Cholinergic anti-inflammatory pathway confers airway protection against oxidative damage and attenuates inflammation in an allergic asthma model.

Asthma is characterized by the influx of inflammatory cells, especially of eosinophils as well as reactive oxygen species (ROS) production, driven by the release of the T helper 2 (Th2)-cell-associated cytokines. The cholinergic anti-inflammatory pathway (CAP) inhibit cytokines production and controls inflammation. Thus, we investigated the effects of pharmacological activation of CAP by neostigmine on oxidative stress and airway inflammation in an allergic asthma model. After the OVA challenge, mice were treated with neostigmine. We showed that CAP activation by neostigmine reduced the levels of pro-inflammatory cytokines (IL-4, IL-5, IL-13, IL-1ß, and TNF-α), which resulted in a decrease of eosinophils influx. Furthermore, neostigmine also conferred airway protection against oxidative stress, attenuating ROS production through the increase of antioxidant defense, evidenced by the catalase (CAT) activity. We propose, for the first time, that pharmacological activation of the CAP can lead to new possibilities in the therapeutic management of allergic asthma.

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.

Reactive oxygen species are involved in eosinophil extracellular traps release and in airway inflammation in asthma.

In asthma, there are high levels of inflammatory mediators, reactive oxygen species (ROS), and eosinophil extracellular traps (EETs) formation in airway. Here, we attempted to investigate the ROS involvement in EETs release and airway inflammation in OVA-challenged mice. Before the intranasal challenge with ovalbumin (OVA), animals were treated with two ROS inhibitors, N-acetylcysteine (NAC) or diphenyleneiodonium (DPI). We showed that NAC treatment reduced inflammatory cells in lung. DPI and NAC treatments reduced eosinophil peroxidase (EPO), goblet cells hyperplasia, proinflammatory cytokines, NFκB p65 immunocontent, and oxidative stress in lung. However, only the NAC treatment improved mitochondrial energy metabolism. Moreover, the treatments with DPI and NAC reduced EETs release in airway. This is the first study to show that ROS are needed for EETs formation in asthma. Based on our results, NAC and DPI treatments can be an interesting alternative for reducing airway inflammation, mitochondrial damage, and EETs release in asthma.

Mesenchymal stem cells improves survival in LPS-induced acute lung injury acting through inhibition of NETs formation.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are syndromes of acute hypoxemic respiratory failure resulting from a variety of direct and indirect injuries to the gas exchange parenchyma of the lungs. During the ALI, we have an increase release of proinflammatory cytokines and high reactive oxygen species (ROS) formation. These factors are responsible for the release and activation of neutrophil-derived proteases and the formation of neutrophil extracellular traps (NETs). The excessive increase in the release of NETs cause damage to lung tissue. Recent studies have studies involving the administration of mesenchymal stem cells (MSCs) for the treatment of experimental ALI has shown promising results. In this way, the objective of our study is to evaluate the ability of MSCs, in a lipopolysaccharide (LPS)-induced ALI model, to reduce inflammation, oxidative damage, and consequently decrease the release of NETs. Mice were submitted lung injury induced by intratracheal instillation of LPS and subsequently treated or not with MSCs. Treatment with MSCs was able to modulate pulmonary inflammation, decrease oxidative damage, and reduce the release of NETs. These benefits from treatment are evident when we observe a significant increase in the survival curve in the treated animals. Our results demonstrate that MSCs treatment is effective for the treatment of ALI. For the first time, it is described that MSCs can reduce the formation of NETs and an experimental model of ALI. This finding is directly related to these cells modulate the inflammatory response and oxidative damage in the course of the pathology.

Respiratory syncytial virus increases eosinophil extracellular traps in a murine model of asthma.

BACKGROUND: Respiratory viral infections are the leading cause of asthma exacerbations. Eosinophil activation results in the formation of eosinophil extracellular traps (EETs), which release web-like structures of DNA and proteins that bind, disarm and extracellularly kill pathogens. OBJECTIVE: We investigated whether the respiratory syncytial virus (RSV) in vitro could induce EETs in bronchoalveolar lavage fluid eosinophils in a murine model of asthma. METHODS: BALB/cJ mice (6-8 weeks old) were sensitized with 2 subcutaneous injections of ovalbumin (20 µg) on days 0 and 7, followed by three intranasal challenges with ovalbumin (100 µg) on days 14, 15, and 16 of the protocol. The control group received Dulbecco's phosphate-buffered saline. Bronchoalveolar lavage fluid eosinophils of ovalbumin group or control group were stimulated with RSV (103 PFU/mL) in vitro for 3 hours. After that, culture supernatant was collected to perform the analyses proposed in this study. RESULTS: We verified an increase in extracellular DNA concentration in bronchoalveolar lavage fluid eosinophils from ovalbumin group stimulated with RSV (103 PFU/mL) in vitro, which was confirmed by confocal microscopy. We demonstrated that most cells are negative for annexin V and propidium iodide in all groups evaluated. Also, RSV in vitro decreased interferon-É£ in culture supernatant when compared to the ovalbumin group. CONCLUSION: In this study, we demonstrated for the first time that RSV in vitro induces EETs formation in eosinophils from asthmatic mice.

