Reactive Oxygen Species In Neurons Research Articles

Paraoxonase 2 protects against oxygen-glucose deprivation/reoxygenation-induced neuronal injury by enhancing Nrf2 activation via GSK-3β modulation.

Paraoxonase 2 (PON2) is a powerful antioxidant that mediates cell survival under oxidative stress; however, its protection neurons against cerebral ischemia-reperfusion injury-induced oxidative stress remains unclear. This study aimed to determine the precise regulating role of PON2 in neuronal survival under oxidative stress. An in vitro model of cerebral ischemia-reperfusion injury was used to assess the effect of PON2 on oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R). Results showed that PON2 expression in neurons was decreased due to OGD/R exposure. A series of functional experiments revealed that upregulated PON2 improved OGD/R-impaired viability and attenuated OGD/R-induced increases in apoptosis and reactive oxygen species in neurons. Decreased PON2 expression enhanced neuronal sensitivity to OGD/R-induced injury. Overexpressed PON2 markedly enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus and increased the levels of Nrf2-mediated transcriptional activity. Furthermore, PON2 enhanced the Nrf2 activation by modulating glycogen synthase kinase-3β (GSK-3β). Inhibition of GSK-3β substantially abrogated the PON2 knockdown-mediated suppression of Nrf2 activity. Notably, Nrf2 inhibition partially reversed the neuroprotective effects of PON2 overexpression in OGD/R-exposed neurons. These findings indicate that PON2 alleviates OGD/R-induced apoptosis and oxidative stress in neurons by potentiating Nrf2 activation via GSK-3β modulation. This study highlights the potential neuroprotective function of PON2 against cerebral ischemia-reperfusion injury.

Nrf2 mediates the neuroprotective effect of isoflurane preconditioning in cortical neuron injury induced by oxygen-glucose deprivation.

To investigate how nuclear factor-E2-related factor 2 (Nrf2) involved in the protective effect of isoflurane (Iso) preconditioning in oxygen glucose deprivation (OGD)-induced cortical neuron injury. Primary mouse cortical neurons were divided into Control, ML385 (an Nrf2 inhibitor), Iso, Iso + ML385, OGD, ML385 + OGD, Iso + OGD, and Iso + ML385 + OGD groups. Lactate dehydrogenase activity (LDH) release and oxidative stress indexes were quantified. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability, Annexin V-FITC/propidium iodide (PI) staining to measure cell apoptosis, dichloro-dihydro-fluorescein diacetate (DCFH-DA) method to test reactive oxygen species (ROS), and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting to evaluate genes and protein expression. Iso preconditioning reduced LDH release and inhibited cell cytotoxicity in OGD-induced cortical neurons, which was abolished by ML385. Iso preconditioning increased the Nrf2 nuclear translocation in cortical neurons. Meanwhile, Iso decreased the OGD-induced apoptosis with the down-regulations of Bax and Caspase-3 and the up-regulation of Bcl-2, which was reversed by ML385. OGD enhanced the level of ROS and malondialdehyde (MDA) in cortical neurons, but reduced the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), which were aggravated in ML385 + OGD group and mitigated in Iso + OGD group. No observable difference was found between OGD group and Iso + ML385 + OGD group regarding apoptosis-related proteins and oxidative stress-related indexes. Iso preconditioning up-regulated Nrf2 level to play its protective role in OGD-induced mouse cortical neuron injury.

