INTRODUCTION

M ethanol is a colorless alcohol that is commonly used as a component of some industrial products such as antifreeze, perfume and cologne[1]. Methanol intoxication,which may be accidental or intentional as a suicide attempt,is known to cause visual disturbances, central nervous system abnormalities such as confusion, coma or even death[2]. Visual loss associated with methanol toxicity usually starts within 12-48h due to relatively severe, painless, bilateral optic nerve damage; which may be transient or progressive[3-4].Rutin (3,3’,4’,5,7-pentahydroxyflavone-3-rhamnoglucoside)is a flavonoid with anti-inflammatory, anti-oxidant and analgesic effects, found in many vegetables and fruits[5-6].Rutin attenuates oxidative stress by inhibiting membrane lipid peroxidation and increasing antioxidant enzyme levels[7]. Recently, Rutin was reported to have promising effects on chronic painful peripheral neuropathy and diabetic neuropathy[8-9].

总而言之,替格瑞洛应用于急性冠脉综合征治疗中具有重要意义,能够在发挥显著疗效的基础上降低不良反应发生率,值得应用及推广。

In this experimental study, we aimed to determine the effects of Rutin on methanol induced optic neuropathy and compare the results with the effects of ethanol. We also aimed to investigate tissue 8-hydroxy-2 deoxyguanine (8-OHdG)levels as a determinant of DNA damage, inflammation markers such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), oxidative stress parameters including Myeloperoxidase (MPO) and malondialdehyde (MDA) and anti-oxidants glutathione peroxidase (tGSH) and superoxide dismutase (SOD) to elucidate the mechanism of association.To the best of our knowledge, this is the first study in literature investigating the role of Rutin in this type of optic neuropathy.

收集年限均超过10年太极拳锻炼的老龄人作为实验组，身高167.9±6.1cm, 体重67.2±10.9kg,锻炼年限18.6±10.7年；收集年限均在1～3年之间短期锻炼太极拳的老龄人作为对照组，身高168.2±5.9cm，体重69.6±7.4kg。所收集的老龄人均为男性，年龄在60～72岁之间，对照组与实验组年龄、体重、身高差异不显著，每周锻炼均4次以上，每次持续1h左右，锻炼时间均在早上，身体健康、四肢无伤病疼痛、无急慢性疾病。

样式是指用有意义的名称保存的字符格式和段落格式的集合，也就是说将要设置的多个格式命令加以组合、命名，应用一次样式，就可相当于设定这些格式，每个样式都有唯一确定的名称，用户可以将一种样式应用于一个段落或选定的字符上。例如使用系统自带的“标题1”样式，即可将所选文字设置为2号字体、加粗、多倍行距等效果。当鼠标指向“标题1”时，就可以看到应用样式的效果，就是应用了一组格式的集合，这一组格式设置按常规要分几步才能完成，现在只需要应用样式就可一步完成，从而简化了字符、段落的格式排版，节省了时间，提高了工作效率。

MATERIALS AND METHODS

This study was approved by the Ataturk University Animal Care and Use Committee (date: Jul. 28, 2017; number: 75296309). The study was performed between Aug. 1, 2017 and Aug. 8, 2017.

Study Animals Totally 30 albino Wistar male rats (280-295 g)obtained from the Ataturk University Medical Experiments Application and Research Center were used in this study. The rats were housed at room temperature (22℃), fed twice a day and had access to water ad libitum.

IL-1β and TNF-α analysis in serum Serum IL-1β and TNF-α concentrations were measured using rat-specific sandwich enzyme-linked immunosorbent assay: rat IL-1β ELISA kit:(Cat No.YHB0616Ra; Shanghai LZ, China) and rat TNF-α ELISA kits (Cat No.YHB1098Ra; Shanghai LZ, China). Analyses were performed according to the manufacturers’ instructions.

Methanol is an oxidant inducing free radical changes and oxidative damages. In an experimental study, Rajamani et al[17]reported that methanol exposure causes increased generation of free radicals and significant oxidative damage in retina and optic nerve. Clinically, acute methanol intoxication is known to cause visual disturbances that may be transient or persistent.Although the personal susceptibilities may vary regarding the toxic effects of different methanol doses, as little as 10 mL may cause blindness[18]. Zakharov et al[19] reported that, totally 13 of 50 patients diagnosed with acute methanol intoxication were discharged with visual squeal and during follow-ups 20/50(40%) of the patients had long-term visual sequel, with 8% of blindness. In that aspect, clinicians should be aware of longterm visual sequelae associated with methanol intoxication which may be underestimated during discharge.

