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

Rutin is an important flavonoid consumed in the daily diet. It is also known as vitamin P and has been extensively investigated due to its pharmacological properties. On the other hand, neuronal death induced by glutamate excitotoxicity is present in several diseases including neurodegenerative diseases. The neuroprotective properties of rutin have been under investigation, although its mechanism of action is still poorly understood. We hypothesized that the mechanisms of neuroprotection of rutin are associated with the increase in glutamate metabolism in astrocytes. This study aimed to evaluate the protective effects of rutin with a focus on the modulation of glutamate detoxification. We used brain organotypic cultures from post-natal Wistar rats (P7–P9) treated with rutin to evaluate neural cell protection and levels of proteins involved in the glutamate metabolism. Moreover, we used cerebral cortex slices from adult Wistar rats to evaluate glutamate uptake. We showed that rutin inhibited the cell death and loss of glutamine synthetase (GS) induced by glutamate that was associated with an increase in glutamate-aspartate transporter (GLAST) in brain organotypic cultures from post-natal Wistar rats. Additionally, it was observed that rutin increased the glutamate uptake in cerebral cortex slices from adult Wistar rats. We conclude that rutin is a neuroprotective agent that prevents glutamate excitotoxicity and thereof suggest that this effect involves the regulation of astrocytic metabolism.

Microglia cells are the immune effector in the Central Nervous System (CNS). However, studies have showed that they contribute more to glioma progression than to its elimination. Rutin and its aglycone quercetin are flavonoids present in many fruits as well as plants and have been demonstrated to bear anti-inflammatory, antioxidant and antitumor properties also to human glioblastoma cell lines. Previous studies also demonstrated that rutin, isolated from the Brazilian plant Dimorphandra mollis Bent., presents immunomodulatory effect on astrocytes and microglia. In this study, we investigate the antitumor and immunomodulatory properties of rutin and its aglycone quercetin on the viability of glioma cells alone and under direct and indirect interaction with microglia. Flavonoid treatment of rat C6 glioma cells induced inhibition of proliferation and migration, and also induced microglia chemotaxis that was associated to the up regulation of tumor necrosis factor (TNF) and the down regulation of Interleukin 10 (IL-10) at protein and mRNA expression levels, regulation of mRNA expression for chemokines CCL2, CCL5 and CX3CL1, and Heparin Binding Growth Factor (HDGF), Insulin-like growth factor (IGF) and Glial cell-derived neurotrophic factor (GDNF) growth factors. Treatment of human U251 and TG1 glioblastoma cells with both flavonoids also modulated negatively the expression of mRNA for IL-6 and IL-10 and positively the expression of mRNA for TNF characterizing changes to the immune regulatory profile. Treatment of microglia and C6 cells either in co-cultures or during indirect interaction, via conditioned media from glioma cells treated with flavonoids or via conditioned media from microglia treated with flavonoids reduced proliferation and migration of glioma cells. It also directed microglia towards an inflammatory profile with increased expression of mRNA for IL-1β, IL-6, IL-18 and decreased expression of mRNA for nitric oxide synthase 2 (NOS2) and prostaglandin-endoperoxide synthase 2 (PTGS2), arginase and transforming growth factor beta (TGF-β), as well as Insulin-like growth factor (IGF). Treatment of U251 cells with flavonoids also reduced tumorigenesis when the cells were xenotransplanted in rat brains, and directed microglia and also astrocytes in the microenvironment of tumor cell implantation as well as in the brain parenchyma to a not favorable molecular inflammatory profile to the glioma growth, as observed in cultures. Together these results demonstrate that the flavonoid rutin and its aglycone quercetin present antiglioma effects related to the property of modulating the microglial inflammatory profile and may be considered for molecular and preclinical studies as adjuvant molecules for treatment of gliomas.

Astrocytes preserve the brain microenvironment homeostasis in order to protect other brain cells, mainly neurons, against damages. Glial cells have specific functions that are important in the context of neuronal survival in different models of central nervous system (CNS) diseases. Microglia are among these cells, secreting several molecules that can modulate astrocyte functions. Although 1,2-dihydroxybenzene (catechol) is a neurotoxic monoaromatic compound of exogenous origin, several endogenous molecules also present the catechol group. This study compared two methods to obtain astrocyte-enriched cultures from newborn Wistar rats of both sexes. In the first technique (P1), microglial cells began to be removed early 48 h after primary mixed glial cultures were plated. In the second one (P2), microglial cells were late removed 7 to 10 days after plating. Both cultures were exposed to catechol for 72 h. Catechol was more cytotoxic to P1 cultures than to P2, decreasing cellularity and changing the cell morphology. Microglial-conditioned medium (MCM) protected P1 cultures and inhibited the catechol autoxidation. P2 cultures, as well as P1 in the presence of 20% MCM, presented long, dense, and fibrillary processes positive for glial fibrillary acidic protein, which retracted the cytoplasm when exposed to catechol. The Ngf and Il1beta transcription increased in P1, meanwhile astrocytes expressed more Il10 in P2. Catechol decreased Bdnf and Il10 in P2 cultures, and it decreased the expression of Il1beta in both conditions. A prolonged contact with microglia before isolation of astrocyte-enriched cultures modifies astrocyte functions and morphology, protecting these cells against catechol-induced cytotoxicity.

