Navegando por Orientadores "SILVA, Anderson Manoel Herculano Oliveira da"

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Acesso aberto (Open Access)
Adenosina modula os níveis extracelulares de glutamato induzido por hiperosmolaridade em cultura de astrócitos hipotalâmicos
(Universidade Federal do Pará, 2016-04-29) BRAGA, Danielle Valente; DINIZ, Domingos Luiz Wanderley Picanço; http://lattes.cnpq.br/9601463988942971; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Recent studies have shown that glutamate release by hypothalamic glial cells is an important physiological response to hyperosmolarity. Furthermore, previous studies point out an accentuated increase of the adenosine levels in renal interstitial fluid after the intake sodium increases. This study aims to evaluate the possible relationship between the adenosine and glutamate releases in primary cultures of astrocytes exposed to hyperosmolarity conditions. Hypothalamic astrocytes cultures of Wistar rats at the first two days after birth were exposed to hypertonic sodium solution (340mOsm/L) in different times (3, 5, 10 e 15 min). After this stimulus, the incubation medium was harvested and the extracellular levels of glutamate and adenosine were determined by High Performance Liquid Chromatography. In order to evaluate the relationship between these compounds in hyperosmotic conditions, we have used treatment of the cultures with adenosine, with R-PIA (an agonist of the A1 receptor), as well as with glutamate (an agonist of the NMDA receptor). Our results showed a significant increase of the extracellular levels of glutamate after the hyperosmotic stimulus with a peak at 5 min. Similarly, we have seen an increase of the adenosine levels in the incubation medium after 10 and 15 min. The treatment with glutamate induced an increase in extracellular levels of adenosine after 15 and 20 minutes in isosmotic medium. The exposure to the NMDA receptor did not induce the release of adenosine in none of the concentrations utilized. The pretreatment with adenosine and R-PIA A1 agonist blocked the release of glutamate induced by hyperosmolarity. Our results also showed that the effect of the stimulus on the release of glutamate and adenosine is sodium-dependent and presents a specific response for hypothalamic astrocytes, which can be modulated by the adenosine A1 receptor activation.
Acesso aberto (Open Access)
Ativação do receptor canabinóide tipo 1 (CB1r) previne o estresse oxidativo cerebral e inibe o comportamento tipo agressivo em Danio rerio (Zebrafish)
(Universidade Federal do Pará, 2022-08-17) PINHEIRO, Emerson Feio; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Aggression is a set of complex actions that involve several factors of a genetic, neurophysiological, hormonal and behavioral nature. Furthermore, the brain redox state can also influence aggressive behavior in different species. Thus, modulators of this process can influence the expression of aggressive episodes, between them is the Endocannabinoid System that acts as the main neuromodulator of the CNS, in addition to exerting an antioxidant effect in different conditions. However, its participation in the modulation of aggressive-like behavior needs to be better understood. In this context, this study evaluated the role of cannabinoid receptor type 1 (CB1r) in brain redox state and aggressive-like behavior in Danio rerio (Zebrafish). For this, 64 animals were subdivided into groups: (a) Control (n=26), (b) ACEA (n=30) and (c) AM-251 (n=12), all treated with the drugs of interest: (a) Vehicle (NaCl 0.9%); (b) ACEA agonist 1 mg/kg; (c) 1 mg/kg AM-251 antagonist. The animals were isolated in pairs, without physical contact for 24 hours, followed by pre-treatment and after 30 minutes of pharmacokinetics, the fights were filmed for 30 minutes, the individuals were identified as Dominant or Subordinate and the brains were collected for evaluation of the state brain redox of these individuals. Our results demonstrate, for the first time, that the activation of CB1r by the ACEA agonist modulates aggressive-like behavior and, consequently, partially interferes with the establishment of social hierarchy in Zebrafish, through a redox-independent mechanism. We suggest, therefore, that acute treatment targeting CB1r is a useful neuropharmacological tool to elucidate the role of CES in social interaction and aggressive behavior, allowing a translation with numerous pathologies that have aggression as a behavioral disorder.
Acesso aberto (Open Access)
Caracterização da cinética de captação de glutamato por cromatografia líquida de alta eficiência em tecido retiniano
(Universidade Federal do Pará, 2011-08-19) MORAES, Edinaldo Rogério da Silva; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The present study describes a simple and efficient method utilizing high performance liquid chromatography (HPLC) coupled to fluorescence detection for the determination of kinetic parameters of glutamate uptake in nervous tissue. Retinal tissue obtained from 7-day-old chicks was incubated to known concentrations of glutamate (50-2000 μM) for 10 minutes, and the levels of the o-phtaldehyde (OPA)-derivatized neurotransmitter in the incubation medium were measured. By assessing the difference between initial and final concentrations of glutamate in the medium, a saturable uptake mechanism was characterized (Km = 8,2 e Vmax = 9,8 nmol/mg protein/minute). This measure was largely sodium- and temperature-dependent, strongly supporting that the mechanism for concentration decrements is indeed uptake by high-affinity transporters. Added to this, our results also demonstrated that zinc chloride (an inhibitor of glutamate/aspartate transporters) evoked a concentration dependent decrease in the glutamate uptake, demonstrating an elevate specificity of our methodology. Overall, the present work characterizes an alternative methodology to evaluate glutamate uptake in nervous tissue using HPLC. This approach could be an important tool for studies associated to the characterization of glutamate transport related with central nervous system injury
Acesso aberto (Open Access)
Efeito antineoplásico do composto 4,2´,3´,4´-tetrametoxi chalcona em linhagem de neuroblastoma B103 de rato
(Universidade Federal do Pará, 2014-03-28) COSTA, José Elierson Barros; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Neuroblastoma is the most frequently diagnosed malignancy in childhood. The term is commonly used to refer to a wide variety of neuroblastic tumors, including neuroblastomas, ganglioneuromas and ganglioneuroblastomas. Estimates show that 8 million children under 15 years of age per year are affected by this cancer, where 80% of cases are affected in up to 4 years of age, the tumor is malignant cells derived from embryonic arising from primary neuronal cells, since nodes adrenal medulla and sympathetic to other points. In this study, we assessed the cytotoxic potential of the compound 4,2 ', 3', 4'- tetrametoxchalcone in vitro model B103 rat neuroblastoma. Drug stock solutions were prepared at 50mM in dimethylsulphoxide (DMSO) and stored at - 20 ° C for the preparation of new concentrations (150μM, 100 mM, 75 mM and 50 mM). Cell viability was assayed from culture of glial cells from rat cortex. Cell migration assays and colony formation were also conducted. For statistical analysis, analysis of variance criterion (ANOVA) followed by Tukey test using the BioEstat 5.0 program was conducted. In the evaluation of cytotoxic effect of chalcones, it was observed that treatment with the compound 4,2’,3’,4’- tetrametoxchalcone showed no cytotoxic effect against normal cells of rat cortex for the concentrations tested, whereas in cell cultures neuroblastoma B103 was shown that the drug promotes cell death significantly.
