Methylphenidate may improve mental fatigue in individuals with multiple sclerosis: A pilot clinical trial.

BACKGROUND: Fatigue is the most common symptom in multiple sclerosis (MS), previously attributed to dopamine imbalance. Evidence suggests that methylphenidate, a psychostimulant that increases striatal and prefrontal dopamine levels, is effective in reducing fatigue in various disorders. However, its effect on state vs. trait mental fatigue in MS is yet to be examined. METHODS: This pilot study investigates the efficacy of methylphenidate on decreasing self-reported mental fatigue in 12 individuals with MS in a double-blind, placebo-controlled, cross-over randomized clinical trial. RESULTS: Our results show that "state", but not "trait" MS-related fatigue, was reduced after 4 weeks of methylphenidate administration as compared to placebo.

Combined 1-Deoxynojirimycin and Ibuprofen Treatment Decreases Microglial Activation, Phagocytosis and Dopaminergic Degeneration in MPTP-Treated Mice.

Inflammation is a predominant aspect of neurodegenerative diseases and experimental studies performed in animal models of Parkinson's disease (PD) suggesting that a sustained neuroinflammation exacerbates the nigrostriatal degeneration pathway. The central role of microglia in neuroinflammation has been studied as a target for potential neuroprotective drugs for PD, for example nonsteroidal anti-inflammatory drugs (NSAIDs) and matrix metalloproteinases (MMP) inhibitors that regulates microglial activation and migration. The aim of this study was to investigate the neuroprotective response of the iminosugar 1-deoxynojirimycin (1-DNJ) and compare its effect with a combined treatment with ibuprofen. MPTP-treated mice were orally dosed with ibuprofen and/or 1-DNJ 1. Open-field test was used to evaluate behavioral changes. Immunohistochemistry for dopaminergic neurons marker (TH+) and microglia markers (Iba-1+; CD68+) were used to investigate neuronal integrity and microglial activation in the substantia nigra pars compacta (SNpc). The pro-inflammatory cytokines TNF-α and IL-6 were analysed by qPCR. Treatments with either 1-DNJ or Ibuprofen alone did not reduce the damage induced by MPTP intoxication. However, combined treatment with 1-DNJ and ibuprofen prevents loss of mesencephalic dopaminergic neurons, decreases the number of CD68+/ Iba-1+ cells, the microglia/neurons interactions, and the pro-inflammatory cytokines, and improves behavioral changes when compared with MPTP-treated animals. In conclusion, these data demonstrate that the combined treatment with a MMPs inhibitor (1-DNJ) plus an anti-inflammatory drug (ibuprofen) has neuroprotective effects open for future therapeutic interventions. Graphical Abstract MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a protoxicant that, after crossing the Blood Brain Barrier, is metabolized by astrocytic MAO-B to MPDP+, a pyridinium intermediate, which undergoes further two-electron oxidation to yield the toxic metabolite MPP+ (methyl-phenyltetrahydropyridinium) that is then selectively transported into nigral neurons via the mesencephalic dopamine transporter. In this study, we demonstrated that MPTP induced death of dopaminergic neurons, microgliosis, increase of gliapses, motor impairment and neuroinflammation in mice, which were inhibited by combined 1-deoxynojirimycin and ibuprofen treatment.

Effect of somatotropin on survival and diameter of bovine preantral follicles / Efeito da somatotropina na sobrevivência e no diâmetro de folículos pré-antrais de bovinos

The aim of this study was to evaluate the effect of Recombinant bovine somatotropin (rbST) on survival and diameter of bovine preantral ovarian follicles (PAOF) cultured in vitro. Ovaries were collected from adult cows and fragments of ovarian cortex were immediately fixed (non-cultured control) or cultured in vitro in α-MEM+ alone or containing 10, 50, 100 or 1,000ng/mL rbST. The fragments were processed for Classical Histology and Transmission Electron Microscopy. After one and seven days of culture, the percentage of normal follicles in the non-cultured control was superior (P< 0.05) to the follicles cultured in α-MEM+ alone or with different rbST concentrations. The oocyte and follicular mean diameter did not increase during the culture for one and seven days, both in media containing rbST and in the medium without this hormone. The only medium in which there was no reduction in follicular diameter with the time of culture was the medium without rbST. Ultrastructural damage in PAOF cultured in vitro was found. It is concluded that the use of rbST at different concentrations in in situ culture of bovine preantral follicles has no beneficial effects on survival and growth of bovine PAOF.(AU)

