Acute and chronic stress differently alter the expression of cytokine and neuronal markers genes in zebrafish brain.

We report the effects of acute and chronic stress on the expression of selective immune-related genes and markers of neuronal function in the brain of the zebrafish (Danio rerio). Fish were distributed into three groups: the non-stressed control group; the acute stress (AS) group, submitted to a single stressing episode; and the unpredictable chronic stress (UCS) group, submitted to two daily stressing episodes of alternating times and types of stress. The stressing protocols were applied for a period of 14 days. The UCS protocol triggered the expression of the pro-inflammatory cytokine genes IL-1ß and TNF-α, the anti-inflammatory cytokine IL-10 (negative feedback from the immune system), reduction in cFOS gene expression, and caused neuro-inflammation. The AS protocol had no effect on gene expression. Altered expression of cytokine genes, as observed in our study, correlates with several pathologies associated with neuro-inflammation, and the reduction of cFOS gene expression may indicate the occurrence of reduced neuronal plasticity. Our study further extends our knowledge about the interaction of the immune system and the different forms of stress.

Patterns of the innate immune response in tambaqui Colossoma macropomum: Modulation of gene expression in haemorrhagic septicaemia caused by Aeromonas hydrophila.

The production of tambaqui Colossoma macropomum has recently reached a milestone, being considered the main native species produced in South American continental waters. Despite the importance of this fish, its immunity is not well understood. In this study we established some patterns of innate immunity for the species via two experiments. Both studies evaluated the fish in the absence (intraperitoneal saline) or presence (intraperitoneal, 3 x 107 CFU/mL of Aeromonas hydrophila at 0.1 mL/10 g of living weight) of infection at 5 points over time-course of 14 days (0 h, 6 h, 24 h, 7 d, 14 d). In the first experiment, the partial gene sequences and gene expression of IL-1ß, IRAK-1, C3, C4, lysozyme, IL-10, HSP70 and ß-actin were determined in the main secondary lymphoid organs of fish: the spleen and head kidney. The second study was performed to analyse the alternative complement pathway ACH50 in serum to support the elucidation of C3 gene expression. Results of the gene expression assays showed a tendency towards up-regulation of immune genes in infected fish in early phases of infection (mostly around 6 h and 24 h) and in the chronic phase (7 d and 14 d), with the exception of HSP70 which showed a down-regulation in infected fish. Our results also suggested that lysozyme was evolved in both pro- and anti-inflammatory activities. For genes of the complement system, it was demonstrated that C4 regulation followed the tendency of pro-inflammatory genes. However, the C3 gene was, surprisingly, not expressed in most fish and this corroborated with the results of the complement system activity in serum that also did not show activity in most fish. The possible reasons for the regulation of gene expression and association with fish disease are addressed in this paper.

Waterborne agrichemicals compromise the anti-predatory behavior of zebrafish.

Due to human activities, there is an increasing presence of agrochemicals residues in water bodies, which could be attributed to an increased use of these chemicals, incorrect disposal of packaging materials, and crop leaching. The effects of these residues on prey-predator relationship of aquatic animals are poorly known. Here, we show that fish acutely exposed to glyphosate, 2,4-D, and methylbenzoate-based agrichemicals have their anti-predatory responses impaired. We exposed zebrafish to sub-lethal concentrations of agrichemicals and evaluated their behavioral reaction against a simulated bird predatory strike. We observed that agrichemical-exposed fish spent more time in a risky area, suggesting that the pesticides interfered with their ability of risk perception. Our results highlight the impairment and environmental consequences of agrochemical residues, which can affect aquatic life and crucial elements for life (food web) such as the prey-predator relationship.

Sex segregation affects exploratory and social behaviors of zebrafish according to controlled housing conditions.

Zebrafish has become an animal model in research and articles have established ideal conditions for their maintenance. However, little is known regarding the influence of gender and other cues on zebrafish behavior. Thus, here we analyzed the exploratory and social behavior of different sexes (male and female, mixed or segregated) under different housing conditions with various types of stimuli (visual or/and chemical cues and structural environmental enrichment). Segregated females and males were more active than mixed individuals and females were more anxious. Fish that visualized and smelled the opposite sex presented higher activity and were less anxious than individuals that only smelled or visualized the opposite sex. Fish segregated by sex while being allowed to visualize and smell the opposite sex with the presence of structural environmental enrichment exhibited lower activity and anxiety-like behavior than fish without structural environmental enrichment. Thus, we emphasized that these variables should be taken into account in housing conditions and should be detailed for better replicability and reproducibility of experiments performed with zebrafish.

Aripiprazole prevents stress-induced anxiety and social impairment, but impairs antipredatory behavior in zebrafish.

