutions as well as in human plasma. A single internal standard was sufficient to determine all drugs in the mixture (limit of quantification: 1-5 ng/ml). Furthermore, it was possible to determine the active ingredient of a pharmaceutical tablet quickly and accurately. The high salt tolerance of MALDI was demonstrated by the determination of acetylcholine (ACh) and choline (Ch) in microdialysates. Despite the high salt content of the samples (~ 150 mM), a direct measurement was possible. The detection and quantification limit was 0.3 and 1 fmol/µl in the case of ACh, and respectively 20 and 100 fmol/µl for Ch. In vivo microdialysates from the right striatum of different CD1 mice were quantified. By connecting the dialysis probe with a MALDI spotter, the time resolution of the dialysis was improve significantly. In another experiment, melamine in milk and milk powder was determined. After a simple sample preparation, melamine was analysed directly with a limit of quantification of 0.25 ppm in milk powder and 0.625 ppm in milk. Thus, the required limits of 1 ppm for infant formula and 2.5 ppm for other foods were reached easily. Finally, different ingredients in energy drinks, such as caffeine, niacin and pyridoxine were successfully quantified without sample preparation using theophylline as IS. In summary, this work shows that MALDI is also suitable for the analysis of small molecules. In addition to the identification also a quantification with good linearity, reproducibility and accuracy was possible. This could be determined in different sample matrices with various low molecular analytes. A direct comparison of the sensitivity of the presented MALDI-methods was comparable or even better than existing LC-MS methods. MALDI allows for a much higher sample throughput, because the use of LC is not necessary. Moreover, no special matrix, special equipment or time-consuming sample preparation was necessary

Perfil proteômico hipocampal em epilepsia do lobo temporal

In this study we used proteomics approaches to obtain the protein profile of human epileptic hippocampi (N=6) and control hippocampi obtained from autopsy (N=6). We used two-dimensional gel electrophoresis (2-D) coupled to HPLC and Mass spectroscopy (MALDI-TOF) to identify proteins differentially expressed. Nine proteins were differentially expressed comparing the hippocampus of patients with the hippocampus of control. Proteins that were increased in the hippocampus of patients with TLE were: isoform 1 of serum albumin, HSP 70, dihydropyrimidinase-related protein 2, isoform 1 of myelin basic protein, proton ATPase catalytic subunit A, and dihydrolipoyllysine-residue acethyltransferase component of pyruvate dehydrogenase complex. The expression of isoform 3 of spectrin alpha chain (fodrin) was down-regulated while the proteins glutathione S-transferase P and the DJ-1 (PARK7) were detected only in the hippocampus of patients with TLE. Taken together, our results provide evidence supporting a direct link between metabolic disturb and oxidative damage related to pathophysiology of TLE. Besides, indicates biomarkers for further investigations as therapies targeted to epilepsy.No presente estudo empregou-se o método de proteômica para obter a expressão diferencial de proteínas em hipocampo de pacientes com epilepsia do lobo temporal (ELT) (N=6) comparado a hipocampos controle obtidos por meio de autópsia (N=6). O estudo foi feito por meio de eletroforese bidimensional, acoplada a HPLC e espectroscopia de massa. As proteínas que tiveram expressão aumentada no hipocampo de pacientes com ELT foram: isoforma-1 da soroalbumina, HSP70, diidropirimidinase-2, isoforma-1 da proteína básica da mielina, subunidade catalítica A da próton ATPase, glutationa S-transferase P, proteína DJ-1 (PARK7), e resíduo diidropolilisina do componente acetil-transferase do complexo da piruvato desidrogenase. A expressão da isoforma-3 da cadeia alfa da espectrina (fodrina) foi menor no hipocampo de pacientes com epilepsia do lobo temporal e as proteínas glutationa S-transferase P e PARK7 foram detectadas somente no tecido epiléptico. Assim, nossos resultados trazem evidencias sobre a direta relação entre distúrbio metabólico e dano oxidativo na patofisiologia da ELT. Além disto, o estudo indica biomarcadores para futuras investigações como alvos terapêuticos para epilepsia.UNIFESP Departamento de Neurologia e NeurocirurgiaUNIFESP Departamento de Microbiologia, Imunologia e ParasitologiaFaculdade de Medicina da Universidade de São Paulo Instituto do Coração Departamento de Anatomia PatológicaUNIFESP, Depto. de Neurologia e NeurocirurgiaUNIFESP, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Proposta de soluções alternativas na resolução de problemas que surgem na manutenção da edificação predial