20‐HETE synthesis inhibition attenuates traumatic brain injury–induced mitochondrial dysfunction and neuronal apoptosis via the SIRT1/PGC‐1α pathway: A translational study

Objectives20‐hydroxyeicosatetraenoic acid (20‐HETE) is a metabolite of arachidonic acid catalysed by cytochrome P450 enzymes and plays an important role in cell death and proliferation. We hypothesized that 20‐HETE synthesis inhibition may have protective effects in traumatic brain injury (TBI) and investigated possible underlying molecular mechanisms.Materials and methodsNeurologic deficits, and lesion volume, reactive oxygen species (ROS) levels and cell death as assessed using immunofluorescence staining, transmission electron microscopy and Western blotting were used to determine post‐TBI effects of HET0016, an inhibitor of 20‐HETE synthesis, and their underlying mechanisms.ResultsThe level of 20‐HETE was found to be increased significantly after TBI in mice. 20‐HETE synthesis inhibition reduced neuronal apoptosis, ROS production and damage to mitochondrial structures after TBI. Mechanistically, HET0016 decreased the Drp1 level and increased the expression of Mfn1 and Mfn2 after TBI, indicating a reversal of the abnormal post‐TBI mitochondrial dynamics. HET0016 also promoted the restoration of SIRT1 and PGC‐1α in vivo, and a SIRT1 activator (SRT1720) reversed the downregulation of SIRT1 and PGC‐1α and the abnormal mitochondrial dynamics induced by 20‐HETE in vitro. Furthermore, plasma 20‐HETE levels were found to be higher in TBI patients with unfavourable neurological outcomes and were correlated with the GOS score.ConclusionsThe inhibition of 20‐HETE synthesis represents a novel strategy to mitigate TBI‐induced mitochondrial dysfunction and neuronal apoptosis by regulating the SIRT1/PGC‐1α pathway.

Mitochondrial defects in the respiratory complex I contribute to impaired translational initiation via ROS and energy homeostasis in SMA motor neurons

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of lower motor neurons, which leads to proximal muscle weakness and atrophy. SMA is caused by reduced survival motor neuron (SMN) protein levels due to biallelic deletions or mutations in the SMN1 gene. When SMN levels fall under a certain threshold, a plethora of cellular pathways are disturbed, including RNA processing, protein synthesis, metabolic defects, and mitochondrial function. Dysfunctional mitochondria can harm cells by decreased ATP production and increased oxidative stress due to elevated cellular levels of reactive oxygen species (ROS). Since neurons mainly produce energy via mitochondrial oxidative phosphorylation, restoring metabolic/oxidative homeostasis might rescue SMA pathology. Here, we report, based on proteome analysis, that SMA motor neurons show disturbed energy homeostasis due to dysfunction of mitochondrial complex I. This results in a lower basal ATP concentration and higher ROS production that causes an increase of protein carbonylation and impaired protein synthesis in SMA motor neurons. Counteracting these cellular impairments with pyruvate reduces elevated ROS levels, increases ATP and SMN protein levels in SMA motor neurons. Furthermore, we found that pyruvate-mediated SMN protein synthesis is mTOR-dependent. Most importantly, we showed that ROS regulates protein synthesis at the translational initiation step, which is impaired in SMA. As many neuropathies share pathological phenotypes such as dysfunctional mitochondria, excessive ROS, and impaired protein synthesis, our findings suggest new molecular interactions among these pathways. Additionally, counteracting these impairments by reducing ROS and increasing ATP might be beneficial for motor neuron survival in SMA patients.

Dicaffeoylquinic acids alleviate memory loss via reduction of oxidative stress in stress-hormone-induced depressive mice

Chronic stress can lead to depression due to elevated levels of stress hormones such as glucocorticoid. This is accompanied by an increase in reactive oxygen species (ROS) levels in the brain, which can cause dendritic spine loss and atrophy in neurons, followed by memory loss. Dicaffeoylquinic acids (diCQAs) are naturally occurring polyphenolic antioxidant compounds in Arctium lappa extracts (AL). The effects of natural derivatives of cafferoylqunic acid on stress hormone-induced depressive behavior and their underlying mechanisms are uncertain. In the current study, we showed that diCQAs reduced depressive behaviors including memory loss in corticosterone (CORT) treated mice. The mechanism of anti-depressants of diCQAs is likely through reduction of ROS production by inhibiting the activity of monoamine oxidase (MAO) type A and B in neurons and astrocytes. Among diCQAs, 3,4- and 3,5-diCQA significantly inhibited the activity of MAO enzymes followed by the reduction of ROS in neurons and astrocytes and also protected neuronal atrophy and synaptic transmission against stress hormone. These results suggest that 3,4- and 3,5-diCQAs effectively reduced depressive symptoms and inhibited ROS production to alleviate memory loss in stress hormone-induced depressive mice and hence, which provide some potential natural antidepressants.