Neuroinflammation is one of the most frequently studied topics of neurosciences as it is a common feature in almost all neurological disorders. Although the primary function of neuroinflammation is to protect the nervous system from an insult, the complex and sequential response of activated glial cells can lead to neurological damage. Depending on the type of insults and the time post-insult, the inflammatory response can be neuroprotective, neurotoxic, or, depending on the glial cell types, both. There are multiple pathways activated and many bioactive intermediates are released during neuroinflammation. One of the most common one is the kynurenine pathway, catabolizing tryptophan, which is involved in immune regulation, neuroprotection, and neurotoxicity. Different models have been used to study the kynurenine pathway metabolites to understand their involvements in the development and maintenance of the inflammatory processes triggered by infections. Among them, the parasitic infection

Traumatic brain injury (TBI) is a critical health problem worldwide, with a high incidence rate and potentially severe long-term consequences. Depending on the level of mechanical stress, astrocytes react with complex morphological and functional changes known as reactive astrogliosis. In cases of severe tissue injury, astrocytes proliferate in the area immediately adjacent to the lesion to form the glial scar, which is a major barrier to neuronal regeneration in the central nervous system. The flavonoid agathisflavone has been shown to have neuroprotective, neurogenic, and immunomodulatory effects and could have beneficial effects in situations of TBI. In this study, we investigated the effects of agathisflavone on modulating the responses of astrocytes and neurons to injury, using the in vitro scratch wound model of TBI in primary cultures of rat cerebral cortex. In control conditions, the scratch wound induced an astroglial injury response, characterized by upregulation of glial fibrillary acidic protein (GFAP) and hypertrophy, together with the reduction in proportion of neurons within the lesion site. Treatment with agathisflavone (1 μM) decreased astroglial GFAP expression and hypertrophy and induced an increase in the number of neurons and neurite outgrowth into the lesion site. Agathisflavone also induced increased expression of the neurotrophic factors NGF and GDNF, which are associated with the neuroprotective profile of glial cells. These results demonstrate that in an in vitro model of TBI, the flavonoid agathisflavone modulates the astrocytic injury response and glial scar formation, stimulating neural recomposition.

In the central nervous system, astrocytes and microglia contribute to homeostasis, regulating the immune response to infectious agents. Neospora caninum is an obligate intracellular protozoan that infects different animal species and it is encysted in their nervous tissue while triggering an immune response modulated by glia. This study aimed to evaluate the infection of primary cultures of rat glial cells by N. caninum through the catabolites of tryptophan, the expression of inflammatory mediators and the integrity of neural tissue. Infection with this coccidium resulted in morphological and functional changes, particularly astrogliosis and microgliosis, and increased the expression of the inflammatory mediators TNF, IL1β, IL-10, and arginase, as well as mRNA for CCL5 and CCL2, molecules involved in the CNS chemotaxis. The infection with N. caninum in glial cells also triggered the activation of the tryptophan pathway, characterized by increased kynurenine 2,3 monooxygenase (KMO) mRNA expression, and by the production of the excitotoxin quinolinic acid (QUIN). Moreover, glia-neuron co-cultures, when exposed to the secretome derived from N. caninum infected glial cells, presented greater neurons distribution and formation of neurite extensions, associated to morphological changes in astrocytes compatible with neuro-preservation. Considering that the tryptophan catabolism is associated to immune response, these findings suggest that glial activation in N. caninum infection should be responsible for modulating the inflammatory status in an attempt to restore the nervous system homeostasis, since excessive inflammatory response can cause irreversible damage to tissue preservation.

O antígeno eritrocitário canino 1 (AEC 1) é o grupo sanguíneo mais imunogênico em cães, podendo as transfusões sanguíneas desencadearem alguns efeitos indesejáveis nos pacientes veterinários. Estes estão diretamente associados à transfusões incompatíveis. O presente trabalho teve como objetivo estudar a frequência do grupo sanguíneo AEC 1 em cães doadores de sangue de um banco de sangue de Salvador-BA, Brasil, e calcular o risco de administrar sangue incompatível tanto em uma primeira quanto em uma segunda transfusão. Foram avaliados 203 cães de diversas raças, de ambos os sexos, com idade entre 1 e 8 anos, peso a partir de 28 kg, sem nenhum grau de parentesco originários de Salvador – BA, Brasil, para pesquisa da frequência do tipo sanguíneo AEC 1, por meio de testes de imunocromatografia e citometria de fluxo para tipagem sanguínea. E calculado o risco de transfusão sanguínea incompatível tanto em uma primeira como em uma segunda transfusão. A frequência do grupo AEC 1 variou entre as raças estudadas de 0% a 100%, porém com uma positividade média de 62,07% (126/203). O menor risco de um animal AEC 1 negativo receber sangue AEC 1 positivo, dentro do grupo dos animais avaliados foi de 0,92% em uma primeira transfusão e o risco do mesmo animal receber sangue incompatível para o gruo AEC 1 na segunda transfusão foi de 0,008%. Quanto ao maior risco de um animal AEC 1 negativo receber sangue AEC 1 positivo destes animais foi de 69,12% e o risco do mesmo receber sangue incompatível para o AEC 1 foi de 47,77%. A frequência do grupo AEC 1 variou entre as raças estudadas e o risco de transfusões incompatíveis variou de acordo com as raças doadoras e receptoras, mas esse risco pode ser anulado se sempre forem realizados os testes para tipagem sanguínea junto com a prova de reação cruzada para compatibilidade. The dog erythrocyte antigen 1 (DEA 1) is the most immunogenic blood group in dogs, and blood transfusions may trigger some undesirable effects in veterinary patients, which are directly associated with incompatible transfusions. The present study aimed to investigate the frequency of positive DEA 1 blood group in blood donor dogs from a blood bank in Salvador, Bahia, Brazil, and also to calculate the risk of managing incompatible blood in both first and second transfusion. A number of 203 dogs of different breeds, aged between 1 and 8 years, weighing 28 kg, with no degree of kinship and of both sexes in Salvador - BA, Brazil were evaluated to investigate the blood type DEA 1 frequency, by means of chromatography and flow cytometry tests for blood typing. The risk of incompatible blood transfusion in either a first or a second transfusion was also calculated. The frequency of the DEA 1 group ranged from 0% to 100% in various breeds, but with a mean positivity of 62.07% (126/203). And the lowest risk of an DEA 1 negative animal receiving DEA 1 positive blood within the group of animals evaluated was 0.92% at a first transfusion; and the risk of the same animal receiving incompatible blood for the DEA group 1 in the second transfusion was 0.008%. The highest risk of an DEA 1 negative animal receiving DEA 1 positive blood from these animals was 69.12%; and the risk of receiving incompatible blood for DEA 1 was 47.77%. In conclusion, the frequency of the DEA 1 group varied between the studied breeds and the risk of incompatible blood transfusions varies according to donor and recipiente breeds, but this can be overridden if blood typing tests are performed along with the cross-reaction test for compatibility.

