Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation.

BACKGROUND: The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial-glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types. METHODS: Mouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function. RESULTS: Herein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood-brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function. CONCLUSIONS: Ago2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

Correction: Simão, A.Y., et al. Evaluation of the Cytotoxicity of Ayahuasca Beverages. Molecules 2020, 25, 5594.

This paper [...].

Evaluation of the Cytotoxicity of Ayahuasca Beverages.

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages.

Simultaneous Separation of Antioxidants and Carbohydrates From Food Wastes Using Aqueous Biphasic Systems Formed by Cholinium-Derived Ionic Liquids.

The food industry produces significant amounts of waste, many of them rich in valuable compounds that could be recovered and reused in the framework of circular economy. The development of sustainable and cost-effective technologies to recover these value added compounds will contribute to a significant decrease of the environmental footprint and economic burden of this industry sector. Accordingly, in this work, aqueous biphasic systems (ABS) composed of cholinium-derived bistriflimide ionic liquids (ILs) and carbohydrates were investigated as an alternative process to simultaneously separate and recover antioxidants and carbohydrates from food waste. Aiming at improving the biocompatible character of the studied ILs and proposed process, cholinium-derived bistriflimide ILs were chosen, which were properly designed by playing with the cation alkyl side chain and the number of functional groups attached to the cation to be able to create ABS with carbohydrates. These ILs were characterized by cytotoxicity assays toward human intestinal epithelial cells (Caco-2 cell line), demonstrating to have a significantly lower toxicity than other well-known and commonly used fluorinated ILs. The capability of these ILs to form ABS with a series of carbohydrates, namely monosaccharides, disaccharides and polyols, was then appraised by the determination of the respective ternary liquid-liquid phase diagrams at 25°C. The studied ABS were finally used to separate carbohydrates and antioxidants from real food waste samples, using an expired vanilla pudding as an example. With the studied systems, the separation of the two products occurs in one-step, where carbohydrates are enriched in the carbohydrate-rich phase and antioxidants are mainly present in the IL-rich phase. Extraction efficiencies of carbohydrates ranging between 89 and 92% to the carbohydrate-rich phase, and antioxidant relative activities ranging between 65 and 75% in the IL-rich phase were obtained. Furthermore, antioxidants from the IL-rich phase were recovered by solid-phase extraction, and the IL was recycled for two more times with no losses on the ABS separation performance. Overall, the obtained results show that the investigated ABS are promising platforms to simultaneously separate carbohydrates and antioxidants from real food waste samples, and could be used in further related applications foreseeing industrial food waste valorization.

A nanoformulation for the preferential accumulation in adult neurogenic niches.

Stimulation of adult neurogenesis by targeting the endogenous neural stem cells (NSCs), located in hippocampus and subventricular zone (SVZ), with nanoformulations has been proposed for brain repair in cases of neurodegenerative diseases. Unfortunately, it is relatively unknown the nanoformulation properties to facilitate their accumulation in the neurogenic niches after intravenous injection. Here, we have screened different gold-based formulations having variable morphology, surface chemistry and responsiveness to light for their capacity to cross the blood brain barrier (BBB) and accumulate preferentially in the neurogenic niches. Results obtained in a human in vitro BBB model showed that gold nanoparticles (Au NPs) and gold nanorods (Au NRs) conjugated with medium density of transferrin (Tf) peptides (i.e. between 169 and 230 peptides per NP) crossed more efficiently the BBB than the remaining formulations. This is due to a relatively lower avidity of these formulations to Tf receptor (TfR) and lower accumulation in the lysosomes, as compared to the other formulations. We further show that the near infrared light (NIR) irradiation of Au NRs, under a certain concentration and at specific cell culture time, lead to the opening of the BBB. Finally, we demonstrate that Au NRs conjugated with Tf administered intravenously in mice and activated by NIR had the highest accumulation in the neurogenic niches. Our results open the possibility of targeting more effectively the neurogenic niches by controlling the properties of the nanoformulations.