Utility of Reactive Species Generation in Plasma Medicine for Neuronal Development.

Reactive oxygen species (ROS) are critical signaling molecules for neuronal physiology that stimulate growth and development and play vital roles in several pathways when in a balanced state, but they cause neurodegeneration when unbalanced. As ROS levels above a certain threshold cause the activation of the autophagy system, moderate levels of ROS can be used as treatment strategies. Currently, such treatments are used together with low-level laser or photodynamic therapies, photo-bio modulation, or infrared treatments, in different chronic diseases but not in the treatment of neurodegeneration. Recently, non-thermal plasma has been successfully used in biomedical applications and treatments, and beneficial effects such as differentiation, cell growth, and proliferation, stimulation of ROS based pathways have been observed. Besides the activation of a wide range of biological signaling pathways by generating ROS, plasma application can be an effective treatment in neuronal regeneration, as well as in neuronal diseases. In this review, we summarize the generation and role of ROS in neurons and provide critical insights into their potential benefits on neurons. We also discuss the underlying mechanisms of ROS on neuronal development. Regarding clinical applications, we focus on ROS-based neuronal growth and regeneration strategies and in the usage of non-thermal plasma in neuronal and CNS injury treatments.

Selenium and L-carnitine protects from valproic acid-Induced oxidative stress and mitochondrial damages in rat cortical neurons

Oxidative stress and mitochondrial dysfunction have been associated with valproic acid (VPA) induced neurotoxicity. Mitochondria are vulnerable to oxidative damage and are also a major source of superoxide free radicals. Therefore, the need for mitochondrial protective and antioxidant agents for reducing valporic acid toxicity in central nerve system (CNS) is essential. In the present study, we investigated the potential beneficial effects of sodium selenite (SS) and L-carnitine (LC) against valproic acid -induced oxidative stress and mitochondrial dysfunction in isolated rat cortical neurons. Valproic acid (50, 100 and 200 µM) treatment caused a significant decrease in cellular viability, which was accompanied by increases in reactive oxygen species (ROS) generation, GSSG and GSH content, lipid peroxidation and lysosomal and mitochondrial damages. Sodium selenite (1 µM) and L-carnitine (1 mM) pretreatment attenuated valproic acid-induced decrease in cell viability. In addition, sodium selenite (1 µM) and L-carnitine (1 mM) pretreatment significantly protected against valproic acid-induced raise in oxidative stress, mitochondrial and lysosomal dysfunction, lipid peroxidation levels and depletion of GSH content. Our results in the current study provided insights into the protective mechanism by L-carnitine and sodium selenite, which is liked, to neuronal ROS generation and mitochondrial and lysosomal damages.

