Takako Nakanishi-Ueda

In vitro Testing of Antioxidants and Biochemical End-Points in Bovine Retinal Tissue

Lipid peroxidation in aliquots of bovine retina (without rod outer segments, ROS), purified ROS and retinal pigment epithelium (RPE) was initiated with 5 mM ferric iron and 80 mM ADP. After 30 min of oxidation at 37°C, the concentration of thiobarbituric-acid-reacting substances (TBARS) which approximates lipid hydroperoxide (LHP), increased in the ROS from 2.0 ± 3.6 to 90.2 ± 34.5 nmol malondialdehyde (MDA)/mg protein and in the RPE from 0.54 ± 0.2 to 51.5 ± 15.8 nmol MDA/mg protein. Sixteen lipid and aqueous antioxidants (AOX) from natural or synthetic sources, including five flavonoids, were evaluated for their ability to inhibit the oxidative reaction. Palm-oil-derived vitamin E showed significant protection in retina, ROS and RPE (64, 68 and 74%), respectively. Of the flavonoids tested, good protection in the retina was found at 10–5M for epigallocatechin gallate (50%) and at 50 ng/ml for pycnogenol (61%) and catechin (52%). When catechin and palm oil vitamin E, catechin and coenzyme Q10 or coenzyme Q10 and pycnogenol were combined, the individual effect was enhanced. TBARS as an indirect measure of LHP level and hemoglobin-methylene blue determination for direct LHP were used as alternative end-point determinations of lipid peroxidation. These measure different aspects of AOX reactions. The results demonstrate the usefulness of an in vitro model system that can rapidly and accurately determine the capacity of a single AOX against lipid peroxidation or be used to show synergistic efficacy.

Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry.

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [Me-13C, Me-2H(3)]methionine sulfoxide and [Me-13C, Me-2H(3)]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0 +/- 1.0 microM (means +/- SD, n = 8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.

Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells

Abstract The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm2. The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm2 exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm2, respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm2).

Eye damage control by reduced blue illumination.

The aim of this study was to demonstrate that a blue light and ultraviolet cut-off filter (blue filter) could reduce short-wavelength retina/RPE damage threshold by a continuous spectrum source. Sixteen normal eyes of two rhesus monkeys and six cynomolgus monkeys were subjected to macular irradiation of 20, 24, 27.4, 30, 35, 45, 50 and 60 J/cm(2) energy densities. The values of energy density were measured before the blue filter. Lesions were measured before and at 2 and 30 days after irradiation of a 2.8 mm diameter region within the macular arcade. Measures were fundoscopy, fluorescein angiography and long wavelength scanning by the Heidelberg Retinal Tomograph (HRT) unit. The lesions, which were produced, were scored and compared to irradiant energy density of the blue LED (NSPB500S, Nichia, Tokushima, Japan). The exposure at the 20 J/cm(2) produced no detectable result at 2 or 30 days. Exposure at 35 J/cm(2) showed definite lesion production without blue filter. With the filter added there was one indication of minor change. At 60 J/cm(2) there was extensive heavy, enduring damage without the filter and with the filter damage was present but was significantly attenuated. These results strongly support the conclusion that the blue filter attenuation reduces the frequency of damage by exposure. This experimental system is a useful model for normal human eye aging and continuous spectrum environment irradiance.

Activation of AP-1 and Increased Synthesis of MMP-9 in the Rabbit Retina Induced by Lipid Hydroperoxide

Purpose: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. Methods: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by eletrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. Results: AP-1 binding activity was increased at 1–3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. Conclusions: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.