Objectives: to estimate the detection rate on prenatal screening pathologies on paper filter in the South and Southwest in the State of Bahia, as well as to delineate the epidemiological profile of these pregnant women, calculate and estimate the rate of adherence and the coverage of the Program. Methods: a descriptive study was carried out from August 2013 to August 2015, and the data were obtained from the Labimuno/ICS/UFBA. Results: 64,743 pregnant women were included; the mean ages were 25 years for the Southwest macro-region and 23 for the South. The results on the screening tests showed positivity of 0.13% and 0.29% for HBsAg, 0.17% and 0.22% for cytomegalovirus, 0.07% and 0.09% for HCV, 0.13% and 0.38% for HTLV, 0.04% and 0.19% for HIV, 1.2% and 2.84% for syphilis, and 0.54% and 0.73% for toxoplasmosis in the Southwest and South macro-regions, respectively. The estimates on coverage were considered satisfactory. Sickle cell anemia showed positivity of 0.02% and of 0.04% and 5% and 6.3% presented sickle cell trait in the Southwest and South macro-regions, respectively. Conclusions: the frequencies of infections in maternal-fetal health were considered low, highlighting on syphilis and the presence for sickle cell trait. Resumo Objetivos: estimar a taxa de detecção de patologias da TPN em papel de filtro nas regiões Sul e Sudoeste do Estado da Bahia, bem como delinear o perfil epidemiológico dessas gestantes, calcular e estimar a taxa de adesão e abrangência de cobertura do Programa. Métodos: estudo descritivo, de agosto 2013 a agosto de 2015, de dados obtidos do Labimuno/ICS/UFBA. Resultados: foram incluídas 64.743 gestantes; as médias das idades foram de 25 anos para a macrorregião Sudoeste e 23 para Sul. Os resultados de exames de triagem mostraram positividade de 0,13% e 0,29% para AgHBs, 0,17% e 0,22% para citomegalovírus, 0,07% e 0,09% para VHC, 0,13% e 0,38% para HTLV, 0,04% e 0,19% para HIV, 1,2% e 2,84% para sífilis, e 0,54% e 0,73% para toxoplasmose, para a macrorregião Sudoeste e Sul, respectivamente. As estimativas de cobertura foram consideradas satisfatórias. A anemia falciforme mostrou positividade de 0,02% e de 0,04% e 5% e 6,3% apresentaram o traço falcêmico, macrorregião Sudoeste e Sul, respectivamente. Conclusões: as frequências das infecções na saúde materno-fetal foram consideradas baixas, com destaque para sífilis e para a presença do traço falcêmico.

This study aimed to investigate the antitumor and immunomodulatory properties of the flavonoid apigenin (5,7,4'-trihydroxyflavone), which was extracted from Croton betulaster Mull, in glioma cell culture using the high-proliferative rat C6 glioma cell line as a model. Apigenin was found to have the ability to reduce the viability and proliferation of C6 cells in a time-dependent and dose-dependent manner, with an IC50 of 22.8 µmol/l, 40 times lower than that of temozolomide (1000 µmol/l), after 72 h of apigenin treatment. Even after C6 cells were treated with apigenin for 48 h, high proportions of C6 cells entered apoptosis (39.56%) and autophagy (22%) as shown by flow cytometry using annexin V/propidium iodide and acridine orange staining, respectively. In addition, the flavonoid apigenin induced cell accumulation in the G0/G1 phase of the cell cycle and inhibited glioma cell migration efficiently. Moreover, apigenin induced astroglial differentiation and morphological changes in C6 cells, characterized by increased expression of glial fibrillary acidic protein and decreased expression of nestin protein, a typical marker of neuronal precursors. The immunomodulating effects of apigenin were also characterized by a change in the inflammatory profile as evidenced by a significant decrease in interleukin-10 and tumor necrosis factor production and increased nitric oxide levels. Because apigenin can induce differentiation, apoptosis, and autophagy, can alter the profile of cytokines involved in regulating the immune response, and can reduce the survival, growth, proliferation, and migration of C6 cells, this flavonoid may be considered a potential antitumor drug for the adjuvant treatment of malignant gliomas.