Abstract MP45: Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

The activation of the brain renin angiotensin system (RAS) is required for the blood pressure (BP) elevation in models of neurogenic hypertension (HT). However, it remains unclear whether the expression of prorenin and its binding to prorenin receptor (PRR) in particular brain regions is required for the activation of the brain RAS. Compelling new evidence indicates that renin is expressed in the proximity of the rostral ventrolateral medulla (RVLM) and the nucleus ambiguous within the brainstem where RAS genes including PRR, angiotensinogen, and angiotensin receptors were also detected. Thus, we hypothesized that prorenin acts within the brainstem to induce extracellular signal-regulated kinases (ERK1/2) phosphorylation and generation of reactive oxygen species (ROS) resulting in BP elevation through a PRR-dependent mechanism. Neonatal brainstem neurons obtained from mice at postnatal day 1-3 were cultured and subsequently incubated in presence of recombinant prorenin (rProrenin; 100 nM) or vehicle. rProrenin increased the ratio of phosphorylated-to-total ERK1/2 from 0.7±0.1 A.U. at baseline to 1.0±0.1 A.U. at 30 min (54% increase; p=0.004; n=7) and 1.3±0.1 A.U. at 60 min (102% increase; p<0.0001; n=7). Both PRO20, a PRR blocker, and Ro-31, a protein kinase C inhibitor, abrogated prorenin-induced ERK1/2 phosphorylation, suggesting a PRR-dependent component to this response. Treatment with rProrenin increased NADPH oxidase activity by 47.2±9.5 % compared to vehicle control (p<0.05, n=5). RVLM-targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane-anesthetized C57BL/6J mice. Moreover, preliminary data indicate that RVLM-targeted ablation of PRR in PRR-flox mice attenuates the pressor response to deoxycorticosterone (DOCA)-salt in females (WT=132±3 vs KO=120±3 mmHg; p<0.05; n=3-4), but not males (n=5-8), during the first week on DOCA-salt treatment. There was no difference in BP during the 2 nd and 3 rd week of DOCA-salt. We conclude that prorenin induces PRR-mediated downstream signaling involving ERK1/2 phosphorylation and generation of ROS in brainstem neurons, which might contribute to BP elevation in neurogenic HT in sex-dependent manner.

Trifluoperazine an Antipsychotic Drug and Inhibitor of Mitochondrial Permeability Transition Protects Cytarabine and Ifosfamide-Induced Neurotoxicity.

The link between Ca2+ dysregulation, mitochondria damages, oxidative stress and cellular derangement is particularly evident in neurotoxicity induced by chemotherapeutic agents. In the current study, we investigated effects of trifluoperazine (TFP) as an inhibitor of calmodulin against the cytotoxicity induced by cytarabine (Ara-C) and Ifosfamide (IFOS) on isolated rat neurons and also the mechanisms involved in this toxicity. Isolated rat neurons were pretreated with TFP (100 µM) for 5 min at 37°C, then Ara-C (226 µM) and IFOS (290 µM) were added in separate experiments. After 3 h, the cytotoxicity, reactive oxygen species (ROS), lysosomal membrane destabilization, mitochondrial membrane potential (MMP), lipid peroxidation (LP), glutathione (GSH) and glutathione disulfide (GSSG) levels were measured. Ara-C and IFOS treatments caused a significant decrease in cellular viability, which was accompanied by ROS generation, GSSG/GSH ratio, lipid peroxidation and lysosomal and mitochondrial damages. On the other hand, TFP (100 µM) pre-treatment attenuated Ara-C and IFOS -induced decrease in cell viability. In addition, TFP (100 µM) pre-treatment significantly protected against Ara-C and IFOS -induced increase in ROS generation, lysosomal and mitochondrial damages, lipid peroxidation levels and decrease in GSH/GSSG ratio. Our data provided insights into the mechanism of protection by TFP against Ara-C and IFOS neurotoxicity, which is related, to neuronal ROS formation and mitochondrial damages.

Action of bioactive compounds in cellular oxidative response

Nowadays there is a growing demand for biodiesel production which can be made, among other resources, from mushroom biomass. It is well established the continuous growth of the population affected with premature aging and numerous diseases in which reactive oxygen species (ROS) are implicated. This fact leads to an increasing consumption of antioxidant rich nutrients, such as mushrooms, which are effectively produced on lignocellulosic biomass substrate. The aim of this study was to investigate, in brain slices, the effect of polysaccharides extracted from the Boletus edulis and Tricholoma equestre mushroom species, in the formation of neuronal ROS, using the fluorescent indicator H2DCFDA. The polysaccharides compounds from both mushroom species had no effect on ROS signals at 0.1 g/L and 0.5 g/L, but decreased the amplitude of the signal at the concentration of 1.0 g/L. These results are in agreement with the idea that polysaccharides have antioxidant properties. Besides the nutritional value of mushrooms, the biomass residues from their production can be used as raw material for bioethanol and biobutanol production, being the later an even more efficient biofuel than ethanol or methanol.