Impaired Contact Hypersensitivity Reaction and Reduced Production of Vascular Endothelial Growth Factor in Tumor Necrosis Factor-α Gene-Deficient Mice

Tumor necrosis factor (TNF)‐α is an important proinflammatory cytokine in contact hypersensitivity (CHS) reactions. Previous efforts to assay CHS in TNF‐α gene‐deficient (‐/‐) mice have demonstrated a significant reduction in ear skin weight at 24 h following challenge by oxazolone, although the mechanisms of this suppression have not been examined. To further characterize the impaired CHS during evolution of the elicitation phase in TNF‐α ‐/‐ mice and to clarify its mechanisms, focusing on the roles of TNF‐α and vascular endothelial growth factor (VEGF), we used an established method of CHS assay‐sensitization and challenge by trinitrochlorobenzene (TNCB)‐ in TNF‐α ‐/‐ and wild‐type mice. We compared the histopathology of the sequential evolution of CHS between the two groups of mice and assessed both the extent of inflammatory cell infiltration and the degree of dilatation in dermal vessels labeled with CD31. We quantified the production of VEGF in the epidermis at specific time points by using a murine VEGF ELISA kit. The CHS reaction was markedly suppressed in TNF‐α ‐/‐ mice at all time points of the elicitation phase. Histologically, in TNF‐α ‐/‐ mice we observed diminished vascular permeability, reduced numbers of infiltrating inflammatory cells, neutrophils at 12 h, mononuclear cells and eosinophils at 24 h, and a decreased area of dilatation of vessels labeled with CD31. The level of epidermal VEGF in wild type mice increased rapidly after challenge and peaked at 24 h, paralleling the peak of ear swelling. In contrast, the peak level of epidermal VEGF in TNF‐α ‐/‐ mice was significantly reduced. These results suggest that TNF‐α plays an enhancing role in the elicitation phase of the CHS reaction. Diminished degrees of vascular permeability, dilatation of CD31+ vessels, and inflammatory cell infiltration in TNF‐α ‐/‐ mice are likely to be the result of a lack of TNF‐α and reduced production of epidermal VEGF.

Moxibustion at mingmen reduces inflammation and decreases IL-6 in a collagen-induced arthritis mouse model.

The purpose of this study was to compare the effectiveness of moxibustion (MOX) treatment at the GV4 and CV12 acupoints, and to determine the correlations between MOX treatment and interleukin (IL)-6 and corticosterone levels in a collagen-induced arthritis (CIA) mouse model. CIA mice were immunized twice intradermally over a 3-week interval with bovine type II collagen. After the second immunization (day 21), MOX was applied to the mouse equivalent of the GV4 and CV12 acupoints with a 1mg moxa cone five times/day. Clinical symptoms of CIA were observed three times/week until day 35. The concentrations of IL-6 and corticosterone in the blood samples were measured by immunoassay kits. At day 35, the incidence of CIA was significantly decreased in mice treated with MOX at the GV4 acupoint (78%, n=23, p<0.05), compared to untreated CIA mice (100%) and mice treated with MOX at the CV12 acupoint (100%). IL-6 and corticosterone levels were significantly increased by immunization. IL-6 levels significantly decreased in mice treated with MOX at the GV4 acupoint. These results suggest that MOX treatment suppressed CIA at the GV4 acupoint, not at the CV12 acupoint, possibly through inhibition of IL-6 production.

Effects of Green Tea Fractions on Oxygen-Induced Retinal Neovascularization in the Neonatal Rat

This study aimed to investigate the preventive effects of green tea fractions (GTFs) on rat model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were exposed to daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and progressive return to 80% oxygen (3 h) until postnatal day 12 (P12), then the rats were placed in ambient air until P18. The green tea was fractionated by DM-A50, DM-W, M-B, and M-W. The rats were treated once daily from P6 to P17 by gastric gavage of GTFs (0.05 or 0.01 g/ml) or distilled water (DW) at 50 µl/10 g body weight. On P18, the rats were sacrificed and the retinal samples were collected. The retinal neovascularization (NV) was scored and avascular areas (AVAs) were measured as a % of total retinal area (%AVAs) in ADPase stained retinas. The NV scores in 0.01 g/ml M-W were significantly lower than those in DW. The %AVAs in 0.05 g/ml DM-A50 and in 0.05 g/ml and 0.01 g/ml M-W were significantly lower than those in DW. There were less catechins, and less caffeine in M-W fraction compared with other GTFs, suggesting components of green tea except for catechins and caffeine might suppress the neovascularization in rat model of OIR.