Acesso aberto (Open Access)
Efeito modulador da glutationa na liberação de gaba induzida por glutamato em retinas de embrião de galinha
(Universidade Federal do Pará, 2012-06-06) PEREIRA, Tiago de Lima; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The γ-aminobutyric acid (GABA) and glutamate are, respectively, major inhibitory and excitatory neurotransmitters in the central nervous system (CNS) and are essential to the visual processing. Studies show that glutamate induces the release of GABA in the retina, but the mechanisms involved in this release are not well elucidated. Previous work also showed that thiols compounds regulate GABA release, but are not well defined the effects of compounds containing sulfhydryl (-SH) on endogenous levels of this neurotransmitter in the retina. In this context, glutathione (GSH) besides being the most important thiols compounds, have demonstrated perform a neuromodulatory role in the release of neurotransmitters. Thus, the objective of this study was to evaluate a possible modulatory effect of GSH on the release of GABA mediated by glutamate in the retina of chick embryo. For this study, we used as experimental model, retinal tissue intact chick embryo, with seven or eight days of development. In tests of release of GABA, the retinas were treated with GSH (100 and 500 μM), glutamate (50 and 500 μM) and Buthionine Sulfoximine (BSO), an inhibitor of glutathione synthesis, (50 μM) per 15 minutes, and GABA levels released into the extracellular medium were quantified by High Performance Liquid Chromatography (HPLC). For release experiments of thiols compounds, the retinas were incubated with glutamate 100 μM (with or without Na +) per 15 minutes, and their extracellular levels were determined by reaction with DTNB and quantified by spectrophotometry (412 nm). The results show that glutamate, as well as GSH, release GABA. Our data also show that BSO attenuates the release of GABA promoted by glutamate. Furthermore, we demonstrate that glutamate induces release of thiol compounds regardless of sodium. Therefore, it is known that glutamate is able to release thiols and GABA, among them, GSH is most abundant and responsible for also release GABA. It is also known that once inhibited GSH synthesis by BSO, the release of GABA induced by glutamate is attenuated. Then, it is suggested a possible modulation of GSH in the release of GABA induced by glutamate in retina intact chicken embryo.
Acesso aberto (Open Access)
Efeitos do fator de crescimento do nervo sobre os níveis extracelulares de glutamato e compostos tióis na retina embrionária de galinha
(Universidade Federal do Pará, 2011-04-20) GARCIA, Tarcyane Barata; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Nerve growth factor (NGF) belongs to the neurotrophin family and induces its effects through activation of two distinct receptor types. NGF was first described by Rita Levi-Montalcini and collaborators as an important factor involved in nerve differentiation and survival. Another role for NGF has been established in neurotransmitter release in the hippocampus, developing visual cortex and cerebellar neuron. However, this phenomenon has not been demonstrated in retina to date. We therefore investigated whether NGF can modulate the glutamate release in the retinal tissue at its peak of the neurotrophic activity (E10-E12). In addition this, we aimed to study the mechanisms of this effect about its dependence on extracellular Ca2+ and participation of Na+-dependent and Na+-independent glutamate transporters. Since high levels of glutamate signalization have been implicated in the oxidative stress, we also investigated the effects of NGF on the thiols compounds. We used intact retinal tissue from chicken embryos (E11) incubated with NGF (10, 50, 100 ng/ml) for different periods (15, 30, 45, 60, 120 min). Extracellular glutamate and thiols content was measured by HPLC methods and colorimetric assay, respectively. We found that NGF rapidly enhances the release of basal glutamate and it can induce thiol release in a more prolonged time of incubation, as well. Interestingly, the NGF-induced increase in the extracellular levels of glutamate was blocked by Ca2+-free medium only in retina treated for 15 min. Retina incubated for 30 min showed a non-vesicular NGF-induced glutamate release. Since glutamate and thiol release was not blocked by Zn2+, we suggested the possible involvement of system Xc- in both processes.NGF-induced increase in the extracellular thiol could be an important protective mechanism enabling retinal neurons to maintain their redox status during development.
