Reactive Oxygen Species Production In Brain Research Articles

The Behavioral Sequelae of Social Defeat Require Microglia and Are Driven by Oxidative Stress in Mice.

Chronic social defeat (CSD) in male mice can produce anxiety and aberrant socialization. Animals susceptible to CSD show activation of microglia, which have elevated levels of oxidative stress markers. We hypothesized that microglia and reactive oxygen species (ROS) production contribute to the CSD stress-induced changes in affective behavior. First, we selectively depleted microglia (99%) by administering the CSF1R (colony-stimulating factor 1 receptor) antagonist PLX5622 before and during the 14 d CSD procedure. Microglia-depleted mice in contrast to nondepleted mice were protected from the stress effects measured by light/dark and social interaction tests. ROS production, measured histochemically following dihydroethidium administration, was elevated by CSD, and the production was reduced to basal levels in mice lacking microglia. The deleterious stress effects were also blocked in nondepleted mice by continuous intracerebral administration of N-acetylcysteine (NAC), a ROS inhibitor. In a second experiment, at the end of the CSD period, PLX5622 was discontinued to allow microglial repopulation. After 14 d, the brain had a full complement of newly generated microglia. At this time, the mice that had previously been protected now showed behavioral deficits, and their brain ROS production was elevated, both in all brain cells and in repopulated microglia. NAC administration during repopulation prevented the behavioral decline in the repopulated mice, and it supported behavioral recovery in nondepleted stressed mice. The data suggest that microglia drive elevated ROS production during and after stress exposure. This elevated ROS activity generates a central state supporting dysregulated affect, and it hinders the restoration of behavioral and neurochemical homeostasis after stress cessation.SIGNIFICANCE STATEMENT Chronic psychosocial stress is associated with psychiatric disorders such as depression and anxiety. Understanding the details of CNS cellular contributions to stress effects could lead to the development of intervention strategies. Inflammation and oxidative stress are positively linked to depression severity, but the cellular nature of these processes is not clear. The chronic social defeat (CSD) paradigm in mice produces mood alterations and microglial activation characterized by elevated reactive oxygen species (ROS) production. The depletion of microglia or ROS inhibition prevented adverse stress effects. Microglial repopulation of the brain post-CSD reintroduced adverse stress effects, and ROS inhibition in this phase protected against the effects. The results suggest that stress-induced microglial ROS production drives a central state that supports dysregulated affective behavior.

Abstract 113: Glucose Metabolic Disorders in Aging-Associated Microglial NADPH Oxidase 2 Activation and Oxidative Damage of Cerebral Microvasculature and Neurons

Aging has been recognised to be a major risk factor for the development of cardiovascular and neurodegenerative diseases and growing evidence suggests a role for oxidative stress. A Nox2-containing NADPH oxidase has been reported to be a major source of reactive oxygen species (ROS) generation in the vascular system and in the brain. However, the role of Nox2 enzyme in aging-related metabolic disorders and vascular neurodegeneration remains unclear. In this study, we used age-matched wild-type (WT) and Nox2-deficient (Nox2 -/- ) mice on a C57BL/6 background at young (3-4 month) and aging (20-24 month) to investigate the role of Nox2 in aging-related oxidative stress, metabolic disorders and cerebral vascular dysfunction. There was an aging-related increase in blood pressure in WT mice (126 mmHg for young and 148 mmHg for aging) (P<0.05); however the blood pressure was well maintained without significant change in Nox2 -/- aging mice. Compared to young WT mice, WT aging mice had significantly high levels of fasting serum insulin and this was accompanied with delayed clearance of glucose (P<0.05) indicating insulin resistance. In contrast, there was no indication of insulin resistance for Nox2 -/- aging mice. We then examined aging-related brain oxidative stress. Compared to WT young mice, there were significant increases (2.7±0.7 folds) in the levels of ROS production by WT aging brain tissue homogenates as detected by lucigenin-chemiluminescence and DHE fluorescence. Increased ROS production in WT aging brain was accompanied by a significant increase (1.8±0.3 folds) in the Nox2 expression detected mainly in the microglial cells (labelled by Iba-1) and decreases in brain capillaries (labelled by CD31) (2.4±0.8 folds) and neurons (labelled by Neu-N) (2.9±0.5 folds) (all P<0.05). Knockout of Nox2 abolished aging-associated increases in brain ROS production and significantly reduced the aging-related pathophysiological changes in the brain. In conclusion, aging-associated metabolic disorders play a crucial role in aging-associated Nox2 activation and vascular neurodegeneration. Nox2-containing NADPH oxidase represents a valuable therapeutic target for oxidative stress-related brain microvascular damage and neurodegeneration.

The Use of Dichlorofluorescein as a Probe for Monitoring the Effects of Calcium on ROS Production in Mitochondria

Dichlorofluorescein (DCF) is widely applied for monitoring reactive oxygen species (ROS) production in cells and isolated mitochondria. Our purpose was to compare different approaches to the use of DCF for monitoring ROS formation accompanying Ca2+ transport in isolated mitochondria. Ca2+ effects on ROS production was studied in rat brain and liver mitochondria using two modes of DCF application: the first was preloading of stock mitochondrial suspension with DCF precursor dichlorofluorescein diacetate (DCFDA), and the other was the addition of the aliquots of DCFDA directly to the incubation medium. Calcium increased ROS production in brain and liver mitochondria both under steady-state and non-equilibrium conditions caused by the gradual release of cytochrome c and the gating of the electron flow. Using DCF-preloaded mitochondria we have shown that the rate of ROS formation under steady-state conditions was linearly dependent on the rate of Ca2+-cycling and Ca2+-stimulated respiration. Transition to non-equilibrium state resulted in the non-linear rise in ROS formation, which too exhibited dependence on Ca2+ concentration. While in DCF-preloaded mitochondria DCF fluorescence closely followed the time course of Ca2+ transport, in unloaded mitochondria probe loading interfered with the detection of ROS formation because of continuing change of DCF concentration into matrix space. Based on the experiments, we came to the conclusion that DCF preloaded in mitochondria could serve a suitable probe for monitoring the effects of Ca2+ transport on ROS production.