The shortage of dog blood donors in veterinary emergencies can lead to blood transfusions between animals whose blood type has not been identified. The antibody profile serves as a warning sign for animals that require a second blood transfusion, which is only advisable from compatible donor dogs. This article focuses on determination of anti-DEA 1 antibodies using the flow cytometry technique in dogs that have undergone a transfusion using DEA 1-positive blood, compared to results obtained from crossmatching. Blood from 18 DEA 1-positive donors ranked according to the chromatographic technique was used to transfuse thirty-three animals with unknown blood types and which demonstrated negative crossmatching to donors. On post-transfusion days 7, 14, 21 and 28, 45% and 27% of the animals tested positive for the anti-DEA 1 antibody, through crossmatching and flow cytometry, respectively. Detecting antibodies using the flow cytometric technique has high specificity and sensitivity, while crossmatching methods are highly sensitive but manifest low specificity. Following blood transfusion, animals that did not present as positive through crossmatching or flow cytometry were considered different from all other DEA 1-positive blood groups.

Rutin is a glycosylated flavonoid present in many fruits and plants that has been demonstrated to have anti-inflammatory and antioxidant properties. However, little is known about the mechanisms underlying microglial activation and its effects on the regulation of cytokines and chemokines associated with inflammatory responses in the central nervous system. In this study we examined the effect of rutin on resting or lipopolysaccharide (LPS)-stimulated microglia and characterized their modulation to an activated M1 phenotype or an alternatively activated M2 phenotype. Microglial cells were treated with rutin (1–100 μM); alternatively, microglial cells were stimulated with LPS and the cells were then treated with rutin (50 μM). The results revealed that rutin treatment was not toxic to microglial cells and induced a dose-dependent increase in microglial proliferation associated with changes in morphology after 24 h of treatment. Rutin also induced microglial activation characterized by an increase in OX-42 positive cells and a large proportion of cells with a CD150/CD206-positive M2 phenotype. Rutin also induced a decrease in the mRNA levels of TNF, IL1β, IL6 and iNOS, reduced the production of IL6, TNF, and nitric oxide, and increased production of the M2 regulatory cytokine IL10 and arginase. Rutin also significantly inhibited the LPS-induced expression of PTGS2, IL18 and TGFβ mRNA. These findings show that rutin has the ability to promote microglial proliferation and induces microglial polarization to the M2 profile when cells are stimulated with LPS. These results point this flavonoid as a possible alternative in the treatment or prevention of neurodegenerative disorders.

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.

Neospora caninum is a parasite that infects many animal species and has tropism for various tissues, particularly the nervous system, where it generally remains in cysts. Under N. caninum infection, glial cells activate immune responses by a Th2 profile, suggesting an immunologically privileged environment that controls parasite proliferation, with neuronal preservation. In this study, we investigated the role of soluble neurotrophic factors released by glial cells on neuronal integrity during N. caninum infection in vitro. Primary cultures of rat glial cells enriched in astrocytes were infected with N. caninum tachyzoites (1:1) for 24 hr. Neuron-glia co-cultures were cultured for 24 hr with conditioned medium from glial cells infected with N. caninum (CMNc) and from uninfected cultures (control). Cell viability was determined through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test; astrocyte morphology and reactivity were determined through immunocytochemistry for glial fibrillar acid protein (GFAP) and the integrity of neurons through immunocytochemistry for β-tubulin III. Expression of inflammatory cytokines and neurotrophic factors was determined through RT-qPCR. The MTT test demonstrated that 1:1 was the best parasite/host cell ratio, considering that it was enough to increase metabolism of glial cells when compared with control cultures and was not cytotoxic after 48 hr infection. N. caninum-infected glial cultures responded with astrogliosis characterized by an increase in GFAP expression and increase in IL-10 (2-fold), BDNF (1.6-fold), and NGF (1.7-fold) gene expression. In the neuron/glia co-cultures, it was observed that treatment with CMNc induced neuritis outgrowth without toxicity. Together, these results show that modulatory mechanisms by neurotrophic factors derived from glial cells, primarily astrocytes during the N. caninum infection, can favor neuroprotection.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-β antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-β, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-β did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-β, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-β cytokines participate in parasite proliferation control mechanisms.

Studies have shown cases of poisoning with plants from the genus Crotalaria (Leguminosae) mainly in animals. They induce damages in the central nervous system (CNS), which has been attributed to toxic effects of the pyrrolizidine alkaloid (PA) monocrotaline (MCT). Previously we demonstrated that both MCT and dehydromonocrotaline (DHMC), its main active metabolite, induce changes in the levels and patterns of expression of the main protein from astrocyte cytoskeleton, glial fibrillary acidic protein (GFAP). In this study we investigated the effect of MCT on rat cortical astrocyte/neuron primary co-cultures. Primary cultures were exposed to 10 or 100 μM MCT. The MTT test and the measurement of LDH activity on the culture medium revealed that after 24h exposure MCT was not cytotoxic to neuron/astrocyte cells. However, the cell viability after 72 h treatment decreased in 10-20%, and the LDH levels in the culture medium increased at a rate of 12% and 23%, in cultures exposed to 10 or 100 μM MCT. Rosenfeld staining showed vacuolization and increase in cell body in astrocytes after MCT exposure. Immunocytochemistry and Western blot analyses revealed changes on pattern of GFAP and βIII-tubulin expression and steady state levels after MCT treatment, with a dose and time dependent intense down regulation and depolarization of neuronal βIII-tubulin. Moreover, treatment with 100 μM MCT for 12h induced GSH depletion, which was not seen when cytochrome P450 enzyme system was inhibited indicating that it is involved in MCT induced cytotoxicity in CNS cells.

