Biting reduces acute stress-induced oxidative stress in the rat hypothalamus

We investigated the inhibitory effect of para-masticatory activity, namely biting, on restraint stress-induced oxidative stress. A blood brain barrier-permeable nitroxyl spin probe, 3-methoxycarbonyl-2,2,5,5,-tetramethylpyrrolidine-1-oxyl (MC-PROXYL), was administered to rats and L-band electron spin resonance (ESR) and ESR-computerized tomography (ESR-CT) imaging were used to show that the decay rate constant of MC-PROXYL in the hypothalamus of isolated brain after 30 min of restraint stress was more rapid than in unrestrained control rats, suggesting that restraint was associated with oxidative stress. Interestingly, biting during restraint stress caused the decay rate constant of MC-PROXYL in isolated brain to approach that of the control group. These observations suggest that biting suppresses oxidative stress induced by restraint stress, and that the anti-stress effect of masticatory motor activity movements, such as biting, are important for reducing the adverse effects associated with exposure to psychological stressors.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Noninvasive evaluation of in vivo free radical reactions catalyzed by iron using in vivo ESR spectroscopy.

The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 micromol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that "chelatable iron" contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron.     

Concentration dependence of nitroxyl spin probes in liposomal solution: electron spin resonance and overhauser-enhanced magnetic resonance studies

In this work, the detailed studies of electron spin resonance (ESR) and overhauser-enhanced magnetic resonance imaging (OMRI) were carried out for permeable nitroxyl spin probe, MC-PROXYL as a function of agent concentration in liposomal solution. In order to compare the impermeable nature of nitroxyl radical, the study was also carried out only at 2?mM concentration of carboxy-PROXYL. The ESR parameters were estimated using L-band and 300?MHz ESR spectrometers. The line width broadening was measured as a function of agent concentration in liposomal solution. The estimated rotational correlation time is proportional to the agent concentration, which indicates that less mobile nature of nitroxyl spin probe in liposomal solution. The partition parameter and permeability values indicate that the diffusion of nitroxyl spin probe distribution into the lipid phase is maximum at 2?mM concentration of MC-PROXYL. The dynamic nuclear polarization (DNP) parameters such as DNP factor, longitudinal relaxivity, saturation parameter, leakage factor and coupling factor were estimated for 2?mM MC-PROXYL in 400?mM liposomal dispersion. The spin lattice relaxation time was shortened in liposomal solution, which leads to the high relaxivity. Reduction in coupling factor is due to less interaction between the electron and nuclear spins, which causes the reduction in enhancement. The leakage factor increases with increasing agent concentration. The increase in DNP enhancement was significant up to 2?mM in liposomal solution. These results paves the way for choosing optimum agent concentration and OMRI scan parameters used in intra and extra membrane water by loading the liposome vesicles with a lipid permeable nitroxyl spin probes in OMRI experiments.     

Direct observations of the redox states of frozen cherry buds by a unique in vivo ESR

Low temperatures cause cellular damage in flower buds of the sweet cherry (Prunus avium L. cv. Satohnishiki). In this study, the redox states within the cherry buds suffering freezing damage were non-destructively observed by a unique in vivo electron spin resonance (ESR) technique with a spin probe such as carbamoyl-PROXYL. The ESR signals of carbamoyl-PROXYL-treated bud were continuously recorded under freezing and thawing condition, which was decreased to approximately -4 degrees C and maintained for 1.5h, and then returned to room temperature. Most of the buds began to freeze at -2.5 to -3.9 degrees C. The peak areas of the ESR signals significantly increased during the period of temperature rise. These results show that the reduced carbamoyl-PROXYL within the frozen bud was re-oxidized and became ESR-detectable while the bud was thawing. Our in vivo ESR technique has confirmed the oxidative transition of the redox states within the buds during thawing.     

Noninvasive study of radiation-induced oxidative damage using in vivo electron spin resonance

Nitroxyl radicals injected into a whole body indicate the disappearance of signal intensity of in vivo electron spin resonance (ESR). The signal decay rates of nitroxyl have reported to be influenced by various types of oxidative stress. We examined the effect of X-irradiation on the signal decay rate of nitroxyl in the upper abdomen of mice using in vivo ESR. The signal decay rates increased 1 h after 15 Gy irradiation, and the enhancement was suppressed by preadministration of cysteamine, a radioprotector. These results suggest that the signal decay of nitroxyl in whole mice is enhanced by radiation-induced oxidative damage. The in vivo ESR system probing the signal decay of nitroxyl could provide a noninvasive technique for the study of oxidative stress caused by radiation in a living body.     