Acesso aberto (Open Access)
Estressores agudos distintos produzem diferentes magnitudes do comportamento tipo de ansiedade e liberação diferencial de glutamato no cérebro de zebrafish
(Universidade Federal do Pará, 2024-08) MARTINS, Milena Letícia; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247; https://orcid.org/0000-0003-4022-8096
Anxiety disorder is one of the most well characterized behavioral disorder in individuals submitted to acute or chronic stress. However, few studies have demonstrated how different types of stressors can modulate the neurochemical alterations involved in generation of anxiety. In the present study, we hypothesize that subjects exposed to different aversive stimuli (mechanical, chemical and spatial restriction) present varied intensity of anxiety-like behavior response associated with distinct pattern of GABA and Glutamate release in the brain. Sixty adult Danio rerio animals were randomly divided into four experimental groups: Control (CTRL), Acute Restraint Stress (ARS), Conspecific Alarm Substance (CAS) and Chasing with Net (CN). After applying the stress protocols, the animals were individually transferred to the Novel Tank Diving Test for behavioral analysis. Subsequently, their brains were collected and subjected to GABA and Glutamate release assay for quantification by HPLC. Our behavioral results showed that all aversive stimuli were capable of inducing anxiety-like behavior. However, the impact of anxiogenic behavior was more prominent in the CN and CAS groups when compared to ARS. This phenomenon was evident in all analyzed behavioral parameters (time on top, freezing, mean speed, maximum speed and erratic swimming). Our data also have shown that all aversive stimuli induced significant decrease in GABA release when compared to the control group. On the other side, only animals exposed to CN and CAS presented increase in extracellular glutamate levels. Different acute stressors induce different intensity of Anxiety-like behavior in zebrafish as well as they provoke specific alterations on the GABAerigic and Glutamatergic release in the brain. These results demonstrate the complexity of anxiety disorders, highlighting that both behavioral and neurochemical responses are highly context-dependent, supporting our initial hypothesis.
Acesso aberto (Open Access)
Estudo eletrorretinográfico da adaptação à luz de vias de processamento específicas de cone e de oponência em cor e luminância
(Universidade Federal do Pará, 2017-12-15) COSTA, Alódia Brasil; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Visual system has an essential function called light adaptation or photopic adaptation, which consists in the regulation of light sensitivity allowing visual adaptation to a wide range of illumination levels. This phenomenon is not fully elucidated. Therefore, the present study aims to evaluate the magnitude and temporal pattern of the variation of retinal electrical responses for L cone and M cone selective stimuli and for luminance pathway and red-green color pathway specific stimuli, making possible the evaluation of the red-green parvocellular (P pathway) and magnocellular (M pathway) visual pathways activity, during the photopic adaptation. For such purpose, 6 healthy subjects underwent 30 minutes of dark adaptation and then were exposed a light steady background for 16 min. The stimuli were given every 2 min and the electrical retinal response was recorded by full field electroretinogram (ffERG) over 16 min. The triple silent substitution method was used to isolate the responses of pathways originating from the L or M cone. We also used specific stimuli of luminance (Lum) and red-green color (Crom). For each stimulation type was used intermediate (12 Hz, which reflects the activity of the red-green parvocellular pathway - P pathway) and high (36 Hz, which reflects the activity of the magnocellular pathway - M pathway) temporal frequency, resulting in 8 stimulation conditions. Amplitude and phase of the first (F), second (2F) and third (3F) harmonic components were extracted by Fast Fourier Transform. It was observed that amplitude and phase increased over light adaptation time with simple sine wave form in the most of components and stimulation conditions. The relative increases in F amplitude from the M-cone driven responses during the light adaptation were higher than those L-cone driven ERGs at both temporal frequencies, 12 Hz (M = 1.21, L = 0.33) and 36 Hz ( M = 1.94, L = 0.55), as well as they were higher at 36 Hz than at 12 Hz for the two cones. In general, there was slight F phase increase during the light adaptation time (<30 degrees), slightly higher at 36 Hz. Regarding the light adaptation kinetics, F amplitude and phase that seem reflect the activity of the P pathway presented a faster adaptation (L-cone 12 Hz, M-cone 12 Hz, Crom 12 Hz, Lum 12, with a mean of 1.4 min) and those that probably reflect activity of the M-pathway had slower adaptation (Lum 36 Hz, L-cone 36 Hz, M-cone 36 Hz, with a mean of 4.9 min). Thefore, M and P pathways presented different magnitude and kinetics of light adaptation, being the M pathway the pathway of greatest increase and slower adaptation.