建构主义理论认为，学习过程是学习者基于一定情境下，结合原有知识经验，通过社会文化互动而形成的学习者主动对新知识建构理解的过程。所以教师创设生动的学习情境，能促进学生进行意义建构。生物知识的故事化，无疑是创设教学情境的一种手段，因为故事具有天然的“情境性”，往往能吸引人，使学生身临其境，全身心投入其中。

教师要正确认识音乐对学生核心素养的提升效果，明确自己在课堂中扮演的角色，为学生讲述基础音乐知识点，指明学习方向，防止浪费课堂时间，使学生在引导下一步步发现音乐学习的重要性，这也是激发学生音乐学习兴趣的途径之一。教师要尽可能减少对学生音乐思维的限制，构建相对自由和谐的课堂氛围，使学生主动走进音乐课堂，高效完成音乐学习任务，提高音乐综合能力。

Biochemical Analyses

DNA oxidation analysis The levels of 8-OHdG in hydrolyzed DNA samples were measured at various wavelengths by high performance liquid chromatography (HPLC) with HPLCUV and HPLC-ECD electrochemical detectors[12]. The final volume consisted of final hydrolysate, HPLC-ECD (HP, HP 1049A ECD detector, Agilent 1100 modular systems HP 1049A ECD detector, Germany), reverse-phase C18 column(250 mm×4.6 mm×4.0 μm; Phenomenex, Torrance, CA, USA)and a potassium phosphate (pH 5.5) tampon that contains acetonitrile (97:3, v/v) with 1 mL flow velocity per minute as the mobile phase. The 8-OHdG concentration was observed with electrochemical readings by measuring the absorbance at 245 nm. The 8-OHdG amounts, defined as the marker of DNA damage, were identified using 8-OHdG standards (Sigma, St.Louis, MO, USA).

The rats were divided into 5 study groups, with 6 rats in each group as follows: healthy controls (C), methotrexate (MTX),methotrexate+methanol (MTM), methotrexate+methanol+ethanol (MTME) and methotrexate+methanol+rutin (MTMR).

Analysis of histopathological data Histopathological data including destruction, hemorrhage, congestion, edema,and decrease in oligodendrocyte and as trocyte counts were analyzed by the same pathologist and quantified between 0 and 3.

MDA analysis in serum MDA measurements were based on the method used by Ohkawa et al[14] involving spectrophotometrical measurement of absorbance of the complex formed by thiobarbituric acid (TBA) and MDA. The standard curve was obtained by using 1,1,3,3-tetramethoxypropane.

Analysis of myeloperoxidase activity in serum H2O2 involving in phosphate buffer was used as substrate to determine MPO activity in serum[15]. Serum/tissue-homogenate was added to assay buffer and MPO activity was kinetically measurement at 460 nm for 5min.

In experimental models of methanol intoxication, due to the high liver folic acid content and rapid formic acid metabolism,rats do not exhibit intoxication. For that reason, since Methotrexate is a folic acid antagonist, foliate-dependent formic acid metabolism is impaired with MTX treatment as defined in literature before[11].

Analysis of superoxide dismutuse activity in serum Measurements were performed according to the method described by Sun et al[16] depending on the conversion of xanthine into uric acid by xanthine oxidase.

tGSH analysis in serum The 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) disulfide is chromogenic in the medium[13].The yellow color produced during the reduction DTNB by sulfhydryl groups is measured by spectrophotometry at 412 nm.For measurement, a cocktail solution was prepared and added to the supernatant of the centrifuged serum. The standard curve was obtained by using glutathion.

军事的技术容不得半点马虎，微电子技术的精确性足以满足军事中的需求。通信指挥系统追求高速、灵活、准确；不同兵种之间的配合和战略模拟，无不依靠微电子技术。通过微电子技术取胜的战役也是不在少数。比如海湾战争，就是通过摧毁敌方的通信系统，瓦解对方战力最终取得胜利。因此，若想在强强竞争的世界之林获得一席之地，微电子技术就必须发展，而且是大力发展、作为重点发展。

Table 1 Results of biochemical analysis

C: Controls; MTX: Methotrexate group; MTM: Methotrexate+methanol group; MTME: Methotrexate+methanol+ethanol group;MTMR: Methotrexate+methanol+rutin group; 8-OHdG: 8-hydroxy-2 deoxyguanine; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-alpha; MPO: Myeloperoxidase; MDA: Malondialdehyde; tGSH: Glutathione peroxidase; SOD: Superoxide dismutase.aStatistically significantly different when compared with control group; bStatistically significantly different when compared with MTX group; cStatistically significantly different when compared with MTM group; dStatistically significantly different when compared with MTME group; eStatistically significantly different when compared with MTMR group; fP value of ANOVA test.