In this study, we investigated the effects of the flavonoid rutin (3,3',4',5,7-pentahydroxyflavone-3-rutinoside) on glioma cells, using the highly proliferative human cell line GL-15 as a model. We observed that rutin (50-100μM) reduced proliferation and viability of GL-15 cells, leading to decreased levels of ERK1/2 phosphorylation (P-ERK1/2) and accumulation of cells in the G2 phase of the cell cycle. On the other hand, 87.4% of GL-15 cells exposed to 100μM rutin entered apoptosis, as revealed by flow cytometry after AnnexinV/PI staining. Nuclear condensation and DNA fragmentation were also observed, further confirming that apoptosis had occurred. Moreover, the remaining cells that were treated with 50μM rutin presented a morphological pattern of astroglial differentiation in culture, characterised by a condensed cell body and thin processes with overexpression of GFAP. Because of its capacity to induce differentiation and apoptosis in cultured human glioblastoma cells, rutin could be considered as a potential candidate for malignant gliomas treatment.

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-alpha, IFN-gamma, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-gamma was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8+/-0.6 and 3.7+/-0.6 pg TNF-alpha/mg protein and 2.7+/-0.69 and 4.1+/-0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24h post-infection (2.3+/-0.8 pg/mg protein) until 72 h (4.2+/-1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-gamma; high levels of TNF-alpha and NO may represent an effective response by infected glial cells against N. caninum.

Texto completo: acesso restrito. p.199-212 Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-09-05T12:45:29Z No. of bitstreams: 1 Hum Exp Toxicol-2010-de Oliveira-199-212.pdf: 859208 bytes, checksum: bf3926c1528ac250894eed99bcac8cda (MD5) Approved for entry into archive by Patricia Barroso(pbarroso@ufba.br) on 2013-09-06T15:24:44Z (GMT) No. of bitstreams: 1 Hum Exp Toxicol-2010-de Oliveira-199-212.pdf: 859208 bytes, checksum: bf3926c1528ac250894eed99bcac8cda (MD5) Made available in DSpace on 2013-09-06T15:24:44Z (GMT). No. of bitstreams: 1 Hum Exp Toxicol-2010-de Oliveira-199-212.pdf: 859208 bytes, checksum: bf3926c1528ac250894eed99bcac8cda (MD5) Previous issue date: 2010 The exposure to benzene is a public health problem. Although the most well-known effect of benzene is hematopoietic toxicity, there is little information about the benzene and its metabolites effects on the central nervous system (CNS). This study examined the toxic effects of 1,2-dihydroxybenzene (catechol), a benzene metabolite, to human glioblastoma GL-15 cells. GL-15 cell cultures were used as a model to provide more information about the toxic effects of aromatic compounds to the CNS. Catechol induced time- and concentration-dependent cytotoxic effects. Morphological changes, such as the retraction of the cytoplasm and chromatin clumping, were seen in cells exposed to 200 μM catechol for 48 hours. In cells exposed to 600 μM catechol for 48 hours, 78.0% of them presented condensed nuclei, and the Comet assay showed DNA damage. The percentage of cells labeled with annexin V (apoptotic cells) was greater in the group exposed to catechol (20.7%) than in control cells (0.4%). Exposure to catechol at concentrations greater than 100 μM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 μM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 micromol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 micromol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 micromol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld's staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 micromol/L rutin. Enhanced TNFalpha release was observed in astrocyte primary cultures treated with 100 micromol/L rutin and in mixed primary cultures treated with 50 and 100 micromol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFalpha production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.

Rutin is a flavonoid obtained from Dimorphandra mollis (Benth.), a medicinal Brazilian plant used as antioxidative, antihemorrhagic, and blood vessel protector. The present study has examined its effects on the viability and function of immune system cells in vitro. Rat spleen and thymus cells were cultured with 10 nM, 1 microM, and 10 microM of the drug in the presence or absence of PWM, LPS, or ConA mitogens. Cellular proliferation was analyzed by H(3)-thymidin uptake and IFN-gamma and IL-10 were measured by ELISA after 48 and 72 hr. Viability was measured by flow cytometry using Annexin V and PI after 24 and 48 hr. The flavonoid rutin inhibited splenocytes and thymocytes proliferation under ConA stimulation observed by an increase on apoptosis levels of thymocytes stimulated with PWM in 24 hr and on splenocytes stimulated with PWM in 48 hr. Function studies showed a decrease on IFN-gamma production by splenocytes and thymocytes stimulated with PWM or ConA. Spleen cells cultured with LPS and rutin showed a decrease on apoptosis after 24 hr and an increase on the IL-10 levels after 48 hr. There was no significant variation on the necrosis rate, viability, and function of cells treated with rutin in the absence of mitogenic stimulus.