Application of in vivo ESR spectroscopy to measurement of cerebrovascular ROS generation in stroke

This study used an in vivo ESR spectroscopy/spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the brain were analyzed from the enhanced ESR signal decay data of three intra-arterially injected spin probes with different membrane permeabilities. The ESR signal decay of the probe with intermediate permeability was significantly enhanced 30 min after reperfusion following MCAO, whereas no enhancement was observed with the other probes or in the control group. The enhanced in vivo signal decay was significantly suppressed by superoxide dismutase (SOD). Brain damage was barely discernible until 3 h of reperfusion, and was clearly suppressed with the probe of intermediate permeability. The antioxidant MCI-186 completely suppressed the enhanced in vivo signal decay after transient MCAO. These results clearly demonstrate that ROS are generated at the interface of the cerebrovascular cell membrane when reperfusion follows MCAO in rats, and that the ROS generated during the initial stages of transient MCAO cause brain injury.     

Evidence for contribution of vascular NAD(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity

It is well established that oxidative stress is enhanced in diabetes. However, the major in vivo source of oxidative stress is not clear. Here we show that vascular NAD(P)H oxidase may be a major source of oxidative stress in diabetic and obese models. In vivo electron spin resonance (ESR)/spin probe was used to evaluate systemic oxidative stress in vivo. The signal decay rate of the spin probe (spin clearance rate; SpCR) significantly increased in streptozotocin-induced diabetic rats 2 weeks after the onset of diabetes. This increase was completely normalized by treatment with the antioxidants alpha-tocopherol (40 mg/kg) and superoxide dismutase (5000 units/kg), and was significantly inhibited by treatment with a PKC-specific inhibitor, CGP41251 (50 mg/kg), and a NAD(P)H oxidase inhibitor, apocynin (5 mg/kg). Both obese ob/ob mice (10 weeks old) with mild hyperglycemia and Zucker fatty rats (11 weeks old) with normoglycemia exhibited significantly increased SpCR as compared with controls. Again, this increase was inhibited by treatment with both CGP41251 and apocynin. Oral administration of insulin sensitizer, pioglitazone (10 mg/kg), for 7 days also completely normalized SpCR values. These results suggest that vascular NAD(P)H oxidase may be a major source of increased oxidative stress in diabetes and obesity.     

Antioxidant Ability and Lipid Peroxidation in the Hippocampus with Epileptogenesis Induced by Fe3+ Injection into the Amygdaloid Body of Rats

To analyze antioxidant ability and lipid peroxidation in the hippocampus of rats in an interictal state of FeCl₃-induced epileptogenesis, the hippocampal eliminating decay ratio of exogenously applied nitroxide radical (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL)) by electron paramagnetic resonance (EPR) spectroscopy, and the thiobarbituric reactive substances (TBARS) level in the hippocampus were measured. The prolonged half-life of electron paramagnetism of carbamoyl-PROXYL in the hippocampus of rats with chronic FeCl₃-induced epileptogenesis revealed decreased antioxidant ability, which supports the vulnerability against oxidative stress. In addition, TBARS level (marker of lipid peroxidation) was increased in the hippocampus of rats injected with FeCl₃ compared with that of control. This study revealed that repetitive seizures resulted in the decreased hippocampal antioxidant ability with lipid peroxidation and explained the regional vulnerability to oxidative stress in the limbic system with epileptogenesis.     