Acesso aberto (Open Access)
Glutationa modula a liberação de adenosina em cultura primária de astrócitos corticais de camundongo
(Universidade Federal do Pará, 2022-09-06) SILVA, Mateus dos Santos; OLIVEIRA, Karen Renata Herculano Matos; http://lattes.cnpq.br/3032008039259369; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Glutathione (GSH) is one of the main antioxidants in the Central Nervous System (CNS) and a potential gliotransmitter, inducing calcium waves in the cytosol of glial cells. Adenosine (Adn) is a neuromodulator widely expressed in the CNS and its extracellular levels are a critical factor in determining its effect on nervous tissue. It is known that Ca2+- dependent pathways regulate Adn release. Since GSH has the ability to induce Ca2+ waves in the cytosol of glial cells, the present work aims to investigate whether this molecule can regulate extracellular DNA levels. To assess this, we used primary cultures of cortical astrocytes maintained in DMEM+10% SBF in a CO2 oven (37oC, 95% O2/5% CO2) for 12-15 days, when they reached confluence. The cells were incubated with Hank buffer for different time intervals, after which this solution was collected and the neurotransmitters present there were quantified by High Performance Liquid Chromatography. Our data show that GSH induces an 80% increase in extracellular Adn levels at two analyzed times: 5 and 20 minutes. Removal of GSH from the incubation medium returns the Adn concentration to baseline levels. Removal of Na+ or Ca2+ from the medium did not affect the effect of GSH. Blockade of nucleoside equilibrative transporters by dipyridamole (10 µM) significantly decreased the levels of Adn in the medium, but did not interfere with the action of GSH. In order to assess whether the effect of GSH derives from an indirect modulation on the release of glutamate or GABA (two agents described as regulators of Adn release), the quantification of these transmitters was performed. Both were significantly increased in the presence of GSH. However, unlike what was observed with Adn, the removal of Na+ from the incubation medium mitigated the effect of GSH on glutamate release. The incubation of astrocytes with GABA (50 and 100 µM) did not influence the extracellular Adn concentration in our experimental model, ruling out a GABAergic modulation behind the effect of GSH. The evaluation of redox agents showed that thiol compounds reproduce the effect of GSH, while the non thiol antioxidant alpha-tocopherol did not regulate extracellular Adn levels. Thus, the present work concludes that astrocytes express a GSH-sensitive component that can be modulated by its sulfhydryl group.
Acesso aberto (Open Access)
A indução do comportamento tipo ansiedade e estresse oxidativo pela indometacina no cérebro do Danio rerio (Zebrafish) é prevenida pelo alfa-tocoferol
(Universidade Federal do Pará, 2021-01) PINHEIRO, Jéssica Souza; OLIVEIRA, Karen Renata Herculano Matos; http://lattes.cnpq.br/3032008039259369; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The non-steroidal anti-inflammatory drugs are among the most used and prescribed in the world, however this type of drug has several side effects at the neural level. Studies related to neurobehavioral and neurochemical damage of this class of drug are still necessary for a better understanding of all the possible damages that they can cause. As a result, indomethacin, which is an NSAID, has been widely used to treat pathologies such as rheumatoid arthritis, musculoskeletal injuries, osteoarthritis and postoperative pain. Indomethacin non-selectively blocks the enzymes COX-1 and COX-2, acting to decrease the production of prostaglandins. Therefore, this study proposed that the indomethacin could be generated anxiogenic effects and oxidative stress in the brain, and whether the antioxidant α-tocopherol exercised protection against the possible damage caused by indomethacin in zebrafish. The animals used were fish of the species Danio rerio (n=160), subdivided into the following groups: Control - Saline 0.9%; Indomethacin - INDO 0.5 mg/kg; INDO 0.75 mg/kg; INDO 1.0 mg/kg; INDO 2.0 mg/kg; INDO 3.0 mg/kg; α-Tocopherol - TF; TF + INDO 1 mg/kg; TF + INDO 2 mg/kg and were subjected novel tank diving test, the parameters time on top, freezing, erratic swimming and crossed quadrants were analyzed. The statistical analysis of the results was performed using one-way ANOVA with a post-test bonferroni or tukey for comparison between groups, with values with p <0.05 being considered significant. The results regarding the behavioral parameters and oxidative stress were expressed as mean ± standard error or standard deviation. The parameters that showed statistical differences were the time at the top and freezing, where the animals of the groups INDO 0.5 mg/kg, INDO 0.75 mg/kg, INDO 1 mg/kg and INDO 2 mg/kg explored for less the top of the apparatus compared to the CTRL group. In the freezing parameter the groups treated with indomethacin INDO 0.5 mg/kg, 0.75 mg/kg and 2 mg/kg did not show statistical differences with the CTRL group, however there was a difference between the CTRL and INDO groups 1 mg/kg. In the freezing parameter, the animals in the INDO 1 mg/kg group showed a longer time without movement compared to the CTRL group. In the other parameters, there were no significant differences between the groups treated with the control group. The analysis of lipid peroxidation, the INDO 1 mg/kg and INDO 2 mg/kg groups showed an increase in MDA production compared to the CTRL group, thus inferring that there was an increase in oxidative stress when animals were treated with indomethacin. The α-tocopherol exercised protection when animals were previously treated in both the TF + INDO 1 mg/kg group and the TF + INDO 2 mg/kg group compared to the INDO 1 mg/kg and INDO 2 mg/kg groups, respectively. Therefore, indomethacin is involved in inducing anxiety-like behavior and oxidative stress in zebrafish brains.
Acesso aberto (Open Access)
Inibição nitrérgica local favorece o processo de regeneração do tendão de aquiles em ratos submetidos à tenotomia
(Universidade Federal do Pará, 2011-05-27) MORAES, Suellen Alessandra Soares de; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The tendon injuries prevalence has been increased usually by repetitive movements in the occupational and recreational activities of society. The main features this injury involve healing dysfunction and loss of extracellular matrix. At this tissue, the extracellular matrix is basically made of type I collagen and the loss of this protein associated with problems in the remodeling have been considered the major responsible for the symptomatic and functional effects of injury. Additionally the literature reports the nitric oxide synthase expression was up-regulated throughout the repair process. At this context the propose of this work is investigated the nitrergic local inhibition in tissue and functional recovery after tendon injury. We used a experimental model of Achilles tendon rupture for establish pathophysiological changes similar to chronic tendinopathy. The animals were divided into experimental groups control, injury+vehicle (saline, 0,9%), injury+L- Nω-nitro-L-arginine methyl ester (L-NAME, 5mM) and injury+sodium nitroprusside (SNP, 3mM), which were followed until the 21º day, when were killed to collect samples. In order to access nitric oxide production Griess nitrite assay was used. Functional recovery was calculated by Achilles Functional Index (AFI) in 0, 7, 14 and 21 days. Tendons were also processed for histomorphological and autofluorescence analysis. The body weight gain was also evaluated. We characterized the effect of drugs about tissue without stronger systemic influences by maintenance of body weight and plasmatic levels of nitrite. The L-NAME led to significant decrease in the cellular density, vessel formation between collagen network, accelerated the tissue organization and restored early the functional pattern at 21 days after injury. On the other hand, the treatment with SNP and vehicle remained the tissue disorganization and low functional performance. Taken together our results suggest a positive effect of local suppression of NOS after rupture for tissue remodeling, contributing to tendon regeneration.