Effect of Lipid-Hydroperoxide-Induced Oxidative Stress on Vitamin E, Ascorbate and Glutathione in the Rabbit Retina

Background: It is possible that oxidative stress causes several retinal diseases. However, the natural biogenic role of antioxidants in the retina is not clear. Purpose: This study investigates the change in concentration of vitamin E (VE), ascorbate and glutathione (GSH) in the retina following vitreous injection of 600 µg 18:2 linoleic acid hydroperoxide (LHP) in male New Zealand rabbits. Method: LHP was injected above the retinal surface. The animals were sacrificed and the eyes enucleated before LHP injection, 1, 3, 6, 12, 24 h and 4 and 7 days after LHP injection. Retinas were removed, VE and ascorbate measured by HPLC, and GSH determined by a fluorometric method. Results: The concentration of VE in the retina decreased from pretreatment levels of 154.6 ± 29.7 nmol/g wet weight (n = 7) and was lowest at 6 h (61.1 ± 18.1 nmol/g wet weight, n = 4, p < 0.05), then increased gradually, returning slowly to pre-LHP levels by 7 days. The concentration of ascorbate in control retinas decreased at 6 h from pretreatment levels of 7.33 ± 0.93 µmol/g wet weight (n = 7) to 2.74 ± 0.16 µmol/g wet weight (n = 4, p < 0.05) and returned to pretreatment levels rapidly by 24 h after injection. The concentration of GSH in retinas decreased from baseline levels of 109.53 ± 8.19 µg/g wet weight (n = 9), was lowest at 12 h (72.40 ± 11.17 µg/g wet weight, n = 5, p < 0.05) and returned to pretreatment levels by 7 days. Conclusion: The results suggest that intravitreous LHP injection is a contributor to oxidative stress in the rabbit retina by causing a reduction in antioxidant capacity.

Lipid hydroperoxide induced corneal neovascularization in hyperglycemic rabbits

Purpose. To compare the effect of hyperglycemia on corneal neovascularization (NV) induced by linoleic acid hydroperoxide (LHP) in a rabbit model. Methods. Male New Zealand rabbits received 80mg/kg alloxan i.v. and subsequently developed hyperglycemia. Four weeks later, 10 µl of LHP (40mM) was injected into corneal stroma 5mm from the superior limbus with a 30 gauge needle. Vessel growth area from the limbal vasculature was measured over a period of 2 weeks and was correlated with plasma levels of insulin, HbA lc, and corneal vascular endothelial growth factor (VEGF). Results. Two days after alloxan, blood glucose was increased from 97 ± 4mg/dl in the untreated control group to 413 ± 3mg/dl. At 24 and 72 hours after LHP injection, VEGF in cornea of hyperglycemic rabbits was elevated 2 to 4 times above that of normoglycemic rabbits. At 14 days after LHP injection, the normoglycemic rabbits vessel growth area measured 2.42 ± 0.31mm², but in the hyperglycemic group, vessel growth area was significantly increased to 7.96 ± 2.26mm² (p < 0.05). At the end of the experimental period, HbA 1c was elevated from 3.9 ± 0.8 % to 8.4 ± 0.6 % and insulin was decreased from 440 ± 123.9pg/ml to 24 ± 11.0pg/ml. Conclusions. These data suggest that hyperglycemia may sensitize corneal and vascular endothelial cells, perhaps by glucose derived radicals, which enhance production of additional LHP through endogenous propagation reactions, and raise in turn the concentration of VEGF levels to induce an enhanced, sustained NV response.