ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection.

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-ß, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Zika Virus affects neurobehavioral development, and causes oxidative stress associated to blood-brain barrier disruption in a rat model of congenital infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with several neurodevelopmental outcomes after in utero infection. Here, we studied a congenital ZIKV infection model with immunocompetent Wistar rats, able to predict disabilities and that could pave the way for proposing new effective therapies. We identified neurodevelopmental milestones disabilities in congenital ZIKV animals. Also, on 22nd postnatal day (PND), blood-brain barrier (BBB) proteins disturbances were detected in the hippocampus with immunocontent reduction of ß_Catenin, Occludin and Conexin-43. Besides, oxidative stress imbalance on hippocampus and cortex were identified, without neuronal reduction in these structures. In conclusion, even without pups' microcephaly-like phenotype, congenital ZIKV infection resulted in neurobehavioral dysfunction associated with BBB and oxidative stress disturbances in young rats. Therefore, our findings highlighted the multiple impact of the congenital ZIKV infection on the neurodevelopment, which reinforces the continuity of studies to understand the spectrum of this impairment and to provide support to future treatment development for patients affected by congenital ZIKV.

Effects of methylphenidate after a long period of discontinuation include changes in exploratory behavior and increases brain activities of Na+,K+-ATPase and acetylcholinesterase.

Methylphenidate (MPH) has been widely misused by children and adolescents who do not meet all diagnostic criteria for attention-deficit/hyperactivity disorder. Since it is not yet known whether MPH can be administered in childhood without consequences in adulthood, in the present study we proposed to investigate the effects of chronic early treatment with MPH after a long period of discontinuation. Wistar male rats were injected with MPH (2 mg/kg, intraperitoneally) or saline solution once daily from 15th to 44th day of life. Two months after the last MPH administration, we evaluated the animal's performances on a battery of behavior tests. We also tested Na+,K+-ATPase and acetylcholinesterase activities in prefrontal cortex and hippocampus, which may be associated with behavior. Rats treated with MPH during peri-adolescence show changes in exploratory behavior in adulthood in the open field but not in the elevated plus maze and light-dark transition tests. MPH-treated rats showed a lower latency to find the platform in the training phase, as well as a better performance in the test phase in the Morris water maze test. No differences were observed in the object recognition index and working memory. Acetylcholinesterase was increased in prefrontal cortex and hippocampus, while Na+,K+-ATPase was increased only in hippocampus. These findings provide additional evidence that early-life exposure to MPH can have complex effects in adulthood and new basis for understanding the behavioral and neurochemical consequences associated with chronic use of MPH during the development of central nervous system.

Airway inflammation induces anxiety-like behavior through neuroinflammatory, neurochemical, and neurometabolic changes in an allergic asthma model.

Allergic asthma is characterized by chronic airway inflammation and is constantly associated with anxiety disorder. Recent studies showed bidirectional interaction between the brain and the lung tissue. However, where and how the brain is affected in allergic asthma remains unclear. We aimed to investigate the neuroinflammatory, neurochemical, and neurometabolic alterations that lead to anxiety-like behavior in an experimental model of allergic asthma. Mice were submitted to an allergic asthma model induced by ovalbumin (OVA) and the control group received only Dulbecco's phosphate-buffered saline (DPBS). Our findings indicate that airway inflammation increases interleukin (IL) -9, IL-13, eotaxin, and IL-1ß release and changes acetylcholinesterase (AChE) and Na+,K+-ATPase activities in the brain of mice. Furthermore, we demonstrate that a higher reactive oxygen species (ROS) formation and antioxidant defense alteration that leads to protein damage and mitochondrial dysfunction. Therefore, airway inflammation promotes a pro-inflammatory environment with an increase of BDNF expression in the brain of allergic asthma mice. These pro-inflammatory environments lead to an increase in glucose uptake in the limbic regions and to anxiety-like behavior that was observed through the elevated plus maze (EPM) test and downregulation of glucocorticoid receptor (GR). In conclusion, the present study revealed for the first time that airway inflammation induces neuroinflammatory, neurochemical, and neurometabolic changes within the brain that leads to anxiety-like behavior. Knowledge about mechanisms that lead to anxiety phenotype in asthma is a beneficial tool that can be used for the complete management and treatment of the disease.

Purple grape juice consumption during the gestation reduces acetylcholinesterase activity and oxidative stress levels provoked by high-fat diet in hippocampus from adult female rats descendants.

The enzyme acetylcholinesterase participates in the end of cholinergic transmission and it has been shown that its activity is increased in some diseases that affect the brain, including Alzheimer disease. The objective of this study was to investigate the effect of purple grape juice consumption with or without high-fat diet in the gestational and lactation period on acetylcholinesterase activity and oxidative stress parameters in the hippocampus of female descendants. During pregnancy and lactation, 40 female Wistar rats received a control diet or a high-fat diet, with half of them receiving grape juice. After lactation, the female descendants received water and control diet in ad libitum until euthanasia on the 120 postnatal day. Hippocampus from were removed for analysis of AChE activity, protein oxidation and lipid peroxidation. It was observed that high-fat diet consumption during the pregnancy increased the AChE activity and the grape juice reduced this activity in descendants. The same was observed in protein oxidation, the descendants from high-fat diet had significantly highest values, and grape juice decreased the levels. We conclude that dietary choices during pregnancy can alter the acetylcholinesterase levels and grape juice is an important alternative to improve this function in adulthood.

