Simple, ultrasensitive detection of superoxide anion radical mutations in melanoma mice with SERS microneedles.

Elevated levels of superoxide anion radicals (O2·-) have been implicated in the pathogenesis of a variety of diseases, such as cancer, inflammatory diseases and autoimmune diseases. To determine the O2·- concentration for assisting disease detection, a method based on surface-enhanced Raman scattering (SERS) combined with transparent polymer microneedles has been developed. Photocrosslinked NOA61 is used to prepare microneedles with sulfhydryl group, which can contribute to anchor gold nanoparticles (Au NPs) functionalized by p-mercaptobenzoic acid (PATP). This work successfully constructed SERS microneedles for in situ detection. A REDOX reaction occurred between PATP and O2·-, resulting in the formation of dimethylaminoborane (DMAB) and a subsequent change in Raman signal. Based on the quantitative relationship between the change of peak area ratio at 1042 cm-1 and 1077 cm-1 and the concentration change of O2·-, a standard curve with a linear range of 0-480 ng/mL was constructed. The SERS microneedles were effectively employed to track melanoma progression in mice, establishing a fundamental correlation between O2·- concentration and melanoma stage, as confirmed by ELISA. The benefits of this approach, including convenience, in situ applicability, and low cost, are anticipated to offer novel insights for non-invasive in situ detection, potentially enhancing disease monitoring and diagnosis.

A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress.

Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.

Phytochemical Properties and In Vitro Biological Activities of Phenolic Compounds from Flower of Clitoria ternatea L.

Phenolic compounds from the flower of Clitoria ternatea L. (PCFCTL) were extracted using a high-speed shearing extraction technique and purified by AB-8 macroporous resins, and the phytochemical composition of the purified phenolic compounds from the flower of Clitoria ternatea L. (PPCFCTL) was then analyzed. Subsequently, its bioactivities including antioxidant properties, enzyme inhibitory activities, and antiproliferative activities against several tumor cell lines were evaluated. Results indicated that the contents of total phenolics, flavonoids, flavonols, flavanols, and phenolic acids in PPCFCTL were increased by 3.29, 4.11, 2.74, 2.43, and 2.96-fold, respectively, compared with those before being purified by AB-8 macroporous resins. The results showed PPCFCTL have significant antioxidant ability (measured by reducing power, RP, and ferric reducing antioxidant power method, FRAP) and good DPPH, ABTS+, and superoxide anion radical scavenging activities. They can also significantly inhibit lipase, α-amylase, and α-glucosidase. In addition, morphological changes of HeLa, HepG2, and NCI-H460 tumor cells demonstrated the superior antitumor performance of PPCFCTL. However, the acetylcholinesterase inhibitory activity was relatively weak. These findings suggest that PPCFCTL have important potential as natural antioxidant, antilipidemic, anti-glycemic and antineoplastic agents in health-promoting foods.

A review of pesticide phototransformation on the leaf surface: Models, mechanism, and influencing factors.

Phototransformation is an important environmental fate of pesticides on plant leaves. This review found that the photodegradation rates of pesticides on leaves might be faster or slower than those in organic solvents or on glass because of the different spectral patterns and light fluxes on the model surface. Wax was found to play an important role in pesticide phototransformation because it has photosensitizing properties, which might be stimulated under light irradiation to produce reactive species, such as hydroxyl radicals, singlet oxygen, methyl radicals, alkyl radicals, and superoxide radicals. These reactive species could accelerate pesticide photodegradation by several times. Wax can also decrease the photodegradation rate of pesticides by quenching reactive species or light-shielding effects. The environmental conditions and phytochemical properties of leaves play important roles in pesticide phototransformation primarily because the composition of wax varies with plant species and environmental factors. The phototransformation of pesticides on leaves was promoted by a low dosage of adjuvant because they act as photosensitizers and improve the dispersity of pesticides, while it was inhibited at a high concentration of adjuvant because of their light shielding effect. Finally, recommendations for future research were discussed, including (1) distinguishing the direct and indirect photodegradation of pesticides; (2) developing model, molecular level visualization and analysis techniques; (3) conducting more field research; and (4) considering the effect of climate change, especially the interaction of climatic factors. This review gives a comprehensive overview of the current knowledge of pesticide phototransformation on leaves and provides suggestions for future studies.

