Takeshi Shiga

Detection of nitric oxide production in lipopolysaccharide-treated rats by ESR using carbon monoxide hemoglobin

Release of nitric oxide (NO), from macrophages activated with E. coli lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO2-. The present study was aimed to prove in vivo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO2- and HbCO. HbNO was, therefore, derived from NO, not from NO2-. These results show that free NO is produced in vivo by the stimulation of LPS.

Electron spin resonance study on peroxidase- and oxidase-reactions of horse radish peroxidase and methemoglobin

The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction of methemoglobin, were studied by electron spin resonance spectroscopy. n nThe difference between the peroxidase- and oxidase-reactions of HRP are demonstrated, i.e., the ferro horse radish peroxidase-O2 system attacks both phenols and benzoate yielding unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish peroxidase-H2O2 system reacts only with phenols and naphthols producing the phenoxy-and naphthoxy-radicals. n nPhenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H2O2 and methemoglobin-H2O2, occurs independently of the molecular sizes of phenols but dependently on their redox-potentials. n nOn the basis of kinetic studies on methemoglobin-H2O2 system, the existence of a reactive intermediate complex between methemoglobin and H2O2 is proposed, which may be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the hemepocket of methemoglobin.

Elevation of plasma nitric oxide end products during focal cerebral ischemia and reperfusion in the rat

We investigated the alterations in the stable end products of nitric oxide, i.e., nitrate and nitrite, in the plasma during and after rat focal cerebral ischemia by an automated procedure based on the Griess reaction. At 2 h of middle cerebral artery (MCA) occlusion, plasma nitrate/nitrite levels were significantly higher (53 ± 8 μM, mean ± SD, n = 5, p < 0.05) than in rats with sham operation (36 ± 9 μM, n = 5), and were mildly elevated at 4 h of MCA occlusion (42 ± 9 μM, n = 5, n.s.). At 30 min of reperfusion after 2 h of MCA occlusion, plasma nitrate/nitrite levels were more markedly elevated (72 ± 7 μM, n = 5, p < 0.01 vs. sham operation), but were moderately elevated at 2 h of reperfusion after 2 h of MCA occlusion (61 ± 10 μM, n = 5, p < 0.05). Plasma nitrite levels were not changed during these experimental periods. Administration of 20 mg/kg of NG-nitro-l-arginine methyl ester (l-NAME) significantly decreased plasma nitrate/nitrite as well as nitrite at 30 min of reperfusion after 2 h of MCA occlusion (n = 5), but 2 mg/kg of l-NAME did not (n = 3). The effect of 20 mg/kg of l-NAME on plasma nitric oxide end products was reversed by the simultaneous administration of 200 mg/kg of l-arginine (n = 3), but not d-arginine (n = 3). The present study suggests that the l-arginine-nitric oxide pathway is activated during acute cerebral ischemia and reperfusion.

Automatic extraction and measurement of leukocyte motion in microvessels using spatiotemporal image analysis

This paper describes a computer vision system for the automatic extraction and velocity measurement of moving leukocytes that adhere to microvessel walls from a sequence of images. The motion of these leukocytes can be visualized as motion along the wall contours. The authors use the constraint that the leukocytes move along the vessel wall contours to generate a spatiotemporal image, and the leukocyte motion is then extracted using the methods of spatiotemporal image analysis. The generated spatiotemporal image is processed by a special-purpose orientation-selective filter and a subsequent grouping process newly developed for this application. The orientation-selective filter is designed by considering the particular properties of the spatiotemporal image in this application in order to enhance only the traces of leukocytes. In the subsequent grouping process, leukocyte trace segments are selected and grouped among all the segments obtained by simple thresholding and skeletonizing operations. The authors show experimentally that the proposed method can stably extract leukocyte motion.

Generation of phenoxy radicals by methemoglobin-hydrogen peroxide studied by electron paramagnetic resonance

Abstract The free radical intermediates of phenol derivatives, produced by the methemoglobin-hydrogen peroxide system at pH 5 and 7, are detected by electron paramagnetic resonance equipped with a continuous-flow apparatus. All the radicals from phenols are the phenoxy radicals, as identified by analyzing the observed hyperfine structures of the spectra with the aid of SCF-LCAO MO calculations. Comparing with the reaction of Fentons reagent, it is concluded that free OH radical, even if it exists, is not liberated into the solution in the methemoglobin-hydrogen peroxide system.