International audience; The malignant gliomas are very common primary brain tumors with poor prognosis, which require more effective therapies than the current used, such as with chemotherapy drugs. In this work, we investigated the effects of several polyhydroxylated flavonoids namely, rutin, quercetin (F7), apigenin (F32), chrysin (F11), kaempferol (F12), and 3',4'-dihydroxyflavone (F2) in human GL-15 glioblastoma cells. We observed that all flavonoids decreased the number of viable cells and the mitochondrial metabolism. Furthermore, they damaged mitochondria and rough endoplasmic reticulum, inducing apoptosis. Flavonoids also induced a delay in cell migration, related to a reduction in filopodia-like structures on the cell surface, reduction on metalloproteinase (MMP-2) expression and activity, as well as an increase in intra- and extracellular expression of fibronectin, and intracellular expression of laminin. Morphological changes were also evident in adherent cells characterized by the presence of a condensed cell body with thin and long cellular processes, expressing glial fibrillary acidic protein (GFAP). Therefore, these flavonoids should be tested as potential antitumor agents in vitro and in vivo in other malignant glioma models.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-06-20T15:48:31Z No. of bitstreams: 1 17_v.14_3.pdf: 967437 bytes, checksum: d1eec427a1bb32fa43a0dbf456d81280 (MD5) Made available in DSpace on 2017-06-20T15:48:31Z (GMT). No. of bitstreams: 1 17_v.14_3.pdf: 967437 bytes, checksum: d1eec427a1bb32fa43a0dbf456d81280 (MD5) Previous issue date: 2015-09 Introdução: sangue tipificado e compatível são parâmetros importantes para uma transfusão sanguínea segura. Transfusão realizada apenas após a prova de compatibilidade não garante igualdade de tipos sanguíneos entre receptor e doador, devido ao fato da não evidência de aloanticorpos naturais nos cães contra os vários tipos sanguíneos. O tipo AEC (Antígeno Eritrocitário Canino) 1.1, por ser o mais imunogênico, causando sensibilização em animais que não possuam esse tipo, apresenta relevância pós-transfusional. Objetivo: o estudo objetivou avaliar o perfil hematológico e bioquímico dos animais receptores de sangue AEC 1.1 positivo, através de hemograma, dosagem de proteínas plasmáticas totais e dosagens séricas de creatinina e gama-glutamiltransferase, e monitorar a presença de aloanticorpos no período pós-transfusional através da prova de reação cruzada. Metodologia: cães com tipo sanguíneo desconhecido foram submetidos à prova de reação cruzada e, a seguir, transfundidos com sangue AEC 1.1 positivo; prova de reação cruzada e os testes laboratoriais foram avaliados nos dias 7, 14, 21 e 28 pós-transfusão. Resultados: no período avaliado, os animais apresentaram índices normais de leucograma, plaquetas, proteínas plasmáticas totais, creatinina e gama-glutamiltransferase. A média dos valores de eritrogramas estiveram abaixo da normalidade, enquanto as provas de reação cruzada apresentaram valores progressivos a cada semana avaliada, evidenciando presença de aloanticorpos ou autoanticorpos em alguns receptores. Conclusão: os parâmetros hematológicos e bioquímicos avaliados nos animais pós-transfundidos foram adequados independentemente do tipo sanguíneo do receptor ter sido igual ou não ao do doador. E a produção de aloanticorpos observada em alguns cães não interferiu em sua normalidade. Salvador

Highlight the relevance of topics studied for professional practice and associate approaches provided by different areas of knowledge are pointed as essential aspects for significant learning. Contextualize the study of metabolic pathways, linking the clinical use and expanding the vision with the approach of cellular and molecular biology discipline was the motivation for the development of the strategy described and evaluated here. In this work, starting from the concept of active methodology of teaching and learning was developed a methodological strategy to approach the carbohydrate and lipid metabolism. This strategy included: questioning the content through the clinical case study on diabetes mellitus and hypercholesterolemia, individual and collective study in the external space the classroom with the help of Moodle tool, classroom discussion accompanied by the teacher, preparation of seminar, evaluation of the content through individual written test and evaluation of the method. Analysis of student involvement with method indicates an average frequency of 98% in the practical class of Biochemistry discipline, effective participation in the preparation of seminars, an increase of 2 points in average of individual written evaluation. As for the fact that the cases were studied in two curricular components, the answers show that 92% of students feel more compression. Only 6% of students think unnecessary to interdisciplinary approach. As for the different steps of the method, the answers show that 99% of students consider how relevant the initial self-study and discussions in class. However, only 50% of students appreciated the use of Moodle tool. Thus, student responses indicated the perception of the effectiveness of the method for their ability to: stimulate interest in learning, stimulate the search for answers through research and the building of learning.

Submitted by Ramon El-Bachá (ramon@ufba.br) on 2016-09-30T20:33:12Z No. of bitstreams: 1 de Jesus et al 2014.pdf: 915416 bytes, checksum: 4d7c829f8d4091e11a0ce03d46824d54 (MD5) Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-10-07T11:54:37Z (GMT) No. of bitstreams: 1 de Jesus et al 2014.pdf: 915416 bytes, checksum: 4d7c829f8d4091e11a0ce03d46824d54 (MD5) Made available in DSpace on 2016-10-07T11:54:37Z (GMT). No. of bitstreams: 1 de Jesus et al 2014.pdf: 915416 bytes, checksum: 4d7c829f8d4091e11a0ce03d46824d54 (MD5) Previous issue date: 2014-10-27 CNPq/CAPES Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-g decreased the parasite proliferation and down regulated nitric oxide (NO) production in astrocyte/microglia cultures. However,it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-gamma or 1.0 mg/mL of LPS on infected rat neuron/glial co-cultures. After 72 h of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-gamma decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-gamma and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-gamma induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not), meanwhile PGE2 release was improved in IFN-gamma/infected or LPS/infected cells. Although IFN-gamma significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%). The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-gamma stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in central nervous system (CNS).