Redox evaluation in sepsis model mice by the in vivo ESR technique using acyl-protected hydroxylamine

In vivo electron spin resonance (ESR) spectroscopy is a noninvasive technique that measures the oxidative stress in living experimental animals. The rate of decay of the ESR signal right after an injection of nitroxyl radical has been measured to evaluate the oxidative stress in animals, although the probe’s disposition could also affect this rate. Because the amount of probes forming the redox pair of hydroxyl amine and its corresponding nitroxyl radical was shown to be nearly constant in most organs or tissues 10 min after the injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in mice, we evaluated the oxidative stress in sepsis model mice induced by lipopolysaccharide (LPS) by intravenously injecting ACP as a precursor of redox probes. The in vivo ESR signal increased up to 7–8 min after the ACP injection and then decreased. Decay of the in vivo signal in LPS-treated mice was significantly slower than that in healthy mice, whereas no significant difference was observed in the rate of change in the total amount of redox probes in the blood and liver between these groups. ESR imaging showed that the in vivo signals observed at the chest and upper abdomen decayed slowly in LPS-treated mice. Suppression of the decay in LPS-treated mice was canceled by the administration of a combination of pegylated superoxide dismutase and catalase, or an inhibitor of nitric oxide synthase, or gadolinium chloride. These results indicate that the LPS-treated mouse is under oxidative stress and that reactive oxygen species, such as superoxide and peroxynitrite, related to macrophages are mainly involved in the oxidative stress.     

In Vivo Evaluation of Hippocampal Anti-Oxidant Ability of Zonisamide in Rats

We evaluated the anti-oxidant property of zonisamide (ZNS) in the rat brain under freely moving conditions by means of in vivo microdialysis of two exogenous nitroxide radicals, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and 3-methoxy carbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM). Time-dependent changes in the signal intensities of these exogenous nitroxide radicals obtained from the hippocampal perfusates were observed using an X-band ESR spectrometer at 20-min intervals. The ESR signal intensities of nitroxide radicals decreased exponentially in all animals, which indicates that their half-life could be used as a parameter to estimate the decay rate of nitroxide radicals. Nitroxide radicals lose their paramagnetism when exposed to reductants in a biological system. Thus, half-life reflects the in vivo reducing ability. Although the half-life of carbamoyl-PROXYL, which could not pass the blood-brain barrier (BBB), was not changed when compared with the controls, pre-treatment with ZNS significantly shortened the half-life of PCAM, which could pass through the BBB. These findings suggest that the ZNS-induced increase in reducing ability did not occur within the extracellular space, but rather mainly at the neural cell membrane. This study is the first in vivo evaluation of the reducing ability of ZNS in freely moving animals.     

Special issue on “AGEs and ALEs: chemistry,physiopathology and molecular strategies for their inhibition”

Methamphetamine (METH)-induced neurotoxicity is associated with mitochondrial dysfunction and enhanced oxidative stress. The aims of the present study conducted in the mouse brain repetitively treated with METH were to (1) examine the redox status using the redox-sensitive imaging probe 3-methoxycarbonyl-2,2,5,5-tetramethylpiperidine-1-oxyl (MCP) and (2) non-invasively visualize the brain redox status with electron paramagnetic resonance (EPR) imaging. The rate of reduction of MCP was measured from a series of temporal EPR images of mouse heads, and this rate was used to construct a two-dimensional map of rate constants called a “redox map.” The obtained redox map clearly illustrated the change in redox balance in the METH-treated mouse brain that is a known result of oxidative damage. Biochemical assays also showed that the level of thiobarbituric acid-reactive substance, an index of lipid peroxidation, was increased in mouse brains by METH. The enhanced reduction in MCP observed in mouse brains was remarkably suppressed by treatment with the dopamine synthase inhibitor, α-methyl-p-tyrosine, suggesting that enhancement of the reduction reaction of MCP resulted from enzymatic reduction in the mitochondrial respiratory chain. Furthermore, magnetic resonance imaging (MRI) of METH-treated mice using a blood–brain barrier (BBB)-impermeable paramagnetic contrast agent revealed BBB dysfunction after treatment with METH for 7 days. MRI also indicated that the impaired BBB recovered after withdrawal of METH. EPR imaging and MRI are useful tools not only for following changes in the redox status and BBB dysfunction in mouse brains repeatedly administered METH, but also for tracing the drug effect after withdrawal of METH.     

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.     

