Keiji Yasukawa

Aripiprazole inhibits superoxide generation from phorbol-myristate-acetate (PMA)-stimulated microglia in vitro: implication for antioxidative psychotropic actions via microglia.

Altered antioxidant status has been implicated in schizophrenia. Microglia, major sources of free radicals such as superoxide (•O(2)(-)), play crucial roles in various brain pathologies. Recent postmortem and imaging studies have indicated microglial activation in the brain of schizophrenic patients. We previously demonstrated that atypical antipsychotics including aripiprazole significantly inhibited the release of nitric oxide and proinflammatory cytokines from interferon-γ-stimulated microglia in vitro. Antioxidative effects of antipsychotics via modulating microglial superoxide generation have never been reported. Therefore, we herein investigated the effects of antipsychotics on the •O(2)(-) generation from phorbol-myristate-acetate (PMA)-stimulated rodent microglia by the electron spin resonance (ESR) spectroscopy and also examined the intracellular mechanism by intracellular Ca(2+) imaging and immunostaining. Neuronal damage induced by microglial activation was also investigated by the co-culture experiment. Among various antipsychotics, only aripiprazole inhibited the •O(2)(-) generation from PMA-stimulated microglia. Aripiprazole proved to inhibit the •O(2)(-) generation through the cascade of protein kinase C (PKC) activation, intracellular Ca(2+) regulation and NADPH oxidase activation via cytosolic p47(phox) translocation to the plasma/phagosomal membranes. Formation of neuritic beading, induced by PMA-stimulated microglia, was attenuated by pretreatment of aripiprazole. D2R antagonism has long been considered as the primary therapeutic action for schizophrenia. Aripiprazole with D2R partial agonism is effective like other antipsychotics with fewer side effects, while aripiprazoles therapeutic mechanism itself remains unclear. Our results imply that aripiprazole may have psychotropic effects by reducing the microglial oxidative reactions and following neuronal reactions, which puts forward a novel therapeutic hypothesis in schizophrenia research.

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.

The detrimental effect of nitric oxide on tissue is associated with inflammatory events in the vascular endothelium and neutrophils in mice with dextran sodium sulfate-induced colitis

Abstract Nitric oxide (NO) is thought to be a key molecule in the progression of ulcerative colitis and experimental colitis induced by dextran sodium sulfate (DSS). However, the detrimental effect of DSS-induced NO production on the colonic mucosa is incompletely understood. Increases in the expression of adhesion molecules in the vascular endothelium and activated neutrophils (thereby releasing injurious molecules such as reactive oxygen species) are reportedly associated with the pathogenesis of DSS-induced colitis. We investigated if the detrimental effect of NO production on the colonic mucosa was attributable to the activation of neutrophil infiltration by NO in mice with DSS-induced colitis. NO2−/NO3− content in the middle and distal colon was increased on days 5 and 7, but alterations in the proximal colon were not observed. Myeloperoxidase (MPO) activity and expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) were significantly increased in the entire colon, whereas TNF-α levels were significantly increased only in the middle and distal colon on day 7. The pathology of colitis and increases in colonic MPO activity, P-selectin, ICAM-1, and TNF-α levels were suppressed by the inducible NO synthase (iNOS)-specific inhibitor aminoguanidine and NO scavenger c-PTIO, whereas all but TNF-α levels were increased by the non-specific NOS inhibitor L-NAME. These findings suggest that iNOS-derived NO increases TNF-α levels in the middle and distal colon and increased TNF-α levels induce expression of P-selectin and ICAM-1, thereby promoting the infiltration of activated neutrophils, which leads to damage to colonic tissue.

Non-invasive Analysis of Reactive Oxygen Species Generated in NH4OH-induced Gastric Lesions of Rats using a 300 MHz In Vivo ESR Technique

Free radicals are reportedly involved in mucosal injury, including NH4OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH4OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300 MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30 min after NH4OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to •OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH4OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH4OH-treated living rats that •OH produced from O2•- derived from neutrophils caused gastric lesion formation, while O2•- or H2O2 derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.