Oleic acid enhances the production of reactive oxygen species in neuronal tissue

One of the major current goals is to obtain clean energy sources and to replace petroleum-derived fuel by biodiesel, which is made from biodegradable and renewable biomass such as vegetable oils. In particular, olive oil contains large amounts of oleic acid (OA), which is a monounsaturated fatty acid used for energy production and also for biosensors, nutritional products, pharmaceuticals and cosmetics. Since oleic acid is particularly abundant in olive oil, large concentrations of this fatty acid may exist in the effluents of olive mill production units, which need to be treated in order to avoid water contaminations and undesirable effects in the ecosystem. Indeed, this type of organic waste, which is potentially useful in the production of biodiesel, in excess may be toxic and cause neurodegeneration, through the production of cellular reactive oxygen species (ROS). The effect of 10–100 μM of oleic acid on neuronal ROS production was evaluated in brain slices incubated with the permeant fluorescent ROS indicator H2DCFDA. The experiments revealed that the amplitude of the ROS signals, corrected for the autofluorescence, increased slightly for the OA concentrations in the range 60–100 μM.

JNK Signalling Mediates Context-Dependent Responses to Reactive Oxygen Species in Neurons

Reactive oxygen species (ROS) are generated during physiological bouts of synaptic activity and can also contribute to pathological conditions in the central nervous system. How neurons respond to and distinguish between ROS in these different contexts is currently unknown. During synaptic activity, a JNK-independent c-Fos/c-Jun AP-1 heterodimer activates transcription of the SRXN1 antioxidant response. Under conditions of oxidative stress, an adaptive JNK-dependent c-Jun/c-Jun AP-1 homodimer is selectively activated by accumulation of ROS to drive SRXN1 expression. This fundamental signaling pathway is conserved in the Drosophila nervous system. In Drosophila highwire mutants, which have constitutively active JNK, basal ROS levels are lower than in wild-type flies, but can be reinstated by pan-neuronal JNK inhibition. We have identified a conserved adaptive role for neuronal JNK signaling in directing the composition of AP-1 dimers to mount an antioxidant response and maintain redox homeostasis during ROS accumulation that is relevant to several neurodegenerative diseases.

Influence of Mitochondrial ATP-Sensitive Potassium Channels on Toxic Effect of Amyloid-β 25–35

Amyloid-β (Aβ) is the main component of senile plaques, one of the hallmarks of Alzheimer,s disease. Is been shown that Aβ25–35 decreased neuronal viability while it increased generation of reactive oxygen species (ROS), and albumin (BSA) prevented ROS production and neuronal death in a dose-and time-dependent manner. One of the major sources of ROS is mitochondrion, and is believed that Mitochondrial ATP-regulated potassium channels (mitoKATP) protect synapses and neurons against oxidative and metabolic stress by modulating inner membrane potential and ROS production. Here we investigate the possible participation of MitoKATP channels on toxic effect of Aβ and the protective effect of BSA, by studying the influence of diazoxide (DIAZ) and tolbutamide (TOLB) on the effect of Aβ25–35 in neuronal morphology, cell viability and ROS generation in presence and absence of BSA. DIAZ decreased ROS generation induced by Aβ25–35 in a concentration dependent manner, but increased with the addition of BSA. TOLB increased Aβ25–35 effect on ROS production in a concentration dependent manner, but only in presence of BSA. Neither DIAZ nor TOLB rescued neurons from morphological damage and cell death induced by Aβ25–35. Hence, it could be proposed that MitoKATP channels participate on toxic effects of Aβ25–35, but not in protective effect of BSA, which seems to go through an extraneuronal mechanism.

Longevity-related molecular pathways are subject to midlife "switch" in humans.