Insight into the pulmonary molecular toxicity of heated tobacco products using human bronchial and alveolar mucosa models at air-liquid interface.

Heated tobacco products (HTP) are novel nicotine delivery products with limited toxicological data. HTP uses heating instead of combustion to generate aerosol (HTP-smoke). Physiologically relevant human bronchial and alveolar lung mucosa models developed at air-liquid interface were exposed to HTP-smoke to assess broad toxicological response (n = 6-7; ISO puffing regimen; compared to sham; non-parametric statistical analysis; significance: p < 0.05). Elevated levels of total cellular reactive oxygen species, stress responsive nuclear factor kappa-B, and DNA damage markers [8-hydroxy-2'-deoxyguanosine, phosphorylated histone H2AX, cleaved poly-(ADP-Ribose) polymerase] were detected in HTP-smoke exposed bronchial and/or alveolar models. RNA sequencing detected differential regulation of 724 genes in the bronchial- and 121 genes in the alveolar model following HTP-smoke exposure (cut off: p ≤ 0.01; fold change: ≥ 2). Common enriched pathways included estrogen biosynthesis, ferroptosis, superoxide radical degradation, xenobiotics, and α-tocopherol degradation. Secreted levels of interleukin (IL)1ꞵ and IL8 increased in the bronchial model whereas in the alveolar model, interferon-γ and IL4 increased and IL13 decreased following HTP-smoke exposure. Increased lipid peroxidation was detected in HTP-smoke exposed bronchial and alveolar models which was inhibited by ferrostatin-1. The findings form a basis to perform independent risk assessment studies on different flavours of HTP using different puffing topography and corresponding chemical characterization.

Degradation of tetracycline by UV/Fe3+/persulfate process: Kinetics, mechanism, DBPs yield, toxicity evaluation and bacterial community analysis.

As a widely produced and used antibiotic, tetracycline (TC) has been frequently found in rivers, soil and drinking water. In this study, the degradation of TC was investigated by UV/Fe3+/persulfate (PS) coupled process. The degradation behavior was well fitted with pseudo-first-order model. Hydroxyl radicals (·OH), sulfate radicals (SO4-·) and superoxide radical (O2-·) were identified as the primary reactive oxygen species (ROS) in UV/Fe3+/PS process, the contribution to TC degradation were found to be 41.94%, 33.94% and 17.44% at pH 3.0, respectively. Fe(IV) generated from the system also played a crucial role in TC removal. The effects of process parameters (PS/Fe3+ dosages, pH, humic acid, Cl-, HCO3-, NO3- and CO32-) on degradation were investigated. It was found that the degradation of TC was highly pH-dependent, and the optimal performance was obtained at pH 3.0. Except for Cl-, the presence of HA, HCO3-, NO3- and CO32- inhibited TC degradation. The possible transformation pathway involving the hydroxylation, N-demethylation, hydrogenation and dehydroxylation was proposed. Furthermore, the toxicity and mutagenicity of TC and transformation products (TPs) were estimated using ECOSAR and TEST softwares, demonstrating that the toxicity level of most TPs was lower/equal to their precursors. The evaluation of DBPs showed that UV/Fe3+/PS process could reduce the potential of DBPs formation, especially for TCAA and TCM. Microbial community composition was analyzed by 16 S rDNA sequencing, and the relative abundance of ARG-carrying opportunistic pathogens was significantly declined after UV/Fe3+/PS treatment. In general, this study provides an economical, efficient and safe strategy for TC removal.

A Plate Reader-Based Measurement of the Cellular ROS Production Using Dihydroethidium and MitoSOX.

Intracellular reactive oxygen species (ROS) act as an important signaling transductor in cells, regulating almost every aspect of cell biology. Measurements of ROS production thus, offer links between oxidative stress and cell pathophysiology. Here, we describe a simple screening assay in intact adherent cells by fluorescence microplate readers, using dihydroethidium (DHE) and MitoSOX to measure cytosolic superoxide and mitochondrial superoxide production, respectively. This assay enables a quick and reliable assessment of ROS generation in a well-controlled environment.

Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring.

The use of gold nanoparticles/superoxide dismutase (AuNP/SOD) bioconjugates is described as building blocks in SOD biosensor development for the quantification of superoxide in cell culture media. AuNP functionalization with 11-mercaptoundecanoic acid (MUA) and 4-mercaptobenzoic acid (MBA) (AuNPMUA and AuNPMBA) was used to improve SOD immobilization through EDC/NHS coupling using their -COOH terminus, leading to the formation of more stable bioconjugates. AuNP and AuNP/SOD bioconjugates were characterized by SEM to determine their size and morphology, UV-Vis for optical properties, FT-IR, and Raman spectroscopies for chemical functional group analysis and EDX for elemental analysis. Electrochemical methods were used to characterize the Au/AuNP-modified electrodes. For the optimization of the biosensor architecture, different AuNP/enzyme bioconjugates were prepared by varying the amount of both enzyme and AuNP, as well as their incubation time. Finally, the biosensors incorporating the bioconjugates were characterized by fixed potential amperometry and voltammetric analysis in order to establish the enzymatic mechanism and to elucidate the best biosensor architecture for monitoring superoxide in cell culture media. The best sensitivity value for superoxide detection corresponded to 41.2 nA µM cm-2, achieved by a biosensor based on AuNPMBA/SOD bioconjugates monitored through fixed potential amperometry at 0.3 V vs. Ag/AgCl, with a limit of detection of 1.0 µM, and overall very good operational stability, maintaining 91% of the initial sensitivity after 30 days. Finally, the optimized biosensor was employed for the quantification of successive additions of superoxide in cell culture media, with excellent recovery values.

Development of a Deep-Sea Submersible Chemiluminescent Analyzer for Sensing Short-Lived Reactive Chemicals.

Based on knowledge of their production pathways, and limited discrete observations, a variety of short-lived chemical species are inferred to play active roles in chemical cycling in the sea. In some cases, these species may exert a disproportionate impact on marine biogeochemical cycles, affecting the redox state of metal and carbon, and influencing the interaction between organisms and their environment. One such short-lived chemical is superoxide, a reactive oxygen species (ROS), which undergoes a wide range of environmentally important reactions. Yet, due to its fleeting existence which precludes traditional shipboard analyses, superoxide concentrations have never been characterized in the deep sea. To this end, we have developed a submersible oceanic chemiluminescent analyzer of reactive intermediate species (SOLARIS) to enable continuous measurements of superoxide at depth. Fluidic pumps on SOLARIS combine seawater for analysis with reagents in a spiral mixing cell, initiating a chemiluminescent reaction that is monitored by a photomultiplier tube. The superoxide in seawater is then related to the quantity of light produced. Initial field deployments of SOLARIS have revealed high-resolution trends in superoxide throughout the water column. SOLARIS presents the opportunity to constrain the distributions of superoxide, and any number of chemiluminescent species in previously unexplored environments.

Facile synthesis of nickel phosphate nanorods as biomimetic enzyme with excellent electrocatalytic activity for highly sensitive detection of superoxide anion released from living cells.

In this work, the superoxide dismutase (SOD) biomimetic enzyme based on nickel phosphate nanorods [Ni(PO4)NRs] was prepared by a simple and eco-friendly hydrothermal method. After further introduction of carboxylated multi-walled carbon nanotubes (C-MWCNTs), the obtained Ni(PO4)NRs/C-MWCNTs nanocomposites were utilized as the novel electrode materials to construct the electrochemical superoxide anion radicals (O2•-) biosensor with excellent electrical conductivity and unprecedented catalytic performance. The morphology of Ni(PO4)NRs/C-MWCNTs nanocomposite was examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Raman microscope, respectively. Under the optimum conditions, the proposed biosensor exhibits high sensitivity (5.67 × 104 µA/mM/cm2), low detection limit (0.097 µM at S/N = 3) and good selectivity to O2•-. In addition, the sensor can monitor O2•- released from MCF-7 cells with satisfactory results, which provides a great opportunity to apply it in the field of fundamental research and clinical diagnostics. Our results would promote Ni(PO4)NRs as the SOD biomimetic enzyme for designing biosensor and expand its various applications in biocatalysis and bioanalysis.