O objetivo deste trabalho foi avaliar o efeito da somatotropina recombinante bovina (rbST) sobre a sobrevivência e o diâmetro de folículos ovarianos pré-antrais (FOPA) bovinos cultivados in vitro. Ovários foram coletados de vacas adultas e fragmentos do córtex ovariano foram imediatamente fixados (controle não cultivado) ou cultivados in vitro em α-MEM + sozinho ou contendo 10, 50, 100 ou 1.000ng/mL de rbST. Os fragmentos foram processados para histologia clássica e microscopia eletrônica de transmissão. Após um e sete dias de cultivo, o percentual de folículos normais no controle não cultivado foi superior (P<0,05) aos cultivados em α-MEM + sozinho ou acrescido de diferentes concentrações de rbST. Os diâmetros médios oocitário e folicular não aumentaram durante o cultivo por um e sete dias, tanto nos meios contendo rbST, como no meio sem esse hormônio (α-MEM + ). O único meio em que não houve redução no diâmetro folicular com o tempo de cultivo foi o sem rbST. Verificaram-se ainda danos ultraestruturais em FOPA cultivados in vitro. Conclui-se que o uso de rbST em diferentes concentrações no cultivo in situ de folículos pré-antrais bovinos não tem efeitos benéficos na sobrevivência e no crescimento de FOPA bovinos.(AU)

IDO, COX and iNOS have an important role in the proliferation of Neospora caninum in neuron/glia co-cultures.

Central nervous system (CNS) is the main site for encystment of Neospora caninum in different animal species. In this tissue, glial cells (astrocytes and microglia) modulate responses to aggression in order to preserve homeostasis and neuronal function. Previous data showed that when primary cultures of glial cells are infected with N. caninum, they develop gliosis and the immune response is characterized by the release of TNF and IL-10, followed by the control of parasite proliferation. In order to elucidate this control, three enzymatic systems involved in parasite-versus-host interactions were observed on a model of neuron/glia co/cultures obtained from rat brains. Indoleamine 2,3-dioxygenase (IDO), induced nitric oxide synthase (iNOS) responsible for the catabolism of tryptophan and arginine, respectively, and cycloxigenase (COX) were studied comparing their modulation by respective inhibitors with the number of tachyzoites or the immune response measured by the release of IL-10 and TNF. Cells were treated with the inhibitors of iNOS (1.5 mM L-NAME), IDO (1 mM 1-methyl tryptophan), COX-1 (1 µM indomethacin) and COX-2 (1 µM nimesulide) before infection with tachyzoites of N. caninum (1:1 cell: parasite). After 72 h of infection, immunocytochemistry showed astrogliosis and a significant increase in the number and length of neurites, compared with uninfected co-cultures, while an increase of IL-10 and TNF was verified. N. caninum did not change iNOS activity, but the inhibition of the basal levels of this enzyme stimulated parasite proliferation. Additionally, a significant increase of about 40% was verified in the IDO activity, whose inhibition caused 1.2-fold increase in parasitic growth. For COX-2 activity, infection of cultures stimulated a significant increase in release of PGE2 and its inhibition by nimesulide allowed the parasitic growth. These data indicate that iNOS, IDO and COX-2 control the proliferation of N. caninum in this in vitro model. On the other hand, the release of IL-10 by glia besides modulating the inflammation also allow the continuity of parasitism.

Altered Effective Connectivity during a Processing Speed Task in Individuals with Multiple Sclerosis.

OBJECTIVES: Processing speed impairment is the most prevalent cognitive deficit in individuals with multiple sclerosis (MS). However, the neural mechanisms associated with processing speed remain under debate. The current investigation provides a dynamic representation of the functioning of the brain network involved in processing speed by examining effective connectivity pattern during a processing speed task in healthy adults and in MS individuals with and without processing speed impairment. METHODS: Group assignment (processing speed impaired vs. intact) was based on participants' performance on the Symbol Digit Modalities test (Parmenter, Testa, Schretlen, Weinstock-Guttman, & Benedict, 2010). First, brain regions involved in the processing speed task were determined in healthy participants. Time series from these functional regions of interest of each group of participants were then subjected to the effective connectivity analysis (Independent Multiple-Sample Greedy Equivalence Search and Linear, Non-Gaussian Orientation, Fixed Structure algorithms) that showed causal influences of one region on another during task performance. RESULTS: The connectivity pattern of the processing speed impaired group was significantly different from the connectivity pattern of the processing speed intact group and of the healthy control group. Differences in the strength of common connections were also observed. CONCLUSIONS: Effective connectivity results reveal that MS individuals with processing speed impairment not only have connections that differ from healthy participants and MS individuals without processing speed impairment, but also have increased strengths of connections.

Effect of sequential medium with fibroblast growth factor-10 and follicle stimulating hormone on in vitro development of goat preantral follicles.