Acesso aberto (Open Access)
A intensificação do comportamento tipo ansiedade induzido por cafeína em Daniorerio(zebrafish) é prevenida pelo tratamento com α-Tocoferol e L-NAME
(Universidade Federal do Pará, 2015-01-23) CARVALHO, Tayana Silva de; OLIVEIRA, Karen Renata Matos; http://lattes.cnpq.br/3032008039259369; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The growing consumption of beverages with high caffeine content can result in the appearance of symptoms from anxiety disorder induced by this drug. Currently, it has been used as a pharmacological caffeine inductor anxiety-like behavior and this induction may help facilitate better understanding of the relationship between behavioral changes and the mechanisms involved in this effect. Therefore, this study proposed that the nitrergic pathway could be a key mechanism to explain the behavioral effects produced by caffeine and that these effects could be reversed by an antioxidant, hence, the present work we had to evaluate the possible effect of L –NAME and - tocopherol expanded in anxiety-like behavior by caffeine in light/dark preference (LDP) test and novel tank dividing (NTD) test in Daniorerio. Zebrafish fish species (N=178) used were divided into the following experimental groups: SAL - 0.9% saline; CAF - Caffeine 100 mg/kg; DMSO - dimethylsulfoxide 0.1% ; L-NAME - (N -nitro -L - arginine methyl ester hydrochloride) 10 mg/kg; TF - -tocopherol 1 mg/kg (received only one injection by i.p.); SAL + SAL; DMSO + SAL; SAL + CAF , L-NAME + SALT , L-NAME + CAF ; TF + CAF, received two followed injections, one injection of each substance in the form of co-treatment, by i.p. The animals were submitted to the light/dark preference test and novel tank dividing test. All tests were filmed and the videos were evaluated using X-PLO-RAT. Data were expressed as mean ± SEM. The normality test was applied using the Shapiro-Wilk test and the ANOVA parametric test one-way with Tukey post-hoc, with the significance level set at p<0.05. The - tocopherol at a dose of 1 mg/kg, reversed all anxiety-like behavior parameters expanded by caffeine in LDP and NTD tests and this effect was similar to that observed when given one inhibitor of the enzyme nitric oxide synthase (NOS), L- NAME. Therefore, this study first demonstrated that the behavioral effect magnified by caffeine in scotaxis test and DVN test can be modulated by the nitrergic pathway and that the -tocopherol reverses completely this anxiety-like behavioral effect induced by caffeine.
Acesso aberto (Open Access)
Método baseado me cromatografia líquida de alta eficiência para analisar a atividade dos transportadores de GABA no sistema nervoso central
(Universidade Federal do Pará, 2022-08) MORAES, Edinaldo Rogério da Silva; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Alterations in GAT activity are implicated in several neurological diseases, including retinopathies. The present study describes an alternative method to determine GAT activity in tissue preparations of the central nervous system, using high performance liquid chromatography (HPLC) with fluorescence detection. The GABA concentration in the medium was determined using the o-phthaldehyde (OPA)-derivation protocol validated by the Brazilian Health Regulatory Agency (ANVISA) and the United States Food and Drug Administration (US-FDA). The GAT activity in the retinal preparations was determined through the evaluation of the GABA uptake, which was measured by assessing the difference between the initial and final concentrations of GABA in the incubation médium (Δ𝐺𝑎𝑏𝑎 = [𝐺𝑎𝑏𝑎] 𝑡_0 − [𝐺𝑎𝑏𝑎] 𝑡_𝑒𝑛𝑑). The evaluation of the GAT kinetics returned values of Km = 382.5 ± 32.2 μM and Vmax = 34 nmol/mg of protein. The data also demonstrated that the GABA uptake was predominantly Na+ and temperature-dependent, and was also inhibited by incubation with nipecotic acid, a substrate of GABA transporters. Taken together, these findings confirm that our approach provided a specific measure of GAT activity in retinal tissue. The data presented here thus validate, for the first time, an alternative, simple and sensitive method for the evaluation of GAT activity using high performance chromatography on preparations of the central nervous system.