Alteration of the Redox State with Reactive Oxygen Species for 5-Fluorouracil-Induced Oral Mucositis in Hamsters

Oral mucositis is often induced in patients receiving cancer chemotherapy treatment. It has been reported that oral mucositis can reduce quality of life, as well as increasing the incidence of mortality. The participation of reactive oxygen species (ROS) in the pathogenesis of oral mucositis is well known, but no report has actually demonstrated the presence of ROS. Thus, the purpose of this study was thus to demonstrate the involvement of ROS and the alteration of the redox state in oral mucositis using an in vivo L-band electron spin resonance (ESR) technique. An oral mucositis animal model induced by treatment of 5-fluorouracil with 10% acetic acid in hamster cheek pouch was used. Lipid peroxidation was measured as the level of malondialdehyde determined by the thiobarbituric acid reaction. The rate constants of the signal decay of nitroxyl compounds using in vivo L-band ESR were calculated from the signal decay curves. Firstly, we established the oral mucositis animal model induced by treatment of 5-fluorouracil with acetic acid in hamster cheek pouch. An increased level of lipid peroxidation in oral mucositis was found by measuring malondialdehyde using isolated hamster cheek pouch ulcer. In addition, as a result of in vivo L-band ESR measurements using our model animals, the decay rate constants of carbamoyl-PROXYL, which is a reagent for detecting the redox balance in tissue, were decreased. These results suggest that a redox imbalance might occur by excessive generation of ROS at an early stage of oral mucositis and the consumption of large quantities of antioxidants including glutathione in the locality of oral mucositis. These findings support the presence of ROS involved in the pathogenesis of oral mucositis with anti-cancer therapy, and is useful for the development of novel therapies drugs for oral mucositis.     

Detection and imaging of superoxide in roots by an electron spin resonance spin-probe method

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Increased lipid peroxidation in Down's syndrome mouse models

Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Non-invasive analysis of reactive oxygen species generated in rats with water immersion restraint-induced gastric lesions using in vivo electron spin resonance spectroscopy

Reactive oxygen species (ROS) are reportedly associated with gastric ulcer. We previously reported the use of an in vivo 300-MHz electron spin resonance (ESR) spectroscopy/nitroxyl probe technique to detect _•OH generation in the stomachs of rats with gastric ulcers induced by NH₄OH. However, this is an acute ulcer model, and the relationship between in vivo ROS generation and lesion formation remains to be clarified. To address this question, the same technique was applied to a sub-acute water immersion restraint (WIR) model. A nitroxyl probe that was less membrane-permeable was orally administered to WIR-treated rats, and the spectra in the gastric region were obtained by in vivo ESR spectroscopy. The signal intensity of the orally administered probe was clearly changed in the WIR group, but no change occurred in the control group. Both enhanced signal decay and neutrophil infiltration into mucosa were observed 2 h after WIR with little formation of any mucosal lesions. The enhanced signal decay was caused by _•OH generation, based on the finding that the decay was suppressed by mannitol, desferrioxamine and catalase. Intravenous treatment with either anti-neutrophil antibody or allopurinol also suppressed the enhanced signal decay, and allopurinol depressed neutrophil infiltration into the mucosa. In rats treated with WIR for 6 h, lesion formation was suppressed by 50% with all antioxidants used in this experiment except anti-neutrophil antibody. These findings suggest that _•OH, which is generated in the stomach via the hypoxanthine/xanthine oxidase system upon neutrophil infiltrated into the mucosa, induces mucosal lesion formation, but that it accounts for only half the cause of lesion formation.     

应用ESR检测探讨急性热暴露大鼠肝脏氧化还原状态的动态变化

目的:通过电子顺磁自旋共振技术(ESR)动态观察大鼠在过热条件下肝脏的氧化还原状态.方法:将52只雄性Wistar大鼠随机分成4组:①加温组:麻醉后进行整体加温到直肠温达(43.0±0.5)℃,持续15 min;②对照组:只进行麻醉处理;③,MPG预处理组:用抗氧化剂MPG预处理后,再进行与上述①同样条件的加温处理;④非MPG预处理组:在③中用生理盐水代替MPG.经过以上处理后在不同时间点取肝脏制备组织匀浆,测定ESR波谱.结果:与对照组比较,加温组热暴露处理后记录的ESR波谱振幅-时间直线斜率增大,2 h达最大值.以后逐渐恢复,24 h接近对照组水平.经抗氧化剂预处理上述反应减弱.结论:过热能诱导肝脏产生活性氧,增强其氧化还原反应.