In vivo imaging of reactive oxygen species in mouse brain by using [3H]hydromethidine as a potential radical trapping radiotracer.

To assess reactive oxygen species (ROS) production by detecting the fluorescent oxidation product, hydroethidine has been used extensively. The present study was undertaken to evaluate the potential of the hydroethidine derivative as a radiotracer to measure in vivo brain ROS production. [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]Hydromethidine) was synthesized, and evaluated using in vitro radical-induced oxidization and in vivo brain ROS production model. In vitro studies have indicated that [(3)H]Hydromethidine is converted to oxidized products by a superoxide radical (O(2)(•)-) and a hydroxyl radical (OH(•)-) but not hydrogen peroxide (H(2)O(2)). In vivo whole-body distribution study showed that [(3)H]Hydromethidine rapidly penetrated the brain and then was washed out in normal mice. Microinjection of sodium nitroprusside (SNP) into the brain was performed to produce ROS such as OH(•)- via Fenton reaction. A significant accumulation of radioactivity immediately after [(3)H]Hydromethidine injection was seen in the side of the brain treated with SNP (5 and 20 nmol) compared with that in the contralateral side. These results indicated that [(3)H]Hydromethidine freely penetrated into the brain where it was rapidly converted to oxidized forms, which were trapped there in response to the production of ROS. Thus, [(3)H]Hydromethidine should be useful as a radical trapping radiotracer in the brain.

The Janus of Oxidative Stress Signaling in Different Pathophysiological Conditions

The Janus of Oxidative Stress Signaling in Different Pathophysiological Conditions

Oxidative Stress Is Related to the Deleterious Effects of Heme Oxygenase-1 in anIn VivoNeuroinflammatory Rat Model

Heme oxygenase-1 (HO-1) induction is associated with beneficial or deleterious effects depending on the experimental conditions adopted and the neurodegenerative rodent models used. The present study aimed first to evaluate the effects of cerebral HO-1 induction in an in vivo rat model of neuroinflammation by intrastriatal injection of quinolinic acid (QA) and secondly to explore the role played by reactive oxygen species (ROS) and free iron (Fe2+) derived from heme catabolism promoted by HO-1. Chronic I.P. treatment with the HO-1 inductor and substrate hemin was responsible for a significant dose-related increase of cerebral HO-1 production. Brain tissue loss, microglial activation, and neuronal death were significantly higher in rats receiving QA plus hemin (H-QA) versus QA and controls. Significant increase of ROS production in H-QA rat brain was inhibited by the specific HO-1 inhibitor ZnPP which supports the idea that ROS level augmentation in hemin-treated animals is a direct consequence of HO-1 induction. The cerebral tissue loss and ROS level in hemin-treated rats receiving the iron chelator deferoxamine were significantly decreased, demonstrating the involvement of Fe2+in brain ROS production. Therefore, the deleterious effects of HO-1 expression in this in vivo neuroinflammatory model were linked to a hyperproduction of ROS, itself promoted by free iron liberation.

Respiration and ROS production in brain and spinal cord mitochondria of transgenic rats with mutant G93a Cu/Zn-superoxide dismutase gene

Mitochondrial dysfunction is involved in the pathogenesis of motor neuron degeneration in the G93A mutant transgenic (tgmSOD1) animal model of ALS. However, it is unknown whether mitochondriopathy is a primary or secondary event. We isolated brain (BM) and spinal cord (SCM) mitochondria from 2 month old presymptomatic tgmSOD1 rats and studied respiration and generation of reactive oxygen species (ROS) using a new metabolic paradigm (Panov et al., Am. J. Physiol., Regul. Integr. Comp. Physiol., 2011). The yields of BM and SCM from tgmSOD1 rats were 27% and 58% lower than normal rats (WT). The rates of the State 3 and State 3U respiration of tgBM and tgSCM were normal with glutamate + pyruvate + malate as substrates but were inhibited with pyruvate + malate in tgBM and glutamate + malate in tgSCM. In tgSCM the State 4 respiration with all substrates was significantly (1.5–2 fold) increased as compared with WT-SCM. Western blot analysis showed that tgSCM had lower contents of complexes III (− 60%) and IV (− 35%), and the presence of mutated SOD1 protein in both tgBM and tgSCM. With glutamate + pyruvate + malate or succinate + glutamate + pyruvate + malate as substrates, tgBM and tgSCM generated 5–7 fold more ROS than normal mitochondria, and tgSCM generated two times more ROS than tgBM. We show that the major damaging ROS species in tgmSOD1 animals is H 2O 2. It is known that mutated SOD1, damaged by H 2O 2, associates with mitochondria, and we suggest that this further increases production of H 2O 2. We also show that the total tissue calcium content remained normal in the brain but was diminished by 26% in the spinal cord of presymptomatic tgmSOD1 rats. Conclusion: In tgSCM abnormally high rates of ROS generation, associated with reverse electron transport, result in accelerated mitochondriopathy, and the Ca 2+-dependent excitotoxic death of motor neurons. Thus mitochondrial dysfunction is a key early element in pathogenesis of motor neuron degeneration in tgmSOD1 rats.

Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduction of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxygen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle's metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20-22 degrees C. Turtle brain ROS production decreased 25% within 10 min of cyanide or N(2)-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of complex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner suggestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.