Acesso aberto (Open Access)
Regeneração tendínea em modelo murino: estudo da plasticidade central e investigação do efeito da modulação nitrérgica na plasticidade periférica
(Universidade Federal do Pará, 2015-08-10) MORAES, Suellen Alessandra Soares de; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Tendon injuries cause strong impact on people due to pain and functional limitation resulting therefrom. After injury, the tissue starts to present a network of nerves. Furthermore, there is evidence for the occurrence of central plasticity after injury. Among the molecular factors involved in injury repair, nitric oxide (NO) is implicated in tissue remodeling, but its effects are not yet well understood. The purpose of this study is to ascertain the existence of central plasticity and the influence of NO in peripheral plasticity, functional limitation and tendon regeneration in murine model. To study the effects of NO in peripheral plasticity, we used control animals (CTRL, without injury) or animals treated with saline (SAL, 0.9% NaCl), L-nitro-arginine-methyl-ester (L-NAME, NO-synthesis inhibitor) and sodium nitroprusside (SNP, NO donor) every other day until the 21st day post injury (DPI). To evaluate central plasticity (L5 segment), an injury was performed alone and the spinal cord collected at 2 or 21 DPI. We analyzed the integrity and tissue organization in tendon samples by H&E, transmission electron microscopy and immunofluorescence, which was also used to evaluate peripheral plasticity. To assess tendon functional recovery, we determined the Achilles functional index, the joint angle and the open field. In spinal cord studies, we investigated glial reactivity and neuron involvement after injury by co-localizations with the cell activation indicator c-Fos. The findings of this research show that NO inhibition promotes tissue organization in association to an increase in collagen synthesis, secretion and deposition. Besides, L-NAME local administration seems to favor cell differentiation to tenocyte-like morphological types and improve the organization of nerve branches in between the collagen mesh in correlation with functional recovery at 21 DPI. On the other hand, increased levels of NO by SNP promoted worsening in almost all parameters analyzed. Our data also show tendon injury triggers a central plasticity process with an increase in glial reactivity at 2 DPI and ipsilateral cell activation at 2 and 21 DPI. Afterall, our findings point out occurrence of central plasticity after tendon injury and favoring of tissue repair and peripheral plasticity through nitrergic blockage, unraveling fundamental aspects of tissue recovery that may represent new targets for a therapeutical approach in tendon injuries.
Acesso aberto (Open Access)
Ruptura do tendão calcâneo induz alterações bioquímicas e histológicas na medula espinhal de camundongos
(Universidade Federal do Pará, 2019-07-08) FRANÇA, Martha de Souza; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
The pathophysiology of the tendons involves release of neuronal mediators that play an active role in regulating tendon pain, inflammation and homeostasis. New directions have pointed out that injury is not restricted to tissue structural changes but indicates a possible involvement of the CNS in the regulation of the lesion. In this way, it is still unknown if the tendon injury affects the CNS, so the present study aims to investigate possible histological and biochemical changes in the spinal cord (L5) caused by the total rupture of the Achilles tendon in murine model. For this, the animals were submitted to tenotomy of the Achilles tendon, and separated in three groups (n = 36): Control; Rupture and Rupture+Suture. The total number cells of the spinal cord gray matter in the L5 vertebral segment was assessed by DAPI labeling. Glial reactivity was assessed by immunohistochemistry for microglia (IBA-1) and astrocytes (GFAP) at 7, 14 and 21 days after tendon rupture. The participation of the nitrergic system was investigated by the quantification of tissue levels of nitrite in lumbar intumescence at 7, 14 and 21 days post-injury and by iNOS (NOS2) immunostaining in L5. Statistical analyzes were performed using the ANOVA-1way test and post-test tukey, considering a significant p <0.05. The results were expressed as mean ± SD. The analysis of the number of cells showed that the Rupture group had a lower number of cells in 7 (1408.33 ± 58.59, p <0.05), 14 (1402.7 ± 72.7, p <0.05) and 21 (1374.5 ± 74.2, p <0.01) days post-injury in relation to the Control group (1668 ± 52.3) and in relation to the Rupture + Suture group on days 7 (1655 ± 66.5 , p <0.05) and 21 (1668.3 ± 14.1, p <0.01). The Suture group did not differ from the Control group. The results of glial reactivity showed that at 14 days after injury the microglia were activated at L5 and that astrocytes were activated at 7, 14 and 21 days after injury. The nitrite quantification showed higher levels of nitrite in the group Rupture in 7 (0.0004 ± 10.8x10-5, p <0.01) and 14 days (0.0006 ± 1.06 x10-5, p <0.01) post-injury in relation to the control group (0.0002 ± 3.45x10-5). Immunostaining for iNOS was identified at 14 days after injury in the Rupture group. Our results showed that the rupture of the Achilles tendon induces changes in the spinal cord in terms of total cell number, activation of glial cells and participation of the nitrergic system in a murine experimental model. In this way, it points to possible degenerative, oxidative, inflammatory and neural plasticity events in the spinal cord resulting from the Achilles tendon injury, highlighting the CNS participation in the repair process of this lesion.
Acesso aberto (Open Access)
Ruptura total do tendão de Aquiles induz resposta inflamatória e ativação glial na medula espinhal de camundongos
(Universidade Federal do Pará, 2022-05) PAULA, Diego Rodrigues de; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Achilles tendon rupture is a common accident that affects both professional and recreational athletes. Acute and chronic pain are commonly seen in patients after rupture, usually associated with local inflammatory activation. The factors leading to hyperalgesia in symptomatic patients are poorly understood. Evidence suggests that Achilles tendon rupture is not restricted to tissue changes, but is able to evoke changes in the central nervous system (CNS). This study aimed to evaluate the impact of Achilles tendon rupture on the biochemical and histological profile in the spinal cord (L5) and on the nociceptive response in a murine model. The animals after Achilles tendon tenotomy surgery were divided into two groups: control (without rupture) and Rupture (tenotomized). Mechanical sensitivity test (von Frey) was assessed on the 7th and 14th day post-tenotomy (dpt). Glial reactivity was assessed by immunohistochemistry for microglia (IBA-1) and astrocytes (GFAP). Inflammatory activation was assessed by immunofluorescence for NOS-2 and COX2 at 7th and 14th dpt. We show, by the mechanical sensitivity test, an increase in the algesic response in the ipsilateral paw of the ruptured group on the 7th and 14th dpt when compared to the control group. This phenomenon was accompanied by hyperactivation of astrocytes and microglia in sensory processing areas of the L5 spinal cord, predominantly on the ipsilateral side to the tendon injury. We show inflammatory activation by expression of COX-2 and NOS-2, exclusively in the 14th dpt. These data were supported by biochemical findings that demonstrated significant nitrite levels increase in the lumbar spinal cord of animals submitted to Achilles tendon rupture at 7 and 14 dpt. The present study demonstrated for the first time that complete rupture of the Achilles tendon induces a neuroinflammatory response associated with glial activation in the spinal cord (L5) of mice.