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-06-20T13:55:24Z No. of bitstreams: 1 18_v.13_3.pdf: 906811 bytes, checksum: d27e0d40ceffc14c3722400a28c02283 (MD5) Made available in DSpace on 2017-06-20T13:55:25Z (GMT). No. of bitstreams: 1 18_v.13_3.pdf: 906811 bytes, checksum: d27e0d40ceffc14c3722400a28c02283 (MD5) Previous issue date: 2014-09 Introdução: A glicoproteína laminina é normalmente encontrada em formas poliméricas que constituem o principal elemento formador das membranas basais. Apresenta diversos domínios de reconhecimento celular, sendo capaz de induzir várias respostas biológicas. Em situações patológicas, como crescimento tumoral ou inflamação, a matriz extracelular torna-se exposta à ação de proteases que, entre outros efeitos, podem degradar elementos da matriz. Objetivo: Neste estudo, serão discutidos os referenciais teóricos que norteiam a construção da hipótese de que a proteólise de polímeros de laminina, no contexto da inflamação ou crescimento tumoral, pode apresentar função reguladora associada à manutenção da saúde tecidual. Metodologia: Trata-se de uma revisão da literatura, realizada a partir da seleção e análise de artigos originais e de revisão, publicados nos últimos dez anos, na base de dados Biblio. Figuras Literatura Internacional em Ciências da Saúde (PubMed). Resultado: A aplicação do descritor “Proteólise e membrana basal” possibilitou a seleção de 87 artigos. Entre estes, apenas 12 referiam a proteólise de elementos da membrana basal com descrição de atividade biológica associada eles. A pesquisa, utilizando como descritor “Proteolysis and laminin”, permitiu a seleção de 70 artigos, dos quais apenas 9 estavam associados à questão de interesse. Entre os 9, 4 foram coincidentes com o resultado da primeira busca. Assim, um total de 17 artigos foi selecionado, sendo apenas 11 encontrados e utilizados para a construção do embasamento teórico da hipótese levantada. Conclusão: O presente artigo apresenta o racional e o embasamento teórico da construção da hipótese de que a proteólise de polímeros de laminina, no contexto da inflamação ou crescimento tumoral, pode apresentar função reguladora associada à manutenção da saúde tecidual. Consequentemente, o artigo constitui-se em ponto de partida para o desenvolvimento de novas pesquisas sobre o tema. O aprofundamento do conhecimento pode ser relevante no campo da biologia celular e do desenvolvimento, como também no campo da bioengenharia, considerando a possibilidade do uso. Salvador

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.

Among the cells that constitute the central nervous system (CNS), astrocytes are the most abundant cells in the brain. They originate from the neural tube, differently from other glial cells of the astrocyte-like, enteric glia, which originate from the neural crest. Cerebral astrocytes interact with other brain cells such as neurons, mediated by cellular contact involving extracellular matrix (ECM) elements and the integrin of astrocytes. Interactions can also occur through exchange of soluble molecules, cytokines, and growth factors, delivered by astrocytes and the interacting cells. Of the growth factors secreted by astrocytes, members of the transforming growth factor (TGF) beta family, especially TGF-β1, are highly important. TGF-β1 acts in brain development, in adult brain homeostasis, and also in orchestrating the brain’s response to injury and/or aging. These cell–cell interactions depend on the cellular membrane plasticity expressed by biophysical forces of interaction. In parallel to the properties of normal astrocytes, a growing astrocytoma develops new malignant behavior and interactions with parenchyma that differ from those of normal astrocytes. In this chapter, we analyze important points that characterize tumor progression and discuss new therapeutic approaches to treat these tumors.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-06-19T18:31:02Z No. of bitstreams: 1 11_v.12_esp..pdf: 551208 bytes, checksum: 7bfb8759c768fa0ea962b92e19e01e2d (MD5) Made available in DSpace on 2017-06-19T18:31:02Z (GMT). No. of bitstreams: 1 11_v.12_esp..pdf: 551208 bytes, checksum: 7bfb8759c768fa0ea962b92e19e01e2d (MD5) Previous issue date: 2013-12 Introdução: O estudo hematológico de doadores de sangue e a organização dos dados de forma a criar um perfil adequado destes são importantes para assegurar compatibilidades e identificar possíveis discrasias sanguíneas como plaquetopenia, anemia, leucopenia ou qualquer alteração dos elementos figurados do sangue. Objetivo: Doadores, especialmente os de repetição, que apresentaram valores hematológicos fora daqueles referenciados como normais para a espécie, ou que apresentaram grandes variações individuais num curto espaço de tempo sem uma causa pré-determinada, devem receber um acompanhamento mais próximo, a fim de instituir medidas diagnósticas e terapêuticas cabíveis ou mesmo afastamento temporário ou definitivo do programa de doadores de sangue. Metodologia: O presente estudo avaliou parâmetros hematológicos (contagem global de hemácias, volume globular, hemoglobina, contagem global e diferencial de leucócitos, contagem global de plaquetas e proteína total plasmática) de 12 cães de raças diversas, pesando entre 28 e 46 Kg, sem distinção de sexo e cujas idades variaram entre 1 e 8 anos. Estes cães doaram sangue com intervalo de 30 dias entre as coletas, num período de 12 a 24 meses, sendo que todos receberam o mesmo padrão nutricional e manejo. Todos os animais passaram por triagens clínicas e laboratoriais para atestarem ausência de doenças infectocontagiosas (leishmaniose, dirofilariose, brucelose, erliquiose e babesiose), bem como estado vacinal adequado quanto às principais doenças infecciosas dos caninos. O volume total médio coletado por cão foi de 450 mL (±20 mL/evento), por meio de venopunção atraumática de jugular. Não foram utilizadas medicações sedativas e/ou anestésicas. Resultados: Os resultados não mostram diferenças estatísticas nos parâmetros avaliados no período de 12 e 24 meses, estando as médias dentro dos padrões de normalidade para a espécie, exceto em contagem plaquetária. Conclusão: Conclui-se, neste estudo, que a produção dos elementos figurados destes animais apresentou um padrão de constância cursando com recuperação pós-doação, e que os cães estão aptos a doar a cada 30 dias, num período de até 24 meses. Novos estudos devem ser realizados, a fim de avaliar o comportamento hematológico de doadores de repetição, estendendo-se o período de observação. Salvador