Emerging evidence indicates that molecular aging may follow nonlinear or discontinuous trajectories. Whether this occurs in human neuromuscular tissue, particularly for the noncoding transcriptome, and independent of metabolic and aerobic capacities, is unknown. Applying our novel RNA method to quantify tissue coding and long noncoding RNA (lncRNA), we identified ~800 transcripts tracking with age up to ~60 years in human muscle and brain. In silico analysis demonstrated that this temporary linear “signature” was regulated by drugs, which reduce mortality or extend life span in model organisms, including 24 inhibitors of the IGF‐1/PI3K/mTOR pathway that mimicked, and 5 activators that opposed, the signature. We profiled Rapamycin in nondividing primary human myotubes (n = 32 HTA 2.0 arrays) and determined the transcript signature for reactive oxygen species in neurons, confirming that our age signature was largely regulated in the “pro‐longevity” direction. Quantitative network modeling demonstrated that age‐regulated ncRNA equaled the contribution of protein‐coding RNA within structures, but tended to have a lower heritability, implying lncRNA may better reflect environmental influences. Genes ECSIT, UNC13, and SKAP2 contributed to a network that did not respond to Rapamycin, and was associated with “neuron apoptotic processes” in protein–protein interaction analysis (FDR = 2.4%). ECSIT links inflammation with the continued age‐related downwards trajectory of mitochondrial complex I gene expression (FDR < 0.01%), implying that sustained inhibition of ECSIT may be maladaptive. The present observations link, for the first time, model organism longevity programs with the endogenous but temporary genome‐wide responses to aging in humans, revealing a pattern that may ultimately underpin personalized rates of health span.

Chemical Basis of Reactive Oxygen Species Reactivity and Involvement in Neurodegenerative Diseases.

Increasing numbers of individuals suffer from neurodegenerative diseases, which are characterized by progressive loss of neurons. Oxidative stress, in particular, the overproduction of Reactive Oxygen Species (ROS), play an important role in the development of these diseases, as evidenced by the detection of products of lipid, protein and DNA oxidation in vivo. Even if they participate in cell signaling and metabolism regulation, ROS are also formidable weapons against most of the biological materials because of their intrinsic nature. By nature too, neurons are particularly sensitive to oxidation because of their high polyunsaturated fatty acid content, weak antioxidant defense and high oxygen consumption. Thus, the overproduction of ROS in neurons appears as particularly deleterious and the mechanisms involved in oxidative degradation of biomolecules are numerous and complexes. This review highlights the production and regulation of ROS, their chemical properties, both from kinetic and thermodynamic points of view, the links between them, and their implication in neurodegenerative diseases.

Ketamine-induced attenuation of reactive oxygen species in zebrafish is prevented by acetyl l-carnitine in vivo

Ketamine, an anesthetic, is a non-competitive antagonist of the calcium-permeable N-methyl-d-aspartate (NMDA) receptor. High concentrations of ketamine have been implicated in cardiotoxicity and neurotoxicity. Often, these toxicities are thought to be mediated by reactive oxygen species (ROS). However, findings to the contrary showing ketamine reducing ROS in mammalian cells and neurons in vitro, are emerging. Here, we determined the effects of ketamine on ROS levels in zebrafish larvae in vivo. Based on our earlier studies demonstrating reduction in ATP levels by ketamine, we hypothesized that as a calcium antagonist, ketamine would also prevent ROS generation, which is a by-product of ATP synthesis. To confirm that the detected ROS in a whole organism, such as the zebrafish larva, is specific, we used diphenyleneiodonium (DPI) that blocks ROS production by inhibiting the NADPH Oxidases (NOX). Upon 20 h exposure, DPI (5 and 10 μM) and ketamine at (1 and 2 mM) reduced ROS in the zebrafish larvae in vivo. Using acetyl l-carnitine (ALCAR), a dietary supplement, that induces mitochondrial ATP synthesis, we show elevated ROS generation with increasing ALCAR concentrations. Combined, ketamine and ALCAR counter-balanced ROS generation in the larvae suggesting that ketamine and ALCAR have opposing effects on mitochondrial metabolism, which may be key to maintaining ROS homeostasis in the larvae and affords ALCAR the ability to prevent ketamine toxicity. These results for the first time show ketamine’s antioxidative and ALCAR’s prooxidative effects in a live vertebrate.