Advantageous application of a surface coil to EPR irradiation in overhauser‐enhanced MRI

The present study describes the advantageous application of a surface coil to electron paramagnetic resonance (EPR) irradiation in Overhauser‐enhanced MRI (OMRI). OMRI is a double‐resonance method for imaging free radicals based on the Overhauser effect. Proton NMR images are recorded without and with EPR irradiation of the free radical resonance, which results in a difference proton image that shows signal enhancement in spatial regions that contain the free radical. To obtain good signal enhancement in OMRI, very high RF power and a long EPR irradiation time are required. To improve sensitivity and shorten the image acquisition time, especially for localized (and topical) applications, we developed and tested a surface‐coil‐type EPR irradiation coil. Theoretical calculations and experimental data showed that EPR irradiation through the surface coil could ameliorate the localized Overhauser enhancement, which was related to the ratio of B1 surface coil/B1 volume coil in the region of interest (ROI), as expected. The increased sensitivity could also be converted into a shortened EPR irradiation time, resulting in fast data acquisition. For biomedical applications, the use of a surface coil (as opposed to a conventional volume coil) could decrease the total RF power deposition in the sample required to obtain the same Overhauser enhancement in the ROI. Magn Reson Med 57:806–811, 2007.

Olmesartan reduces oxidative stress in the brain of stroke-prone spontaneously hypertensive rats assessed by an in vivo ESR method

We previously showed that oxidative stress in the brain is involved in the neural mechanisms of hypertension. Therefore, olmesartan, an angiotensin type 1 receptor blocker, might affect oxidative stress in the brains of stroke-prone spontaneously hypertensive rats (SHRSP). Here, we evaluated the effects of olmesartan treatment using an in vivo electron spin resonance (ESR)/spin probe technique. Two groups of SHRSP were treated with either olmesartan (10 mg kg−1 day−1) or hydralazine (Hyd, 20 mg kg−1 day−1)/hydrochlorothiazide (HCT, 4.5 mg −1kg day−1) for 30 days (n=5 for each). Systolic blood pressure decreased similarly in both groups after treatment. Heart rate and urinary norepinephrine (NE) excretion increased in rats treated with Hyd/HCT, but not in those treated with olmesartan. The in vivo ESR signal decay rates of the blood–brain barrier-permeable spin probe methoxycarbonyl-PROXYL were significantly higher in SHRSP brains than in age-matched normotensive Wistar–Kyoto rat brains (P<0.01; n=6 for each). Olmesartan attenuated the ESR signal decay rates in SHRSP brains, but Hyd/HCT did not. Intracerebroventricular infusion of active form of olmesartan, RNH-6270, reduced blood pressure and NE excretion, and the ESR signal decay rate was reduced in SHRSP brains. These findings indicate that olmesartan has anti-oxidative property in the brain without stimulating reflex-mediated sympathetic activity in SHRSP.

In vivo imaging of tumor physiological, metabolic, and redox changes in response to the anti-angiogenic agent sunitinib: longitudinal assessment to identify transient vascular renormalization.

AIMS The tumor microenvironment is characterized by a highly reducing redox status, a low pH, and hypoxia. Anti-angiogenic therapies for solid tumors frequently function in two steps: the transient normalization of structurally and functionally aberrant tumor blood vessels with increased blood perfusion, followed by the pruning of tumor blood vessels and the resultant cessation of nutrients and oxygen delivery required for tumor growth. Conventional anatomic or vascular imaging is impractical or insufficient to distinguish between the two steps of tumor response to anti-angiogenic therapies. Here, we investigated whether the noninvasive imaging of the tumor redox state and energy metabolism could be used to characterize anti-angiogenic drug-induced transient vascular normalization. RESULTS Daily treatment of squamous cell carcinoma (SCCVII) tumor-bearing mice with the multi-tyrosine kinase inhibitor sunitinib resulted in a rapid decrease in tumor microvessel density and the suppression of tumor growth. Tumor pO2 imaging by electron paramagnetic resonance imaging showed a transient increase in tumor oxygenation after 2-4 days of sunitinib treatment, implying improved tumor perfusion. During this window of vascular normalization, magnetic resonance imaging of the redox status using an exogenously administered nitroxide probe and hyperpolarized (13)C MRI of the metabolic flux of pyruvate/lactate couple revealed an oxidative shift in tumor redox status. INNOVATION Redox-sensitive metabolic couples can serve as noninvasive surrogate markers to identify the vascular normalization window in tumors with imaging techniques. CONCLUSION A multimodal imaging approach to characterize physiological, metabolic, and redox changes in tumors is useful to distinguish between the different stages of anti-angiogenic treatment.