An easy-to-use liquid chromatography method with fluorescence detection for the simultaneous determination of five neuroactive amino acids in different regions of rat brain.

INTRODUCTION: To comprehend the normal function and pathological characteristics of certain neurological disorders it is important to evaluate the neuroactive amino acids levels in animal models. METHODS: This work describes a simple liquid chromatography-fluorescence detection (LC-FLD) method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), taurine (Tau) and γ-aminobutyric acid (GABA), using methyl-L-arginine as internal standard, in samples of rat brain tissue. The five target analytes (Asp, Glu, Gln, Tau and GABA) were determined in a single chromatographic run of less 11 min after a derivatization step with o-phthalaldehyde. The derivatives were separated on a reversed-phase C18 column and detected by fluorescence at excitation and emission wavelengths of 340 and 448 nm, respectively. RESULTS: The method was validated in accordance with international guidelines on bioanalytical methods validation and it presented limits of quantification in the range of 25-50 ng mL-1 and calibration curves with determination coefficients (r2) equal to or higher than 0.9920. In addition, the precision (coefficient variation, %) and accuracy (bias, %) of the method meet the established criteria, and the stability of the analytes at the sample handling and storage conditions was demonstrated. DISCUSSION: Unlike other similar bioanalytical assays, the current method was validated using diluted biological matrix, which is advantageous in order to ensure the derivatization process integrity. Moreover, this LC-FLD method was successfully applied for the determination of the compounds of interest in different rat brain tissue regions (frontal cortex, amygdala, hippocampus, cerebellum and striatum). Thus, this bioanalytical assay represents a useful tool to support multiple nonclinical studies in the field of neurosciences, requiring the quantitative profiling and pattern analysis of neuroactive amino acids.

Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography.

The profiling analysis of catecholamines and their metabolites in brain tissue offers a crucial key to understand their functions in the body and the opportunity to follow up neural diseases. A rapid and simple liquid chromatography-fluorescence detection (LC-FLD) method was developed and validated for simultaneously measuring several catecholamines and endogenous related compounds in the rat brain tissue samples. The target analytes measured in this bioanalytical assay were levodopa (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-O-methyldopa (3-O-MD), and homovanillic acid (HVA), being the 3,4-dihydroxybenzylamine (DHBA) used as internal standard (IS). The six analytes (L-DOPA, DA, NE, E, 3-O-MD and HVA) can be determined in a single chromatographic run of less than 12min, and all the compounds (analytes and IS) were detected using their native fluorescence and monitored at excitation/emission wavelengths of 279nm/320nm, respectively. The chromatographic and detection conditions were experimentally optimized and then several validation parameters (linearity, limits of quantification and detection, precision and accuracy, recovery, stability and selectivity) were examined. In accordance with the international guidelines of the Food and Drug Administration and European Medicines Agency the method described herein exhibited limits of quantification in the range of 2-25ngmL-1, linearity in wide concentration ranges (r2≥0.994), and acceptable precision (coefficient variation ≤8.76%) and accuracy (bias ±14.65%) levels. Since the bioanalytical procedure does not involve pre-purification or derivatization of the sample, the absolute recovery was found to be around 100%. Moreover, the developed LC-FLD method was successfully applied for the determination of the compounds of interest in tissue samples of different rat brain regions (cerebellum, amygdala, cortex, hippocampus, striatum, mesencephalon, medulla oblongata, substantia nigra and ventral tegmental area). Hence, this assay represents a valuable bioanalytical tool to support several pre(non)clinical studies in the broad field of neurosciences, requiring the quantitative analysis of these bioamines and their metabolites.

Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation.

BACKGROUND: Histamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival. METHODS: The effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice. RESULTS: We found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo. CONCLUSIONS: Overall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson's disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia-induced neuroinflammation. Importantly, our results also open promising new perspectives for the therapeutic use of H1R antagonists to treat or ameliorate neurodegenerative processes.

Retinoic acid-loaded polymeric nanoparticles induce neuroprotection in a mouse model for Parkinson's disease.