Autophagy induces eosinophil extracellular traps formation and allergic airway inflammation in a murine asthma model.

Studies have shown autophagy participation in the immunopathology of inflammatory diseases. However, autophagy role in asthma and in eosinophil extracellular traps (EETs) release is poorly understood. Here, we attempted to investigate the autophagy involvement in EETs release and in lung inflammation in an experimental asthma model. Mice were sensitized with ovalbumin (OVA), followed by OVA challenge. Before the challenge with OVA, mice were treated with an autophagy inhibitor, 3-methyladenine (3-MA). We showed that 3-MA treatment decreases the number of eosinophils, eosinophil peroxidase (EPO) activity, goblet cells hyperplasia, proinflammatory cytokines, and nuclear factor kappa B (NFκB) p65 immunocontent in the lung. Moreover, 3-MA was able to improve oxidative stress, mitochondrial energy metabolism, and Na+ , K+ -ATPase activity. We demonstrated that treatment with autophagy inhibitor 3-MA reduced EETs formation in the airway. On the basis of our results, 3-MA treatment can be an interesting alternative for reducing lung inflammation, oxidative stress, mitochondrial damage, and EETs formation in asthma.

Disruption of Purinergic Receptor P2X7 Signaling Increases Susceptibility to Cerebral Toxoplasmosis.

Toxoplasmosis is a neglected disease that affects millions of individuals worldwide. Toxoplasma gondii infection is an asymptomatic disease, with lethal cases occurring mostly in HIV patients and organ transplant recipients. Nevertheless, atypical strains of T. gondii in endemic locations cause severe pathology in healthy individuals. Toxoplasmosis has no cure but it can be controlled by the proinflammatory immune response. The purinergic receptor P2X7 (P2X7) is involved in many inflammatory events and has been associated with genes that confer resistance against toxoplasmosis in humans. In vitro studies have reported parasite death after P2X7-receptor activation in various cell types. To understand the contribution of P2X7 during cerebral toxoplasmosis, wild-type and P2rx7 knockout mice were infected orally with T. gondii and their pathologic profiles were analyzed. We found that all P2rx7-/- mice died 8 weeks after infection with an increased number of cysts and fewer inflammatory infiltrates in their brains. The cytokines interleukin-1ß, interleukin-12, tumor necrosis factor-α, and reactive oxygen species were absent or reduced in P2rx7-/- mice. Taken together, these data suggest that the P2X7 receptor promotes inflammatory infiltrates, proinflammatory cytokines, and reactive oxygen species production in the brain, and that P2X7 signaling mediates major events that confer resistance to cerebral toxoplasmosis.

Neostigmine treatment induces neuroprotection against oxidative stress in cerebral cortex of asthmatic mice.

During chronic inflammatory disease, such asthma, leukocytes can invade the central nervous system (CNS) and together with CNS-resident cells, generate excessive reactive oxygen species (ROS) production as well as disbalance in the antioxidant system, causing oxidative stress, which contributes a large part to neuroinflammation. In this sense, the aim of this study is to investigate the effects of treatment with neostigmine, known for the ability to control lung inflammation, on oxidative stress in the cerebral cortex of asthmatic mice. Female BALB/cJ mice were submitted to asthma model induced by ovalbumin (OVA). Control group received only Dulbecco's phosphate-buffered saline (DPBS). To evaluate neostigmine effects, mice received 80 µg/kg of neostigmine intraperitoneally 30 min after each OVA challenge. Our results revealed for the first time that treatment with neostigmine (an acetylcholinesterase inhibitor that no crosses the BBB) was able to revert ROS production and change anti-oxidant enzyme catalase in the cerebral cortex in asthmatic mice. These results support the communication between the peripheral immune system and the CNS and suggest that acetylcholinesterase inhibitors, such as neostigmine, should be further studied as possible therapeutic strategies for neuroprotection in asthma.

Methylphenidate disrupts cytoskeletal homeostasis and reduces membrane-associated lipid content in juvenile rat hippocampus.

Although methylphenidate (MPH) is ubiquitously prescribed to children and adolescents, the consequences of chronic utilization of this psychostimulant are poorly understood. In this study, we investigated the effects of MPH on cytoskeletal homeostasis and lipid content in rat hippocampus. Wistar rats received intraperitoneal injections of MPH (2.0 mg/kg) or saline solution (controls), once a day, from the 15th to the 44th day of age. Results showed that MPH provoked hypophosphorylation of glial fibrillary acidic protein (GFAP) and reduced its immunocontent. Middle and high molecular weight neurofilament subunits (NF-M, NF-H) were hypophosphorylated by MPH on KSP repeat tail domains, while NFL, NFM and NFH immunocontents were not altered. MPH increased protein phosphatase 1 (PP1) and 2A (PP2A) immunocontents. MPH also decreased the total content of ganglioside and phospholipid, as well as the main brain gangliosides (GM1, GD1a, and GD1b) and the major brain phospholipids (sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine). Total cholesterol content was also reduced in the hippocampi of juvenile rats treated with MPH. These results provide evidence that disruptions of cytoskeletal and lipid homeostasis in hippocampus of juvenile rats are triggers by chronic MPH treatment and present a new basis for understanding the effects and consequences associated with chronic use of this psychostimulant during the development of the central nervous system.