Whole-body kinetic image of a redox probe in mice using Overhauser-enhanced MRI.

Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes using OMRI provided both anatomic and physiological information. Phantom experiments demonstrated the linear correlation between the enhancement factor and the concentration of a membrane-impermeable probe, carboxy-PROXYL (3-carboxy-2,2,5,5-tetramethyl- pyrrolidine-1-oxyl). Whole-body OMRI images illustrated the in vivo kinetics of carboxy-PROXYL for 25 min. Initial distribution was observed in lung, heart, liver, and kidney, but not brain, corresponding to its minimal lipophilicity. Based on these images (pixel size, 1.33 × 1.33 mm; slice thickness, 50mm), a time-concentration curve with low coefficient of variance (<0.21) was created to assess pharmacokinetic behaviors. A biexponential curve showed a distribution phase from 1 to 10 min and an elimination phase from 15 to 25 min. The α rate constant was greater than the β rate constant in ROIs, confirming that its pharmacokinetics obeyed a two-compartment model. As a noninvasive technique, combining OMRI imaging with redox probes to monitor tissue redox status may be useful in acquiring valuable information regarding organ function for preclinical and clinical studies of oxidative diseases.

Spatially resolved time-course studies of free radical reactions with an EPRI/MRI fusion technique.

Electron paramagnetic resonance imaging (EPRI) using nitroxyl radicals is a useful technique for visualizing reactive oxygen species (ROS) and the pharmacokinetics of probes. To unambiguously identify anatomical locations, coregistration of EPRI with images obtained by MRI is necessary. In this study the feasibility of performing reliable EPRI/MRI fusion imaging using nitroxyl radical fiducial markers was tested. The pharmacokinetics of the nitroxyl radicals were observed after oral or intravenous administration in C57BL6 mice. To fuse both images, the nitroxyl radical was used as fiducial markers. The EPR and MR images corresponded well and clearly illustrated minimal changes in pharmacokinetics between carbamoyl‐PROXYL and carboxy‐PROXYL. These results demonstrate that the EPRI/MRI fused imaging technique is useful for investigating in vivo pharmacokinetics and provides unambiguous anatomic details. Magn Reson Med, 2006.

Involvement of nitric oxide with activation of Toll-like receptor 4 signaling in mice with dextran sodium sulfate-induced colitis

Ulcerative colitis is an inflammatory bowel disease characterized by acute inflammation, ulceration, and bleeding of the colonic mucosa. Its cause remains unknown. Increases in adhesion molecules in vascular endothelium, and activated neutrophils releasing injurious molecules such as reactive oxygen species, are reportedly associated with the pathogenesis of dextran sodium sulfate (DSS)-induced colitis. Nitric oxide (NO) production derived from inducible NO synthase (iNOS) via activation of nuclear factor κB (NF-κB) has been reported. It is also reported that stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide can activate NF-κB. In this study, we investigated the involvement of NO production in activation of the TLR4/NF-κB signaling pathway in mice with DSS-induced colitis. The addition of 5% DSS to the drinking water of male ICR mice resulted in increases in TLR4 protein in colon tissue and NF-κB p65 subunit in the nuclear fraction on day 3, increases in colonic tumor necrosis factor-α on day 4, and increases in P-selectin, intercellular adhesion molecule-1, NO2(-)/NO3(-), and nitrotyrosine in colonic mucosa on day 5. These activated inflammatory mediators and pathology of colitis were completely suppressed by treatment with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as an iNOS inhibitor, aminoguanidine. Conversely, a NO-releasing compound, NOC-18, increased TLR4 levels and nuclear translocation of NF-κB p65 and exacerbated mucosal damage induced by DSS challenge. These data suggest that increases in TLR4 expression induced by drinking DSS-treated water might be directly or indirectly associated with NO overproduction.