Environmental pollution caused by antipsychotic residues is a relevant ecological problem. Studies revealed that residues of these drugs are present in a wide range of different ecosystems and can have adverse effects on non-target organisms even in low environmental concentrations. Among these antipsychotic drugs, aripiprazole (APPZ) is a second-generation atypical antipsychotic that is a partial agonist of dopaminergic and serotoninergic receptors. APPZ is used to treat schizophrenia, bipolar disorder, autism, obsessive-compulsive disorder, and anxiety or panic disorders. Thus, in this study we posed the following question: "What will be the behavioral effects of waterborne APPZ on fish?" To answer this question, we exposed adult zebrafish to different APPZ concentrations (0.556, 5.56, and 556 ng/L) for 15 min and evaluated their exploratory, anxiety-like, social, and anti-predatory behaviors. Our results showed that, despite the apparent beneficial reversal of stress-induced social impairment and anxiety-like behavior, APPZ exposure impaired the anti-predatory reaction of adult zebrafish. Taken altogether, our results show that APPZ-exposed zebrafish may have a decreased perception of predators, even at concentrations lower than those already detected in the environment. A failure to exhibit an antipredatory response may favor the predator, decrease the fitness of the prey species, and, consequently, affect the food chain. Our results highlight the risks and consequences associated with APPZ residues in water, which may affect aquatic life and endanger species that depend on appropriate behavioral responses for survival.

The effects of auditory enrichment on zebrafish behavior and physiology.

Environmental enrichment is widely used to improve welfare and behavioral performance of animal species. It ensures housing of laboratory animals in environments with space and complexity that enable the expression of their normal behavioral repertoire. Auditory enrichment by exposure to classical music decreases abnormal behaviors and endocrine stress responses in humans, non-humans primates, and rodents. However, little is known about the role of auditory enrichment in laboratory zebrafish. Given the growing importance of zebrafish for neuroscience research, such studies become critical. To examine whether auditory enrichment by classical music can affect fish behavior and physiology, we exposed adult zebrafish to 2 h of Vivaldi's music (65-75 dB) twice daily, for 15 days. Overall, zebrafish exposed to such auditory stimuli were less anxious in the novel tank test and less active, calmer in the light-dark test, also affecting zebrafish physiological (immune) biomarkers, decreasing peripheral levels of pro-inflammatory cytokines and increasing the activity of some CNS genes, without overt effects on whole-body cortisol levels. In summary, we report that twice-daily exposure to continuous musical sounds may provide benefits over the ongoing 50-55 dB background noise of equipment in the laboratory setting. Overall, our results support utilizing auditory enrichment in laboratory zebrafish to reduce stress and improve welfare in this experimental aquatic organism.

Characterization of sickness behavior in zebrafish.

In a previous study we showed a clear relationship between immune system and behavior in zebrafish and we hypothesized that the immune system is capable of inducing behavioral changes. To further investigate this subject and to address our main question, here we induced an inflammatory response in one group of fish by the inoculation of formalin-inactivated Aeromonoas hydrophila bacterin and compared their social and exploratory behavior with control groups. After the behavioral tests, we also analyzed the expression of cytokines genes and markers of neuronal activity in fish brain. In the bacterin-inoculated fish, the locomotor activity, social preference and exploratory behavior towards a new object were reduced compared to the control fish while the expression of proinflammatory cytokines in the brain was upregulated. With this study we demonstrated for the first time that the immune system is capable of causing behavioral changes that are consistent with the sickness behavior observed in mammals.

First description of behavior and immune system relationship in fish.

Considering the intriguing relationship between immune system and behavior recently described in mammals, and the lack of information of this relationship in fish, here we describe for the first time the interaction between the immune system and social and exploratory behavior in zebrafish. Fish high responders to novelty (HRN) presented a proinflammatory profile, with increased IL-1ß and reduced IL-10 expression compared to fish low responders to novelty (LRN). Likewise, fish less responsive to social stimuli have a reduced expression of INF-γ. We show that fish with different behavior patterns have differences in the immune response. Our findings indicate that the interplay between immune system and behavior in zebrafish is similar to that found in mammalian models and that zebrafish should be considered as a potential model organism to study the relationship between immune system and behavior.

Reduced expression of selective immune-related genes in silver catfish (Rhamdia quelen) monocytes exposed to atrazine.