Effects of an Acute Vitamin C Infusion on Neurovascular Coupling in Healthy Adults

Increases in neuronal metabolic demand are typically matched by a proportional increase in brain blood flow. This neurovascular coupling (NVC) response is impaired with healthy aging and in Alzheimer's disease and has been attributed to an imbalance of reactive oxygen species (ROS) relative to endogenous antioxidant defenses (oxidative stress) in animal models; however, the role of ROS on NVC in healthy humans is not completely understood. High levels of ROS in the vasculature impairs endothelium‐dependent vasodilatation whereas smaller amounts of ROS in neurons may be necessary for NVC. Therefore, we aimed to investigate the effect of an acute antioxidant infusion (vitamin C; VTC) on NVC and shear‐mediated dilation of the internal carotid artery (ICA) in healthy adults. We hypothesized that, compared with control (saline), acute infusion of the antioxidant VTC would augment NVC in healthy adults. Seven healthy adults (2 males/5 females, age: 38 ± 24 years; BMI: 24.0 ± 3.5 kg/m2; blood pressure (BP): 104 ± 8/62 ± 5 mmHg) participated in this randomized, double‐blind placebo‐controlled intervention. Participants received a 20‐minute priming bolus infusion of either saline or 0.06 g ascorbic acid per kg fat‐free mass (FFM), followed by a drip‐infusion of either saline or 0.02 g ascorbic acid per kg FFM. Middle cerebral artery blood flow velocity (MCAv) was measured using transcranial Doppler (TCD) ultrasound (Spencer Technologies, Redmond, WA) at an average insonation depth of 48.0 ± 6.0 mm. Cerebrovascular conductance (CVC) was calculated as mean MCAV divided by beat‐beat mean arterial pressure (FINAPRES NOVA, Enschede, Netherlands). NVC was assessed as the percent change (from baseline) in CVC (CVC%Δ) during an incrementally difficult cognitive challenge (N‐back task) after acute infusion of VTC vs. saline. Shear‐mediated vasodilation of the ICA to a transient (30 second) hypercapnia stimulus was also assessed at each time point as a measure of vascular function independent of neuronal demand. To quantify the NVC response, post infusion CVC and performance at each stage of the N‐back test were analyzed using a 2×2 repeated measures ANOVA with treatment (saline vs. VTC) and stage of the N‐back test as factors. Post‐infusion vasodilation of the ICA to transient hypercapnia was analyzed between treatment conditions using a paired t‐test. Data are presented as mean ± SD. Post‐infusion CVC%Δ was blunted at all levels of the N‐Back task after the infusion of VTC vs. saline (0‐Back: −3.94 ± 7.23% vs. 0.95 ± 3.23%; 1‐Back,: −4.16 ± 8.15% vs. 2.23 ± 4.72%; 2‐Back: −4.83 ± 14.94% vs. 4.79 ± 6.13%; Treatment effect: P = 0.045; F‐statistic (1,18) = 4.66). While there were no significant differences between stages, the number of correct responses decreased as the difficulty of the test increased. No significant differences in the ICA response to transient hypercapnia were observed after infusion of VTC vs. saline (4.08 ± 1.44% vs. 4.62 ± 0.76%; P = 0.54). These data suggest that ROS may be necessary to elicit an appropriate NVC response in healthy adults.Support or Funding InformationThis work was supported by a pilot grant through the Center of Biomedical Research Excellence in Cardiovascular Health P20GM113125.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila.

Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, we show that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. We identified the highly conserved Parkinson's disease-linked protein DJ-1β as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.