Retinoic acid (RA) plays an important role in the commitment, maturation and survival of neural cells. Recently, RA was pointed as a therapeutic option for some neurodegenerative diseases, including Parkinson's disease (PD). The administration of RA has been defying, and in this sense we have previously developed novel RA-loaded polymeric nanoparticles (RA-NPs) that ensure the efficient intracellular transport and controlled release of RA. Herein, we show that nanoformulation as an efficient neuroprotective effect on dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mouse model for PD. The results showed that the RA-NPs administration induced a significant reduction of DA neuron loss in the substantia nigra (SN) as well as their neuronal fiber/axonal innervations in the striatum. Furthermore, we observed an increase in the expression levels of the transcription factors Pitx3 and Nurr1 induced by RA-NPs, showing its supportive effect on the development and functional maintenance of DA neurons in PD. This is the first study showing that RA-NPs can be an innovative strategy to halt the progression of PD pathogenesis, suggesting that this nanoformulation could be of particular interest for the development of new approaches for PD therapeutics.

Daily consumption of white tea (Camellia sinensis (L.)) improves the cerebral cortex metabolic and oxidative profile in prediabetic Wistar rats.

Diabetes mellitus (DM) is a major public health problem and its incidence is rising dramatically. The brain, particularly the cerebral cortex, is very susceptible to glucose fluctuations and hyperglycaemia-induced oxidative stress. Tea (Camellia sinensis (L.)) is widely consumed; however, the antidiabetic properties of white tea remain largely unexplored. In the present study, we investigated the effects of daily consumption of white tea on the cerebral cortex of prediabetic rats. The cerebral cortex metabolic profile was evaluated, and the expression levels of GLUT, phosphofructokinase-1, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were assessed. LDH activity was also determined. The cerebral cortex oxidative profile was determined by evaluating its antioxidant power, lipid peroxidation and protein oxidation levels. Catalase, glutathione, glutamate, N-acetylaspartate, aspartate, choline, γ-aminobutyric acid, taurine and valine contents were determined. Daily consumption of white tea ameliorated glucose tolerance and insulin sensitivity. Moreover, white tea altered the cortex glycolytic profile, modulating GLUT expression and lactate and alanine contents. Finally, white tea consumption restored protein oxidation and lipid peroxidation levels and catalase expression, and improved antioxidant capacity. In conclusion, daily consumption of white tea improved the cerebral cortex metabolic and oxidative profile in prediabetic rats, suggesting it as a good, safe and inexpensive strategy to prevent DM-related effects in the cerebral cortex.

Rodent models of Parkinson's disease: beyond the motor symptomatology.

Parkinson's disease (PD) is classically characterized by motor symptoms; however, non-motor symptoms (NMS) are increasingly recognized as relevant in disease-state, given the associated alterations in mood (depression and anxiety) and cognition. Here, particularly in regards to NMS, we aimed to compare the motor, emotional and cognitive behavior of three animal models of PD that trigger dopaminergic (DAergic) degeneration on both brain hemispheres: (i) the 6-hydroxydopamine (6-OHDA, 8 or 6 µg) lesion model; (ii) the paraquat (PQ) induced model, and (iii) a genetic model based on α-synuclein overexpression (α-syn). 6-OHDA and α-syn vector were injected bilaterally in the substantia nigra pars compacta (SNpc) of adult male Wistar rats; as for PQ delivery, micro-osmotic pumps were implanted in the interscapular region. Motor deficits were observed in all models, with histological analysis of tyrosine hydroxylase positive cells in the SNpc revealing a significant loss of DAergic neurons in all animal models. In addition, the α-syn animal model also presented a reduction in exploratory activity, and the 6-OHDA and PQ animals displayed a significant increase in both depressive- and anxiety-like behavior. Interestingly, cognitive impairment (working memory) was only observed in the 6-OHDA model. Overall, these PD models are suitable for mimicking the motor symptoms associated to PD, with each encompassing other relevant NMS components of the disorder that may prove beneficial for further studies in PD.