Hypothermia suppresses nitric oxide elevation during reperfusion after focal cerebral ischemia in rats

We aimed to investigate effect of temperature on the jugular levels of nitric oxide (NO) at reperfusion after focal cerebral ischemia. Both nitrosyl hemoglobin (HbNO) (2.5 +/- 0.4 microM) and plasma nitrite plus nitrate levels (61 +/- 5 microM) in rats under normothermia (approximately 37 degrees C) after 30 min of reperfusion following 2 h of left middle cerebral artery occlusion were significantly high, compared with sham operated rats (1.3 +/- 0.1 microM, 40 +/- 4 microM, respectively). Both HbNO (1.5 +/- 0.3 microM) and nitrite plus nitrate levels (43 +/- 7 microM) under moderate hypothermia (approximately 32 degrees C) were significantly low, compared with normothermic rats. HbNO (2.8 +/- 0.8 microM) and nitrite plus nitrate levels (65 +/- 8 microM) under mild hyperthermia (approximately 39 degrees C) were not significantly high. These results firstly demonstrated that hypothermia suppresses the elevation in intrajugular NO after cerebral ischemia-reperfusion.

Nitrosyl hemoglobin production during reperfusion after focal cerebral ischemia in rats

We first detected a definite nitrosyl hemoglobin (HbNO) signal in the jugular blood by electron spin resonance spectroscopy during early reperfusion after cerebral ischemia. A distinct three-line hyperfine structure, characteristic to HbNO, was demonstrated at 30 min of recirculation after 2 h of middle cerebral artery occlusion in rats. Only a weak HbNO signal was observed in animals with 2 h sustained ischemia or with sham operation. The present findings suggest that reperfusion after cerebral ischemia facilitates nitric oxide generation in the brain, which leads to the increased nitrosylation of erythrocyte hemoglobin in the cerebral circulating blood.

Intravital Microreflectometry of Individual Pial Vessels and Capillary Region of Rat

A microscopic reflectance spectrophotometer was constructed to obtain the spectra of single pial vessels and of a region containing only capillaries (capillary region). The difference in the oxygen saturation (So2) of hemoglobin between the regional arteriole and venule [R(A – V)] and that between the regional arteriole or capillaries [R(A – C)] were calculated. The reduction of cytochrome aa3 was also estimated in the capillary region. This method was applied to the brain surface of spontaneously breathing rats subjected to hypoxic and anemic hypoxia. On decreasing the inhaled O2 from 100 to 15%, elevation of R(A – V) and R(A – C) with slight arteriolar dilatation (though statistically not significant) was observed. Below 10% O2 (especially at 4 and 3% O2), the R(A – V) and R(A – C) decreased in spite of significant arteriolar dilatation with progressive reduction of cytochrome aa3, indicating suppression of oxygen transport to mitochondria. In the case of hemodilution down to 37% hematocrit (Ht), elevation of R(A – V) and R(A – C) occurred with a slight tendency toward arteriolar dilatation. Below 32% Ht, the R(A – V) decreased but the R(A – C) remained steady, while reduction of cytochrome aa3 progressed. Altogether, the So2 in the capillary region decreased and the reduction of cytochrome aa3 progressed with the decline of arteriolar O2 supply in both hypoxic and anemic hypoxia.

Effects of NG-nitro-l-arginine and/or l-arginine on experimental pulmonary metastasis in mice

Effects of NG-nitro-L-arginine methyl ester (L-NAME; an inhibitor of nitric oxide (NO) synthase) and/or L-arginine (substrate of NO synthase) on pulmonary metastasis of murine melanoma and Lewis lung carcinoma cells were investigated. L-NAME, L-arginine or both L-NAME and L-arginine was injected i.p. into mice 5, 3, and 1 h before and 1, 3, 5, and 7 h after the injection of tumor cells into mice via a tail vein. The administration of L-NAME (9.3 mumol/mouse) alone or L-arginine alone (46.5 or 186 mumol/mouse) potentiated pulmonary metastasis of highly and poorly metastatic B16 melanoma cells. L-NAME alone also increased the number of pulmonary metastasis of Lewis lung carcinoma cells, but L-arginine (185 mumol/mouse) did not. However, the combination of L-NAME and L-arginine increased the number of pulmonary metastasis of both the melanoma and Lewis lung carcinoma cells synergistically. L-NAME or L-arginine administration enhanced the retention of B16 melanoma cells in the lungs examined 24 h after the tumor cell injection. Synergistic effect of L-NAME and L-arginine was also seen in the tumor cell retention. The present results suggest that the metastatic potentials of the tumor cells do not simply correlate to NO production in vivo.

Synergistic stimulation of nitric oxide hemoglobin production in rats by recombinant interleukin 1 and tumor necrosis factor

Nitric oxide (NO) is formed from arginine in Escherichia coli lipopolysaccharide (LPS) treated rat; however, none of specific cytokine inducing NO generation is yet determined. We studied the effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on NO production in rats by detecting NO-hemoglobin in their blood, using electron spin resonance. Either IL-1 or TNF alone stimulated NO-hemoglobin formation. Combined administration of IL-1 and TNF markedly enhanced NO-hemoglobin generation, demonstrating the synergistic character of both stimuli on NO production. Further, LPS and TNF in combination were more potent stimulator of NO-hemoglobin production in rats than each alone.