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia Civil.Muito tem se falado sobre a durabilidade das construções, mas pouco sobre cuidados de manutenção da edificação. O presente trabalho propõe procedimentos para manutenção de edificações prediais, a fim de prolongar a sua vida útil, evidenciando as patologias mais constantes, que se instalam e proliferam, provocando degradação dos elementos construtivos desde o estágio de projeto. Está fundamentado na experiência do autor em obras de manutenção de alguns prédios. Com esse propósito, aborda vários tópicos de construção na tentativa de compor ao máximo esse universo das edificações construídas pelos métodos tradicionais. Inicialmente, se definem os tipos de manutenção e se faz uma comparação sobre a eficácia relativa da adoção de cada uma delas, além de ordenar a implantação dos seus vários tipos, indicando o momento mais adequado para a sua introdução. Em seguida, abordam-se problemas de edificação mais comuns relativos aos sistemas construtivos, pertencentes à edificação predial e seus elementos característicos. Isso implica planejamento dos materiais que serão adotados para executar a recuperação, na formação do almoxarifado capaz de atender às necessidades básicas. Finalmente, numa abordagem mais gerencial que técnica, são determinadas as condições para a formação da equipe de manutenção e estabelecidas as prioridades de atendimento para orientar essas ações. Espera-se, com essas ações, que haja prolongamento da vida útil da edificação com gastos compatíveis e sem surpresas desagradáveis

The sensitivity to replacement and displacement of the eyes region in early adolescence, young and later adulthood

Recent evidence suggests a rather gradual developmental trajectory for processing vertical relational face information, lasting well into late adolescence (de Heering and Schlitz, 2008). Results from another recent study (Tanaka et al., 2014) indicate that children and young adolescents use a smaller spatial integration field for faces than do adults, which particularly affects assessment of long-range vertical relations. Here we studied sensitivity to replacement of eyes and eyebrows (F), variation of inter-eye distance (H), and eye height (V) in young adolescents (11-12 years), young (21-25 years), and middle-age adults (51-62 years). In order to provide a baseline for potential age effects the sensitivity to all three types of face manipulations was calibrated to equal levels for the young adults group. Both the young adolescents and the middle-age adults showed substantially lower sensitivity compared to young adults, but only the young adolescents had selective impairment for V relational changes. Their inversion effects were at similar levels for all types of face manipulations, while in both adult groups the inversion effects for V were considerably stronger than for H or F changes. These results suggest that young adolescents use a limited spatial integration field for faces, and have not reached a mature state in processing vertical configural cues. The H-V asymmetry of inversion effects found for both adult groups indicates that adults integrate across the whole face when they view upright stimuli. However, the notably lower sensitivity of middle-age adults for all types of face manipulations, which was accompanied by a strong general "same" bias, suggests early age-related decline in attending cues for facial difference

The two-systems account of theory of mind : testing the links to social-perceptual and cognitive abilities

According to the two-systems account of Theory of Mind (ToM), understanding mental states of others involves both fast social-perceptual processes, as well as slower, reflexive cognitive operations (Apperly and Butterfill, 2009; Frith and Frith, 2008). To test the respective roles of specific abilities in either of these processes we administered 15 experimental procedures to a large sample of 343 participants, testing ability in face recognition and holistic perception, language, and reasoning. ToM was measured by a set of tasks requiring ability to track and to infer complex emotional and mental states of others from faces, eyes, spoken language and prosody. We used structural equation modeling to test the relative strengths of a social-perceptual (face processing related) and reflexive-cognitive (language and reasoning related) path in predicting ToM ability. The two paths accounted for 58% of ToM variance, thus validating a general two-systems framework. Testing specific predictor paths revealed language and face recognition as strong and significant predictors of ToM. For reasoning, there were neither direct nor mediated effects, albeit reasoning was strongly associated with language (r = 0.73). Holistic face perception also failed to show a direct link with ToM ability, while there was a mediated effect via face recognition. These results highlight the respective roles of face recognition and language for the social brain (Kennedy and Adolphs, 2012), and contribute closer empirical specification of the general two-systems account