Relined fiberglass post: an ex vivo study of the resin cement thickness and dentin-resin interface.

The aim of this study was to evaluate the thickness of resin cements in the root thirds when using conventional fiberglass posts (CP) and relined fiberglass posts (RP) in weakened roots and to evaluate the morphological characteristics of the dentin-resin interface. Forty human maxillary anterior teeth had the crown sectioned below the cemento-enamel junction. The canals were endodontically treated and weakened with diamond burs. Teeth were divided into four groups (n = 10): Group 1 - CP + RelyX ARC; Group 2 - CP + RelyX U200; Group 3 - RP + RelyX ARC; and Group 4 - RP + RelyX U200. Prior to luting, 0.1% Fluorescein and 0.1% Rhodamine B dyes were added to an adhesive and resin cement, respectively. Slices were obtained from the apical, middle, and cervical thirds of the root. Confocal laser scanning microscopy images were recorded in four areas (buccal, lingual, mesial, distal) of each third. In each area, four equidistant measures of the resin cement were made and the mean value was calculated. The interface morphology was observed. The data were submitted to three-way ANOVA and Tukey's test (α = 0.05). The interaction between fiberglass posts, resin cement, and root thirds was significant (p < 0.0001). The resin cement thicknesses were significantly lower for RP in comparison with CP, except in the apical third. There was no significant difference between the resin cements for RP. There was formation of resin cement tags and adhesive tags along the root for RP. RP favored the formation of thin and uniform resin cement films and resin tags in weakened roots.

Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems.

Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 µg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min(-1) to 36 ± 1.3 min(-1) (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca(2+) influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential.

The antiepileptic activity of JSTX-3 is mediated by N-methyl-D-aspartate receptors in human hippocampal neurons.

We analyzed the effect of the acylpolyaminetoxin JSTX-3 on the epileptogenic discharges induced by perfusion of human hippocampal slices with artificial cerebrospinal fluid lacking Mg2+ or N-methyl-D-aspartate. Hippocampi were surgically removed from patients with refractory medial temporal lobe epilepsy, sliced in the surgical room and taken to the laboratory immersed in normal artificial cerebrospinal fluid. Epileptiform activity was induced by perfusion with Mg2+-free artificial cerebrospinal fluid or by iontophoretically applied N-methyl-D-aspartate and intracellular and field recordings of CA1 neurons were performed. The ictal-like discharges induced by Mg2+-free artificial cerebrospinal fluid and N-methyl-D-aspartate were blocked by incubation with JSTX-3. This effect was similar to that obtained with the N-methyl-D-aspartate receptor antagonist DL (-)2-amino-5 phosphonovaleric acid. Our findings suggest that in human hippocampal neurons, the antiepileptic effect of JSTX-3 is mediated by its action on N-methyl-D-aspartate receptor.

Altered distribution of striatal activity-dependent synaptic plasticity in the 3-nitropropionic acid model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary choreiform movements, neuropsychiatric disturbances and cognitive decline. The hyperkinetic phenomenology has commonly been attributed to a disturbance of the basal ganglia function, mainly the neostriatum, but its pathophysiology mechanisms remain unclear. Activity-dependent long-term changes in synaptic efficacy, such as long-term potentiation (LTP) and long-term depression (LTD), are widely considered to be the cellular models for acquisition and storage of information in neuronal networks. Both LTP and LTD have been described at the corticostriatal pathway and they might be probably involved not only in physiological motor behavior processing but also in disease states affecting that pathway. Systemic injection of 3-nitropropionic acid (3-NP) induces excitotoxic striatal lesions and abnormal movements in rodents, resembling those seen in HD. We examined synaptic plasticity in dorsolateral striatum slices prepared from both control and 3-NP-treated rats by recording extracellular field potentials. Our results reinforce the idea that both forms of activity-dependent synaptic plasticity can be recorded at the dorsolateral region of striatum by the same stimulating protocol in control rats and suggest that 3-NP-induced striatal lesions may be associated with suppression of LTD expression in the sensorimotor striatum.