Effect of carbon monoxide postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration in rat hippocampal neurons: the relationship with mPTP/ROS signaling pathway

Objective To evaluate the effect of carbon monoxide(CO) postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration(OGD/R) in rat hippocampal neurons and the relationship with mitochondrial permeability transition pore(mPTP)/reactive oxygen species(ROS) signaling pathway. Methods Primary hippocampal neurons were cultured in vitro, seed in 6-well or 96-well plates, and divided into 5 groups(n=24 each) using a random number table method: control group(C group), OGD/R group, CO postconditioning group(CO group), specific mPTP opener atractyloside plus CO postconditioning group(ACO group), and specific ROS inducer antimycin A plus CO postconditioning group(KCO group). Neurons were subjected to O2-glucose deprivation(OGD) for 16 h followed by restoration of O2 -glucose supply for 24 h to establish the model of OGD/R injury. In group CO, neurons were exposed to 2% CO-5% CO2 for 3 h at 37 ℃ starting from the end of OGD, followed by normal culture for 21 h. In ACO and KCO groups, atractyloside 20 μmol/L and antimycin A 50 μmol/L were added at the end of OGD, respectively, and the other treatments were similar to those previously described in group CO. Neuronal pyroptosis rate was determined using double immunofluorescent staining cleaved caspase-1-AlexaFluor 568/DAPI after the end of treatments in each group. The neuronal survival rate was determined by MTT, opening of mPTP by Calcein-AM fluorescence, ROS content by DCFH-DA, and expression of interleukin-1beta(IL-1β) and IL-18 by Western blot. Results Compared with C group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in the other groups(P<0.05). Compared with OGD/R group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly decreased, the neuronal survival rate was increased, and the expression of IL-1β and IL-18 was down-regulated in CO, ACO and KCO groups(P<0.05). Compared with CO group, neuronal pyroptosis rate and ROS content were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in ACO and KCO groups, and opening of mPTP was significantly inctreased in ACO group(P<0.05). Conclusion CO postconditioning can inhibit OGD/R-induced pyroptosis in rat hippocampal neurons, and the mechanism is related to inhibiting mPTP/ROS signaling pathway. Key words: Carbon monoxide; Neurons; Cell death; Reperfusion injury; Reactive oxygen species; Mitochondrial membrane transport proteins

A novel Nrf2 activator, RS9, attenuates secondary brain injury after intracerebral hemorrhage in sub-acute phase

The poor prognosis of intracranial hemorrhage (ICH) is attributed to secondary brain injury (SBI), which is caused by oxidative stress. Blood components induce reactive oxygen species (ROS) over-production and cause cytotoxicity. We focused on the antioxidant system and investigated nuclear factor-erythroid 2-related factor 2 (Nrf2), which is a transcription factor that controls several antioxidant enzymes. We examined the effects of a novel Nrf2 activator, RS9, on SBI after ICH. ICH was induced by injecting autologous blood collected from the jugular vein (25 µL) into the striatum of mice. RS9 (0.2 mg/kg, i.p.) was administrated 0, 24, and 48 h after the induction of ICH. Using the ICH model, we measured brain edema, neurological function, neuronal damage and antioxidant proteins expression. We then investigated the mechanisms responsible for the effects of RS9 in vitro using the SH-SY5Y cell line. We used zinc protoporphyrin (ZnPP), a heme oxygenase-1 (HO-1) inhibitor, to elucidate the relationship between HO-1 expression and cell death in vitro in a hemin injury model. RS9 decreased brain edema, improved neurological deficits, decreased neuronal damage area and up-regulated HO-1 and superoxide dismutase 1 (SOD) expressions in the ICH mouse model. RS9 also suppressed neuronal cell death and ROS over-production in vitro. These protective effects were cancelled by the ZnPP co-treatment. Our results suggest that the activation of Nrf2 by RS9 exerts neuroprotective effects that are mediated by the attenuation of oxidative stress, and also that RS9 is an effective therapeutic candidate for the treatment for SBI after ICH.