Acesso aberto (Open Access)
Sensibilização dependente de tempo em paulistinhas adultos como modelo de transtorno de estresse pós-traumático: papel do óxido nítrico
(Universidade Federal do Pará, 2015-08-11) LIMA, Monica Gomes; OLIVEIRA, Caio Maximino de; http://lattes.cnpq.br/7758963790962240; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Post-traumatic stress disorder (PTSD) is classified as a trauma- and stressor-related disorder, a set of severely debilitating neuropsychiatric disorders characterized by the disregulation of stress responses after a traumatic event. Zebrafish (Danio rerio Hamilton 1822) emerged as an important model organism for the study of genetic, neuropharmacological and behavioral functions, such as the study of anxiety and stress. Nitric oxide (NO) is a gaseous transmitter that appears to have an important role in the regulation of neurobehavioral responses to stresss, including in zebrafish. In this scenario, we propose a behavioral model for PTSD in the evaluation of the time-dependent sensitization of behavior in zebrafish as a consequence of the exposure to conspecific alarm substance (AS) – a potent stressor. Using this model, we will verify the role of the nitrergic system in this process of sensitization. Animals will be exposed to AS and kept stress-free for 24 h; after this interval, animals' behavior will be analyzed. 5 experiments will be made to investigate: i) the delayed effect of alarm substance on different behavioral tasks in zebrafish, ii) a comparison of time-dependent sensitization on shortfin and longfin phenotypes; a comparação da sensibilização dependente de tempo nas linhagens shortfin e longfin, iii) the application of Behavioral Cutoff Criteria on timedependent sensitization, iv) the quantification of extracellular glutamate and tissue nitrite in the telencephalon after exposure to alarm substance, and v) the participation of NO on the initiation and consolidation of time-dependent sensitization. Our results revealed that: i) alarm substancce produces a delayed sensitization of anxiety (increased geotaxis, decreased habituation, increased erratic swimming and thrashing in the novel tank test; decreased time on white, increased erratic swimming, risk assessment and thigmotaxis on the ligh/dark test) and arousal (increased swim distance on the first trial and increased habituation slope in the startle reactivity test). ii) In relation to shortfin animals, exposure of longfin zebrafish to AS sensitized time on white, risk assessment and thigmotaxis more, while shortfin animals had more erratic swimming. Iii) 25.74% of AS-exposed animals reached criteria for Extreme Behavioral Response (EBR), and 20% reached criteria for Minimal Behavioral Response VII (MBR); in non-exposed animals, only 4% reached criteria for EBR and 96% reached criteria for MBR. Animals classified as EBR spent less time in the white compartment, with shorter entries, more thigmotaxis and more erratic swimming than animals classified as MBR and non-exposed controls. iv) treatment with L-NAME 30 minutes before AS exposure did not block the behavioral sensitization in the light/dark test; v) treatment with L-NAME 30 minutes after AS exposure blocked the sensitization of scototaxis and risk assessment; vi) treatment with L-NAME 90 minutes after AS exposure blocked the sensitization of risk assessment, erratic swimming and thigmotaxis. Theses results suggest that time dependent sensitization is a good model to study PTSD and point to NO as a important mediator in this process.
Acesso aberto (Open Access)
O tratamento com ácido ascórbico acelera o processo de reparo do tendão calcâneo em modelo de lesão tendínea em ratos
(Universidade Federal do Pará, 2015-02-24) SOUZA, Martha Costa de; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Tendon rupture affects a large part of the population in special seniors and athletes.The repair process requires more studies which can indicate the possibility of new treatments. The ascorbic acid (AA) is a well known substance by its requirement for proline and lysine hydroxylase activity during the collagen synthesisis and the efficiency of this vitamin for various treatments, because of its antioxidant properties. So, the aim of the present study is to evaluate the local treatments effects with this substance on tissue and functional repair the Achilles tendon from rats. The study was approved by the ethics committee of the institution (CEPAE-UFPA) according to the license 161-13. The animals were submitted to rupture of Achilles tendon, divided into three groups (n=27): control, injury+AA (30 mM), injury+vehicle (0.9% NaCl). All treatments were performed by local injection, from the second day after injury and every other day until day 14 or 21. The walk of the animals was assessed by functional index of Achilles (IFA) on days 7(n=6), 14(n=6) e 21(n=3), cells number was assessed by staining with DAPI and tissue organization by staining with HE and autofluorescence, at 14(n=9) and 21(n=9) days of injury. The animals did not differ in body mass gain. The injury+AA group (-39.51±15.3) showed functional improvement especially at day 14 when compared to the injury+vehicle (-89.22±16.57, p<0.01). The histological examination demonstrated in counting the number of cells that the injury+AA group (762±29.6) showed a smaller number of cells on day 21 (762±29.6) in relation to the groups injury+vehicle (916±57.0, p<0.01). The analysis of autofluorescence of collagen and HE showed that the injury+AA group achieved better ECM organization on day 14 and 21 in relation to the groups injury+vehicle, in turn, differs significantly from the control group. Our results suggest that AA accelerates the healing process of tendon injury, presenting tissue and functional improvements 21 days after injury.