Antiepileptic effect of acylpolyaminetoxin JSTX-3 on rat hippocampal CA1 neurons in vitro.

The Joro spider toxin (JSTX-3), derived from Nephila clavata, has been found to block glutamate excitatory activity. Epilepsy has been studied in vitro, mostly on rat hippocampus, through brain slices techniques. The aim of this study is to verify the effect of the JSTX-3 on the epileptiform activity induced by magnesium-free medium in rat CA1 hippocampal neurons. Experiments were performed on hippocampus slices of control and pilocarpine-treated Wistar rats, prepared and maintained in vitro. Epileptiform activity was induced through omission of magnesium from the artificial cerebrospinal fluid (0-Mg2+ ACSF) superfusate and iontophoretic application of N-methyl-D-aspartate (NMDA). Intracellular recordings were obtained from CA1 pyramidal neurons both of control and epileptic rats. Passive membrane properties were analyzed before and after perfusion with the 0-Mg2+ ACSF and the application of toxin JSTX-3. During the ictal-like activity, the toxin JSTX-3 was applied by pressure ejection, abolishing this activity. This effect was completely reversed during the washout period when the slices were formerly perfused with artificial cerebrospinal fluid (ACSF) and again with 0-Mg2+ ACSF. Our results suggest that the toxin JSTX-3 is a potent blocker of induced epileptiform activity.

Relined fiberglass post: an ex vivo study of the resin cement thickness and dentin-resin interface

Abstract The aim of this study was to evaluate the thickness of resin cements in the root thirds when using conventional fiberglass posts (CP) and relined fiberglass posts (RP) in weakened roots and to evaluate the morphological characteristics of the dentin-resin interface. Forty human maxillary anterior teeth had the crown sectioned below the cemento-enamel junction. The canals were endodontically treated and weakened with diamond burs. Teeth were divided into four groups (n = 10): Group 1 - CP + RelyX ARC; Group 2 - CP + RelyX U200; Group 3 - RP + RelyX ARC; and Group 4 - RP + RelyX U200. Prior to luting, 0.1% Fluorescein and 0.1% Rhodamine B dyes were added to an adhesive and resin cement, respectively. Slices were obtained from the apical, middle, and cervical thirds of the root. Confocal laser scanning microscopy images were recorded in four areas (buccal, lingual, mesial, distal) of each third. In each area, four equidistant measures of the resin cement were made and the mean value was calculated. The interface morphology was observed. The data were submitted to three-way ANOVA and Tukey's test (α = 0.05). The interaction between fiberglass posts, resin cement, and root thirds was significant (p < 0.0001). The resin cement thicknesses were significantly lower for RP in comparison with CP, except in the apical third. There was no significant difference between the resin cements for RP. There was formation of resin cement tags and adhesive tags along the root for RP. RP favored the formation of thin and uniform resin cement films and resin tags in weakened roots.

Estudo eletrofisiológico das propriedades neuronais intrínsicas passivas de CAI em fatias hipocampais de ratos e tecido humano / Electrophysiological study of the intrinsic passive properties neurons of the CAI in slices hippocampal rats and hippocampal human tissue

O presente estudo tem como objetivo verificar a utilização de ratos como controle experimentais para estudos In vitro com tecido hipocampal humano. As fatias obtidas de hipocampo de ratos Wistar machos (espessura de 400 um) e amostras de tecido hipocampal humano (espessura 500um) removidos durante o procedimento cirúrgico para o tratamento de epilepsia de lobo temporal refratária ao tratamento medicamentoso. As fatias mantidas imersas numa cuba com solução de Ringer em temperatura ambiente continuamente oxigenadas e posteriormente transferidas para uma cãmara de interface para registro eletrofisiológico. Registros intracelulares foram obtidos a partir dos neurônios piramidais de CA1 de ratos controles (CR), epilépticos no modelo da pilocarpina (ER) e tecido hipocampal humanos (HHT). Foram avaliadas as propriedades passivas (potencial de membrana, resistência de entrada, constante de tempo) antes e depois da perfusão com o Ringer O-Mg2+. A análise entre CR (n: 40) e ER (n:22) não demonstrou diferenças significativas nas propriedades estudadas. Entre CR e HHT (n:30) não foram observadas diferenças significativas nas propriedades intrínsecas passivas. A análise entre ET e HHT não revelou diferença no potencial de membrana e resistência de entrada, porém a constante de tempo antes da perfusão com o Ringer O-Mg2+ apresentou diferença. Nossos achados sugerem que neurônios hipocampais de CAI de ratos são válidos como grupo controle para estudos experimentais com tecido hipocampal humano