The effect of atrazine (ATZ) and its metabolites on aquatic vertebrate species has been a matter of concern to researchers and environmentalist. In this study we exposed head kidney monocytes to sublethal concentrations of atrazine (1 and 10 µg/ml-1), corresponding to 1% and 10% of the LC50-96h, to evaluate the expression of immune-related genes central to immune stimulation. The mRNA levels of TNF-α, Mieloperoxidase and Mx genes were significantly reduced following 24 h exposure to both concentrations of ATZ. The mRNA levels of iRAK4 were reduced only at the higher ATZ concentration and the mRNA levels of IL-1ß were not affected. The results reported here support our previous findings on the immunosuppressive effect of ATZ indicating its potential to interfere with the expression of immune-related genes, and strengthen the need to regulate ATZ usage aiming to preserve animal and human health.

Effects of Campomanesia xanthocarpa on inflammatory processes, oxidative stress, endothelial dysfunction and lipid biomarkers in hypercholesterolemic individuals.

In Brazil, the edible plant Campomanesia xanthocarpa Berg. (Myrtaceae), popularly known as "guavirova," has been studied in hypercholesterolemic individuals. The present work investigated the effects of C. xanthocarpa on inflammatory processes, oxidative stress, endothelial dysfunction and lipid biomarkers in hypercholesterolemic individuals. A total of 156 individuals were selected in a double-blind fashion and randomly divided into two groups in accordance with the intervals used in the criteria for hypercholesterolemia: individuals with total cholesterol (TC) levels between 200 and 240 mg/dL (undesirable level individuals - UL) and individuals with TC levels >240 mg/dL (hypercholesterolemic individuals - HL). Both groups had a control group (CG), which received placebo treatment, an encapsulated excipient group (lactose) and an experimental group that received 500 mg (EG 500), 750 mg (EG 750) or 1000 mg (EG 1000) of encapsulated C. xanthocarpa. The inflammatory processes (high-sensitivity C-reactive protein - hs-CRP), oxidative stress (advanced oxidation protein products-AOPPs; ischemia-modified albumin-IMA), endothelial dysfunction (nitric oxide - NOx) and biochemical (TC, triglycerides, high-density lipoprotein - HDL, low-density lipoproteins - LDL, and very low-density lipoprotein - VLDL) parameters were measured before and 90 days after the initiation of treatment. A significant decrease in TC and LDL levels was observed in HL individuals from the EG 500 group (reduction of 29 ± 3% and 41 ± 5% to levels before treatment) compared to the CG group individuals. A significant reduction in oxidative stress and inflammatory process components (reduction of 52 ± 11% in AOPPs, 32 ± 10% in IMA and 57 ± 7% in hs-CRP) and a significant increase in NOx (increase of 84 ± 27%) was observed in HL individuals in the EG 1000 group when compared to the CG group individuals. Treatment with encapsulated C. xanthocarpa reduced blood TC and LDL levels in hypercholesterolemic individuals. In addition to its effect on cholesterol levels, this plant reduced oxidative stress in hypercholesterolemic individuals and improved the levels of NOx.

Mortalidade infantil e Saúde da Família nas unidades da Federação brasileira, 1998-2008 / Infant mortality and Family Health units in the Brazilian Federation, 1998-2008

Objetivo: Analisar a relação entre mortalidade infantil e cobertura populacional da Saúde da Família nas unidades da Federação brasileira. Métodos: Estudo ecológico cuja unidade de análise foi composta pelos 26 Estados brasileiros, na série histórica de 1998 a 2008. Foram descritos o percentual de cobertura populacional da Estratégia Saúde da Família (ESF) e a taxa de mortalidade infantil e construídas curvas de regressão linear das variáveis estudadas. A associação estatística foi realizada por meio do teste de correlação de Pearson e da regressão linear simples. Resultados: Houve aumento de 857% nas equipes da ESF, ampliação de 43% na cobertura populacional e diminuição da mortalidade infantil em 58% no Brasil. A expansão da cobertura da ESF esteve associada à redução das taxas de mortalidade infantil em 73% dos Estados brasileiros (p<0,05). Conclusão: Os achados permitem explorar uma possível associação entre o aumento da cobertura populacional da ESF e a redução das taxas de mortalidade infantil no Brasil. .

Objective: To analyze the relationship between infant mortality and population coverage of the Family Health units of the Brazilian Federation. Methods: An ecological study the unit of analysis was composed by 26 Brazilian States, in the historical series from 1998 to 2008. The percentage of population coverage Family Health Strategy (FHS) were described, the rate of infant and built linear regression analysis of the variables studied mortality. The statistical association was performed using the Pearson correlation test and linear regression. Results: There was an increase of 857% in FHS teams, expansion of 43% in population coverage and reduce infant mortality by 58% in Brazil. The expansion in coverage FHS was associated with reduced rates of infant mortality in 73% of Brazilian States (p<0.05). Conclusion: The findings explore a possible association between the increase in population coverage FHS with the reduction of infant mortality rates in Brazil. .