Acesso aberto (Open Access)
O tratamento com meio condicionado em cultura primária de tenócitos acelera o reparo tendíneo em modelo de lesão total do tendão calcâneo
(Universidade Federal do Pará, 2022-10-14) MACIEL, Analú Alves; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Conventional treatments for tendinopathies are ineffective and most clinical interventions do not provide adequate recovery leaving this tissues more likely to suffer reinjures. Recently, cell based therapies has been shown to be effective for the treatment in connective tissue injuries, such as tendons. Our aim is to evaluate if local treatment with tenocytes conditioned medium promoves tissue and functional improvements in the calcaneal tendon of tenotomized mice. The calcaneal tendon cells of Swiss mice were cultured for conditioning culture medium that will be used as a treatment. The animals were subjected to right calcaneal tenotomy and treated with saline solution (SAL), DMEM without serum (DMEM) and DMEM conditioned in primary tenocyte culture (MC) and compared to the control group (CTRL). Tendon functionality was measured using the Achilles Functional Index (AFI) and mechanical sensitivity through the paw withdrawal threshold (PWT) using the Von Frey test. All analyzes were performed at 7, 14 and 21 days post-injury (dpi). For histological analysis, tissues were stained with HE. Statistics were performed by ANOVA-2 followed by Tukey's post test, p<0.01. The MC group showed functional improvement at 7° and 14°dpi (-40.4±12.6; -36.6±10.4) compared to the DMEM groups (-76.5±11.7; -71, 6±7.9, p<0.01) and SAL (-88.8±15; -71.4±12.6 p<0.01). The MC group showed improvement in the paw withdrawal threshold at 7° and 14°dpl (2.24±1.15; 2.66±1.06) compared to the DMEM groups (0.15±0.07; 0 .45±0.76 p<0.01) SAL (0.13±1.15; 0.77±0.95 p<0.01). In the histological analysis, the MC group showed better tissue organization with cells presenting a format more similar to the control group, while the SAL and DMEM groups were more different from this one. We conclude that treatment with tenocytes conditioned medium accelerates tendon recovery, promoving improvement in mechanical sensitivity, functionality and tissue organization in the proposed injury model.
Acesso aberto (Open Access)
Variação temporal dos níveis de gaba e glutamato no cérebro de Danio rerio (Zebrafish) submetidos a ambientes ansiogênicos
(Universidade Federal do Pará, 2018-07-13) SILVA, Waldo Lucas Luz da; OLIVEIRA, Karen Renata Herculano Matos; http://lattes.cnpq.br/3032008039259369; SILVA, Anderson Manoel Herculano Oliveira da; http://lattes.cnpq.br/8407177208423247
Anxiety-like behavior can be defined as a state of apprehension where the danger is imminent and may occur from exposure to new environments or uncontrollable aversive stimuli. Many neurotransmission systems may be involved in the modulation of anxiety states in mammals as well as in teleosts. Among these, the GABAergic and glutamatergic systems are the main modulation pathways of anxiety-like behavior. Therefore, the present work aims to evaluate the extracellular levels of GABA and glutamate throughout the process of anxiety generation in Danio rerio exposed in Dark/light Preference (DLP) and Novel Tank (NT) tests. Sixty animals (Danio rerio, (wild type, longfinn) were used, which were exposed to DLP and NT at times 5, 10 and 15 minutes. The parameters analyzed for DLP were: time in the white compartment, number of quadrants crossed in the white compartment and transitions between compartments; for the NT, the parameters were: Time in the upper half, number of squares crossed, entrances at the top, maximum speed, average speed and total distance traveled. Then, the brains were dissected and incubated with Hank/Na+ for further quantification of GABA and Glutamate by High Performance Liquid Chromatography (HPLC). All tests were filmed and videos evaluated using Zebtrack software. One-way ANOVA test with Tukey post-test was applied, considering significant values p<0.05. Data were expressed as mean ± standard error mean and all experiments were previously approved by the local ethics committee (CEPAE UFPA 213-14). Our results demonstrate exploration of the white compartment in the LDT test, with no difference between the times (F [3, 20] = 17.10) and no change in the number of midleline crossings between the compartments between minutes 5 and 10 (F [3,20] = 6.28, p <0.05). There was an increase in the number of squares crossed in the time of 15 minutes in relation to the other exploration times (F [3, 20] = 13.04, p <0.03).In addition, there was an increase in extracellular glutamate content during the test (F [4, 10] = 8.98) and decrease of GABA in the last minutes compared to animals not exposed to the test (F [4,9] = 20,83; <0.05). The behavioral patterns of the animals exposed to the NT test also vary according to the time of exposure, where, as time increases, there is an increase in the time of the top exploration (F [3, 28] = 15.99, p <0.01), (F [3, 22] = 16.86 p <0.05), increase in the number of squares crossed (F [3, 21] = 38.70, p <0.01), increase in the total distance traveled [3, 27] = 61.44, p <0.01), with no change in maximum speed (F [3, 28] = 19.73, p <0.01) and mean speed at all times. Glutamate levels increase on exposure to the test (F [4, 10] = 24.62) and GABA levels remain unchanged (F [4,9] = 1.76). We conclude, therefore, that glutamatergic and gabaergic systems modulate the anxiety-like behavior in Danio rerio differently.