Detection of superoxide radical in all biological systems by Thin Layer Chromatography.

The study presents a new method that detects O2•-, via quantification of 2-hydroxyethidium (2-ΟΗ-Ε+) as low as ∼30 fmoles by High-Performance Thin Layer Chromatography (HPTLC). The method isolates 2-ΟΗ-Ε+ after its extraction by the anionic detergent SDS (at 18-fold higher than its CMC) together with certain organic/inorganic reagents, and its HPTLC-separation from di-ethidium (di-Ε+) and ethidium (Ε+). Quantification of 2-OH-E+ is based on its ex/em maxima at 290/540 nm, and of di-E+ and E+ at 295/545 nm. The major innovations of the present method are the development of protocols for (i) efficient extraction (by SDS) and (ii) sensitive quantification (by HPTLC) for 2-OH-E+ (as well as di-E+ and E+) from most biological systems (animals, plants, cells, subcellular compartments, fluids). The method extracts 2-ΟΗ-Ε+ (by neutralizing the strong binding between its quaternary N+ and negatively charged sites on phospholipids, DNA etc) together with free HE, while protects both from biological oxidases, and also extracts/quantifies total proteins (hydrophilic and hydrophobic) for expressing O2•- levels per protein quantity. The method also uses SDS (at 80-fold lower than its CMC) to extract/remove/wash 2-ΟΗ-Ε+ from cell/organelle exterior membrane sites, for more accurate internal content quantification. The new method is applied on indicative biological systems: (1) artificially stressed (mouse organs and liver mitochondria and nuclei, ±exposed to paraquat, a known O2•- generator), and (2) physiologically stressed (cauliflower plant, exposed to light/dark).

[Generation, Detection and Bio-protection of Reactive Oxygen Species/Free Radicals].

In the present paper, generation, detection and protection of reactive oxygen species (ROS)/free radicals in relation to the author's research over about 20 years are reviewed. ROS/free radicals are generally generated physically, chemically and biologically, and they are harmful to living organisms by inducing various disorders and diseases. To prevent the harmful effects of ROS/free radicals, antioxidants are believed to be useful. Among many methods to detect ROS/free radicals, ESR technique is a direct method and is described in detail in this review. Several topics such as the production of ROS/free radicals by low temperature atmospheric pressure plasma, the evaluation of antioxidant activity using hemolysis of erythrocytes and the protective effects of antioxidants against X-ray induced damage to mice, are presented.

Virulence of Vibrio alginolyticus Accentuates Apoptosis and Immune Rigor in the Oyster Crassostrea hongkongensis.

Vibrio species are ubiquitously distributed in marine environments, with important implications for emerging infectious diseases. However, relatively little is known about defensive strategies deployed by hosts against Vibrio pathogens of distinct virulence traits. Being an ecologically relevant host, the oyster Crassostrea hongkongensis can serve as an excellent model for elucidating mechanisms underlying host-Vibrio interactions. We generated a Vibrio alginolyticus mutant strain (V. alginolyticus△vscC ) with attenuated virulence by knocking out the vscC encoding gene, a core component of type III secretion system (T3SS), which led to starkly reduced apoptotic rates in hemocyte hosts compared to the V. alginolyticusWT control. In comparative proteomics, it was revealed that distinct immune responses arose upon encounter with V. alginolyticus strains of different virulence. Quite strikingly, the peroxisomal and apoptotic pathways are activated by V. alginolyticusWT infection, whereas phagocytosis and cell adhesion were enhanced in V. alginolyticus△vscC infection. Results for functional studies further show that V. alginolyticusWT strain stimulated respiratory bursts to produce excess superoxide (O2•-) and hydrogen peroxide (H2O2) in oysters, which induced apoptosis regulated by p53 target protein (p53tp). Simultaneously, a drop in sGC content balanced off cGMP accumulation in hemocytes and repressed the occurrence of apoptosis to a certain extent during V. alginolyticus△vscC infection. We have thus provided the first direct evidence for a mechanistic link between virulence of Vibrio spp. and its immunomodulation effects on apoptosis in the oyster. Collectively, we conclude that adaptive responses in host defenses are partially determined by pathogen virulence, in order to safeguard efficiency and timeliness in bacterial clearance.