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-06-19T18:26:28Z No. of bitstreams: 1 7_v.12_esp..pdf: 377750 bytes, checksum: 0c424a1e56a427392213a1ba26e85538 (MD5) Made available in DSpace on 2017-06-19T18:26:28Z (GMT). No. of bitstreams: 1 7_v.12_esp..pdf: 377750 bytes, checksum: 0c424a1e56a427392213a1ba26e85538 (MD5) Previous issue date: 2013-12 Introdução: A toxicidade do Catecol está relacionada com a formação de quinonas seguido de promoção de apoptose, ambos os efeitos suprimidos pela superóxido dismutase. Objetivo: Neste trabalho foi estudada a ação antioxidante do ácido ascórbico em células GL-15 após o estresse oxidativo induzido pelo catecol, a fim de comparar os dados e gerar mais informações sobre a citotoxicidade do catecol nesta linhagem celular. Metodologia: Uma vez que trabalhos anteriores mostraram que o valor de IC50 nestas células foi de 230 μM, após 72 horas de exposição, foram utilizadas as concentrações de catecol desde 300 μM até 600 μM. Conclusão: As concentrações sub-tóxicas de ácido ascórbico (600 μM - 3000μM) protegeram as células GL-15 contra danos oxidativos induzidos pelo catecol e reverteram completamente a formação de quinonas, confirmando que a toxicidade do catecol contra células GL-15 está essencialmente relacionada com a formação de quinonas reativas. Salvador

The effects of arborinine, an alkaloid extracted from Erthela bahiensis and of rutin, a flavonoid obtained from Dimorphandra mollis (Benth.), Brazilian medicinal plants, on the viability and function of a murine B-cell hybridoma as a tumor model were investigated. The flavonoid rutin at 50 microM induced an increase in the number of apoptotic cells of one- to fivefold and reductions in cellular proliferation and monoclonal antibody production. Less but still significant necrosis was also induced by rutin under the same experimental conditions. On the other hand, the alkaloid arborinine exerted no significant effects on the studied parameters.

Plants of Crotalaria genus (Leguminosae) present large amounts of the pyrrolizidine alkaloid monocrotaline (MCT) and cause intoxication to animals and humans. Therefore, we investigated the MCT-induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model. The comet test showed that 24h exposure to 1-500microM MCT and 500microM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42-64% and 53%, respectively. Cells exposed to 100-500microM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1-10microM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1-10microM). Apoptosis was also evidenced in cells treated with 100-500microM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500microM MCT. The data obtained provide support for heterogenic and multipotential effects of MCT on GL-15 cells, either interfering on cell growth and cytoskeletal protein expression, or inducing DNA damage and apoptosis and suggest that the response of glial cells to this alkaloid might be related to the neurological signs observed after Crotalaria intoxication.

Dehydromonocrotaline (DHMC) is the main monocrotaline active cytochrome P450's metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 microM DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 microM, and induced membrane damages at 500 microM. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 microM DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 microM), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/-30%) of the astrocytes exposed to 100-500 microM DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependent effect of DHMC on cultures of rat cortical astrocytes, leading to apoptotic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria.

Since the biological activity of [Ru III (NH 3 ) 4 (catechol)] + has never been tested, its cytotoxicity to glial cells was assayed and correlated with its redox properties. Coordinated catechol oxidizes faster than catechol in the presence of oxygen, but controlled potential electrolysis showed that its oxidation involves only one-electron. However, the oxidation of the free ligand by oxygen involves two electrons, which could generate more reactive oxygen species. Indeed, catechol was more cytotoxic than [Ru III (NH 3 ) 4 (catechol)] + complex to human glioblastoma GL-15 cells and also to rat astrocytes. [Ru III (NH 3 ) 4 (catechol)]-induced cytotoxicity was related to the generation of reactive oxygen species and [Ru II (NH 3 ) 4 (quinone)] 2+ . However, other mechanisms should be involved since antioxidant enzymes and deferoxamine only partially protected GL-15 cells. INTRODUCTION Ruthenium is an element that has industrial, pharmaceutical and medical applications. Ruthenium can be used in the reduction of noxious oxides from industrial emissions