A sequential medium with fibroblast growth factor-10 (FGF-10) and follicle stimulating hormone (FSH) was evaluated on the survival, ultrastructure, activation and growth rate of caprine preantral follicles submitted to long-term culture, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with FGF-10 and/or FSH added sequentially on different days of culture. Ovarian fragments were cultured during the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+) (cultured control), FSH/FSH, FSH/FGF-10, FSH/FSH+FGF-10, FGF-10/FGF-10, FGF-10/FSH, FGF-10/FSH+FGF-10, FSH+FGF-10/FSH+FGF-10, FSH+FGF-10/FSH and FSH+FGF-10/FGF-10. Follicle morphology, viability and ultrastructure were analyzed. The FSH/FGF-10 treatment showed a higher (P<0.05) percentage of normal follicles compared to all other treatments. In addition, follicles from the FSH/FGF-10 treatment maintained ultrastructural integrity after the culture period. After 16 days of culture, the FSH/FGF-10 and FSH/FSH treatments showed a higher percentage of activation compared to the cultured control (α-MEM(+)/α-MEM(+)). Moreover, the FSH/FGF-10 treatment promoted greater follicular and oocyte diameters compared to the fresh control. In conclusion, this study showed that a sequential medium with FSH followed by FGF-10 (FSH/FGF-10 and FSH/FSH) maintains follicular viability and ultrastructure and promotes transition from the primordial to primary stage (activation) and growth in goat preantral follicles cultured in vitro.

Proteína bruta e energia metabolizável para codornas de corte de um a 14 dias de idade / Crude protein and metabolizable energy for meat quail from 1 to 14 days of age

Com o objetivo de avaliar diferentes níveis de proteína bruta e de energia metabolizável para codornas de corte de um a 14 dias de idade, foi realizado um estudo utilizando 840 codornas de corte, mistas. As aves foram distribuídas em arranjo fatorial (2x5) constituído por 10 tratamentos (dois níveis de energia metabolizável - 2800 e 3000kcal/kg e cinco níveis de proteína bruta - 22,0; 24,0; 26,0; 28,0 e 30,0por cento), sete repetições com 12 aves cada. Foram avaliados os seguintes parâmetros: consumo de ração, ganho em peso aos sete e 14 dias de idade, conversão alimentar aos 14 dias e viabilidade. Verificou-se que codornas alimentadas com dietas com o nível energético mais baixo, 2800kcal/kg, apresentaram maior consumo. As aves que consumiram ração com menor nível de energia apresentaram maior peso aos sete dias de idade independentemente do nível de proteína bruta utilizado. O peso aos 14 dias de idade apresentou resposta quadrática com o acréscimo de proteína bruta na ração, sendo que dietas contendo 28,86 por cento de PB proporcionaram melhor ganho independentemente do nível de energia utilizado. A conversão alimentar apresentou melhores resultados com o aumento do nível de proteína bruta da ração, independentemente do nível de energia metabolizável utilizado. Não foram observados efeitos significativos dos níveis de proteína e energia ou de sua interação sobre a viabilidade das aves. Rações contendo 30,0 por cento de proteína bruta e 2800 ou 3000kcal/kg de energia metabolizável proporcionam satisfatórios resultados de desempenho para codornas de corte de um a 14 dias de idade...

Aiming to evaluate the effect of different levels of crude protein and metabolizable energy for quails from 1 to 14 days of age, a study was conducted using 840 quails, mixed. The birds were distributed in a factorial arrangement (2x5) consisting of 10 treatments (two levels of metabolizable energy - 2,800 and 3,000kcal/kg and 5 levels of crude protein - 22.0, 24.0, 26.0, 28.0 and 30.0 percent), 7 repetitions with 12 birds each. We evaluated the following parameters: feed intake, weight gain at 7 and 14 days of age, feed conversion at 14 days and viability. It was found that quail fed diets with the lowest energy level, 2,800kcal/kg, showed higher consumption. Birds fed diets with lower energy level had greater weight at 7 days of age regardless of the level of crude protein used. The weight at 14 days of age showed a quadratic response with the addition of crude protein in feed, showing that diets containing 28.86 percent CP provide better gain regardless of the level of energy used. The feed showed better results with the increased level of dietary CP, regardless of level of metabolizable energy used. There were no significant effects of protein and energy levels or their interaction on viability. Diets with 30.0 percent crude protein and 2,800 or 3,000kcal/kg metabolizable energy provide satisfactory performance results for quails from 1 to 14 days of age...

Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro / Fator de crescimento transformante-β (TGF-β) mantém a ultraestrutura folicular e estimula o crescimento de folículos pré-antrais caprinos inclusos em tecido ovariano cultivado in vitro

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture...