The Inactivation of Human Aldehyde Oxidase 1 by Hydrogen Peroxide and Superoxide.

Mammalian aldehyde oxidases (AOX) are molybdo-flavoenzymes of pharmacological and pathophysiologic relevance that are involved in phase I drug metabolism and, as a product of their enzymatic activity, are also involved in the generation of reactive oxygen species. So far, the physiologic role of aldehyde oxidase 1 in the human body remains unknown. The human enzyme hAOX1 is characterized by a broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into their corresponding carboxylic acids, and hydroxylating various heteroaromatic rings. The enzyme uses oxygen as terminal electron acceptor to produce hydrogen peroxide and superoxide during turnover. Since hAOX1 and, in particular, some natural variants produce not only H2O2 but also high amounts of superoxide, we investigated the effect of both ROS molecules on the enzymatic activity of hAOX1 in more detail. We compared hAOX1 to the high-O2 .--producing natural variant L438V for their time-dependent inactivation with H2O2/O2 .- during substrate turnover. We show that the inactivation of the hAOX1 wild-type enzyme is mainly based on the production of hydrogen peroxide, whereas for the variant L438V, both hydrogen peroxide and superoxide contribute to the time-dependent inactivation of the enzyme during turnover. Further, the level of inactivation was revealed to be substrate-dependent: using substrates with higher turnover numbers resulted in a faster inactivation of the enzymes. Analysis of the inactivation site of the enzyme identified a loss of the terminal sulfido ligand at the molybdenum active site by the produced ROS during turnover. SIGNIFICANCE STATEMENT: This work characterizes the substrate-dependent inactivation of human aldehyde oxidase 1 under turnover by reactive oxygen species and identifies the site of inactivation. The role of ROS in the inhibition of human aldehyde oxidase 1 will have a high impact on future studies.

Assessment of Mitochondrial Reactive Oxygen Species and Redox Regulation in Stem Cells.

Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.

Rosiglitazone restores nitric oxide synthase-dependent reactivity of cerebral arterioles in rats exposed to prenatal alcohol.

BACKGROUND: Prenatal exposure to alcohol leads to a greater incidence of many cardiovascular-related diseases, presumably via a mechanism that may involve increased oxidative stress. An agonist of peroxisome proliferator-activated receptor gamma (PPARγ; rosiglitazone) has been shown to suppress alcohol-induced neuroinflammation and oxidative stress. The goal of this study was to determine whether acute and chronic treatment with rosiglitazone could restore or prevent impaired nitric oxide synthase (NOS)-dependent responses of cerebral arterioles in male and female adult (14-16 weeks old) rats exposed to alcohol in utero. METHODS: We fed Sprague-Dawley dams a liquid diet with or without 3% ethanol for the duration of their pregnancy (21-23 days). In the first series of studies, we examined the reactivity of cerebral arterioles to eNOS- (ADP), nNOS-dependent (NMDA), and NOS-independent agonists in male and female adult rats before and during acute (1 hour) topical application of rosiglitazone (1 µM). In a second series of studies, we examined the influence of chronic treatment with rosiglitazone (3 mg/kg/day in drinking water for 2-3 weeks) on the responses of cerebral arterioles in male and female adult rats exposed to alcohol in utero. RESULTS: We found that in utero exposure to alcohol similarly reduced responses of cerebral arterioles to ADP and NMDA, but not to nitroglycerin in male and female adult rats. In addition, acute treatment of the male and female adult rats with rosiglitazone similarly restored this impairment in cerebral vascular function to that observed in controls. We also found that chronic treatment with rosiglitazone prevented impaired vascular function in male and female adult rats that were exposed to alcohol in utero. CONCLUSIONS: PPARγ activation may be an effective and relevant treatment to reverse or prevent cerebral vascular abnormalities associated with prenatal exposure to alcohol.

Dual-emission copper nanoclusters-based ratiometric fluorescent probe for intracellular detection of hydroxyl and superoxide anion species.