O objetivo desse estudo foi investigar se o TGF-β afeta a sobrevivência, ativação e crescimento de folículos primordiais caprinos inclusos no córtex ovariano após o cultivo in vitro. Ovários de cabras foram coletados em abatedouro e fragmentos de tecido ovariano foram cultivados por um e sete dias em meio essencial mínimo alfa (α-MEM+) sozinho ou suplementado com TGF-β (1, 5, 10 ou 50ng/mL). Fragmentos ovarianos não cultivados e cultivados foram processados para análise histológica e ultraestrutural. Os resultados mostraram que, comparado ao controle fresco, houve diminuição no percentual de folículos morfologicamente normais em todos os tratamentos somente após sete dias de cultivo. O TGF-β não afetou a ativação folicular independente da concentração testada, contudo, o diâmetro folicular foi superior (P<0.05) no tratamento com 10ng/mL de TGF-β quando comparado ao controle fresco e aos demais tratamentos. Além disso, essa mesma concentração manteve a ultraestrutura normal dos folículos após sete dias de cultivo. Em conclusão, o TGF-β apresentou efeito adicional no crescimento folicular e na manutenção da integridade ultraestrutural de folículos pré-antrais caprinos inclusos no tecido ovariano quando utilizado na concentração de 10ng/mL durante sete dias de cultivo...

Effects of IFN-γ, TNF-α, IL-10 and TGF-ß on Neospora caninum infection in rat glial cells.

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-ß 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-ß, 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-ß 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-ß, 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-ß cytokines participate in parasite proliferation control mechanisms.

Antiproliferative, proapoptotic and morphogenic effects of the flavonoid rutin on human glioblastoma 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.

Catechol cytotoxicity in vitro: induction of glioblastoma cell death by apoptosis.

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 microM catechol for 48 hours. In cells exposed to 600 microM 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 microM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 microM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.

Neospora caninum: early immune response of rat mixed glial cultures after tachyzoites infection.

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.

Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells.

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.

Screening for placental insufficiency in high-risk pregnancies: is earlier better?

OBJECTIVE: To compare a profile of placental function between the first and second trimesters in pregnancies at high risk of adverse perinatal outcomes attributable to placental insufficiency. STUDY DESIGN: Prospective cohort study in 61 singleton pregnancies. Uterine artery Doppler and placental morphology (shape and texture) were determined at 11-13(+6) weeks and at 18-23(+6) weeks. First trimester (pregnancy-associated placental protein-A [PAPP-A]) and second trimester (total hCG and alpha fetoprotein [AFP]) serum biochemistry were determined. The two screening periods were compared for the prediction of a range of severe adverse perinatal outcomes (intrauterine growth restriction [IUGR], abruption, severe pre-eclampsia/HELLP syndrome, delivery<32 weeks, or stillbirth). RESULTS: Adverse perinatal outcomes occurred in 14 (23%) women; 3 (4.9%) losses<20 weeks, 2 (3.3%) stillbirths>20 weeks, 4 (6.6%) IUGR, 7 (11.5%) severe pre-eclampsia/HELLP syndrome, and 10 (16.4%) deliveries<32 weeks. Abnormal second trimester placental morphology was significantly associated with adverse outcome [+LR: 3.6, 95% CI: 1.3-8.5; -LR: 0.63, 95% CI: 0.36-0.93; p=0.025], as was > or = 1 abnormal second trimester tests [+LR: 5.9, 95% CI: 1.6-24; -LR: 0.68, 95% CI: 0.59-0.89; p=0.005] or > or = 2 abnormal second trimester tests [+LR: 3.6, 95% CI: 1.3-7.7; -LR: 0.58, 95% CI: 0.27-0.94; p=0.035]. No combination of first trimester tests significantly predicted severe adverse perinatal outcomes. A study sample size of 822 women with similar high-risk characteristics would be needed in order to refute the conclusion that present methods of first trimester screening are not inferior to second trimester screening for severe placental insufficiency (p=0.05, power 80%, z-test). CONCLUSIONS: In clinically high-risk pregnancies, prediction of adverse perinatal outcomes using placental function testing is more effective in the second compared with the first trimester.

Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures.

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.

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures.

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.

Flavonoid rutin alters the viability and function of mitogen-stimulated splenocytes and thymocytes compared with non stimulated cells.

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.

Alkaloids from Prosopis juliflora leaves induce glial activation, cytotoxicity and stimulate NO production.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS.

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro, with different concentrations of N. caninum. Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-alpha and the neurotoxic-free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.

Neospora caninum: infection induced IL-10 overexpression in rat astrocytes in vitro.

The effect of Neospora caninum, a parasite that causes abortion and neuromuscular changes, has been investigated on a major population of neural cells, the astrocytes. Highly enriched astroglial primary cultures obtained from neonatal rats were infected after 21 days of culture. Astroglial reactivity, IL-10 and IFN-gamma expression, and cell viability (lactate dehydrogenase activity, metabolization of tetrazolium salt, and trypan blue exclusion assay) have been investigated after 24 and 72 h of infection. Astroglial hypertrophy, gliofilament reorganization, metabolic changes suggesting hypoxia and a strong IL-10 release have been observed in the infected cells. These results show that neural cells are targets for the parasite and that astrocytes may contribute to the CNS immune response to the parasite.