A fluorescent nanoprobe based on copper nanoclusters (CuNCs) has been developed for ratiometric detection of hydroxyl radicals (•OH) and superoxide anion radicals (O2•-). Two differently luminescent CuNCs, namely cyan-emissive poly(methacrylic acid)-protected copper nanoclusters (PCuNCs) and orange-emissive bovine serum albumin-protected CuNCs (BCuNCs), were conjugated to obtain a hybrid, dual-emission nanoprobe (PCuNCs-BCuNCs) with the corresponding peaks at 445 nm and 652 nm at an excitation wavelength of 360 nm. In particular, the fluorescence peak at 445 nm gradually enhanced with the incremental addition of •OH and O2•-. However, the fluorescence emission at 652 nm was greatly quenched in the presence of •OH, while in case of O2•-, the fluorescence intensity remained constant. The differential response of the PCuNCs-BCuNCs towards •OH and O2•- formed the basis of ratiometric detection. Under optimal conditions, the PCuNCs-BCuNCs exhibited good sensitivity and linearity towards •OH and O2•- with limits of detection of 0.15 µM and 1.8 µM, respectively. Moreover, the nanoprobe exhibited high selectivity for •OH and O2•- over other potential ROS interferences. Besides, PCuNCs-BCuNCs were eventually applied for qualitative and quantitative ratiometric assessment of intracellular •OH and O2•- in L-132 cells. Therefore, this strategy unveils a new potential for copper nanocluster-based sensing of ROS.

Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy.

Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.

Detection of Superoxide Radical in Adherent Living Cells by Electron Paramagnetic Resonance (EPR) Spectroscopy Using Cyclic Nitrones.

Spin trapping with cyclic nitrones coupled to electron paramagnetic resonance (EPR) enables the detection and characterization of oxygen-derived free radicals, such as superoxide and hydroxyl radicals, in living cells. Detection is usually performed on cell suspensions introduced in glass capillaries, gas-permeable tubing, or flat cells, even when cells normally require attachment for growth. However, radical production may be influenced by cell adhesion, while enzymatic or mechanical cell harvesting may damage the cells and alter their metabolic rates. Here, we describe the detection on adherent cells attached to microscope coverslip glasses. This method preserves cell integrity, ensures near physiological conditions for naturally adherent cells, and is relatively simple to set up. Up to 12 conditions can be screened in half a day using a single batch of culture cells.

Remote Ischemic Post-Conditioning Therapy is Protective in Mouse Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is characterized by visual dysfunction after indirect or direct injury to the optic nerve following blunt head trauma. TON is associated with increased oxidative stress and inflammation resulting in retinal ganglion cell (RGC) death. Remote ischemic post-conditioning (RIC) has been shown to enhance endogenous protective mechanisms in diverse disease models including stroke, vascular cognitive impairment (VCI), retinal injury and optic nerve injury. However, the protective mechanisms underlying the improvement of retinal function and RGC survival after RIC treatment remain unclear. Here, we hypothesized that RIC therapy may be protective following TON by preventing RGC death, oxidative insult and inflammation in the mouse retina. To carry out the study, mice were divided in three different groups (Control, TON and TON + RIC). We harvested retinal tissue 5 days after TON induction for western blotting and histochemical analysis. We observed increased TON-induced retinal cell death compared with controls by cleaved caspase-3 immunohistochemistry. Furthermore, the TON cohort demonstrated increased TUNEL positive cells which were significantly attenuated by RIC. Immunofluorescence data showed that oxidative stress markers dihydroethidium (DHE), NOX-2 and nitrotyrosine expression were elevated in the TON group relative to controls and RIC therapy significantly reduced the expression level of these markers. Next, we found that the proinflammatory cytokine TNF-α was increased and anti-inflammatory IL-10 was decreased in plasma of TON animals, and RIC therapy reversed this expression level. Interestingly, western blotting of retinal tissue showed that RGC marker Brn3a and tight junction proteins (ZO-1 and Occludin), and AMPKα1 expression were downregulated in the TON group compared to controls. However, RIC significantly increased the expression levels of these proteins. Together these data suggest that RIC therapy activates endogenous protective mechanisms which may attenuate TON-induced oxidative stress and inflammation, and improves BRB integrity.