Parameters C MTX MTM MTME MTMR fP 8-OHdG 0.81 ±0.03b,c,d 1.57±0.09a,c,d,e 2.9±0.28a,b,d,e 2.10±0.14a,b,c,e 0.92±0.04b,c,d 0.001 IL-1β 1.65±0.18b,c,d 2.81±0.24a,c,d,e 9.18±0.33a,b,d,e 4.7±0.42 a,b,c,e 1.98±0.23b,c,d 0.001 TNF-α 1.07±0.15b,c,d 2.16±0.21a,c,d,e 7.75±0.30a,b,d,e 4.21±0.27a,b,c,e 1.63±0.25b,c,d,e 0.001 MDA 2.65±0.27b,c,d 3.71±0.54a,c,d,e 7.98±0.37a,b,d,e 4.85±0.28a,b,c,e 3.0±0.31b,c,d 0.001 MPO 3.20±0.30b,c,d 6.35±0.56a,c,d,e 20.33±2.25a,b,d,e 9.43±0.93a,b,c,e 3.52±0.54b,c,d 0.001 tGSH 6.68±0.48b,c,d 4.21±0.41a,c,e 1.85±0.18a,b,d,e 3.73±0.36a,c,e 6.18±0.45b,c,d 0.001 SOD 20.0±2.61b,c,d,e 6.0±0.56a,c,e 2.88±0.23a,b,d,e 5.20±0.61a,c,e 9.95±0.55a,b,c,d 0.001

Table 2 Results of pathological analysis

C: Controls; MTX: Methotrexate group; MTM: Methotrexate+methanol group; MTME: Methotrexate+methanol+ethanol group; MTMR:Methotrexate+methanol+rutin group; 8-OHdG: 8-hydroxy-2 deoxyguanine. aStatistically significantly different when compared with control group; bStatistically significantly different when compared with MTX group; cStatistically significantly different when compared with MTM group; dStatistically significantly different when compared with MTME group; eStatistically significantly different when compared with MTMR group; fP value of Kruskal-Wallis test.

Parameters C MTX MTM MTME MTMR fP Destruction 0b,c 2.0±0.6a,c,d,e 3.0±0.0a,b,d,e 0b,c 0b,c ＜0.001 Hemorrhage 0b,c,d 2.0±0.6a,c,d,e 3.0±0.0a,b,d,e 1.0±0.6a,b,c,e 0b,c,d ＜0.001 Congestion 0b,c 0c,d 3.0±0.0a,b,d,e 1.0±0.6a,b,c,e 0c,d ＜0.001 Edema 0b,c 0c,d 3.0±0.0a,b,d,e 1.0±0.6a,b,c,e 0c,d ＜0.001 Decrease in oligodendrocytes 0b,c 2.0±0.6a,c,d,e 2.8±0.4a,b,d,e 0b,c 0b,c ＜0.001 Decrease in astrocytes 0b,c 2.0±0.6a,c,d,e 3.0±0.0a,b,d,e 0b,c 0b,c ＜0.001

RESULTS

At the end of the study, all rats were evaluated without any failure.Regarding the biochemical results, there were statistically significant differences in 8-OHdG, IL-1β, TNF-α, MDA,MPO, tGSH and SOD levels between groups (Table 1). The highest tissue 8-OHdG and serum IL-1β, TNF-α, MDA, and MPO levels were determined in MTM and MTME groups while the anti-oxidant tGSH and SOD levels were the lowest in those groups. In MTMR group, tissue 8-OHdG, and serum IL-1β, MDA, and MPO levels were similar with the healthy controls but significantly different than other groups. In that aspect Rutin was effective to prevent the increases in systemic oxidative stress and in fl ammation markers.

The results of quantified data in pathological analyses are summarized in Table 2. In that aspect, regarding all parameters,MTMR group was similar with the control group. The worst pathological alterations were determined in MTM group followed by MTME group. In histopathological evaluations; in MTX group, there was moderate focal destruction, hemorrhage and decrease in number of astrocytes and oligodendrocytes;in MTM group, there was severe destruction and edema with decrease in number of astrocytes and oligodendrocytes and dilated blood vessels; in MTME group, there was mild hemorrhage, mild edema, mildly dilated blood vessels with congestion while in MTMR group optic nerve tissue was resembling the healthy controls (Figure 1).

DISCUSSION

In this experimental study we have investigated the role of Rutin in treatment of methanol-induced optic neuropathy and we determined that, Rutin was highly effective in decreasing the inflammatory substances and oxidative stress markers while increasing the antioxidants in rats with methanol toxicity. Histopathological evaluations of optic nerve were also supporting the promising effects of Rutin in treatment of methanol-induced optic neuropathy. To the best of our knowledge, this is the first study in literature evaluating the role of Rutin in optic neuropathy.

Figure 1 Optic nerve of a rat in each group A: Optic nerve of a rat in control group. Square arrow is the meninx, straight arrow is the vessel,bilateral ended arrow is the astrocyte and circular arrow is the oligodendrocyte. H&E×400; B: Optic nerve of a rat in MTX group. There was moderate focal destruction (circular arrow), hemorrhage (straight arrow) and decrease in number of astrocytes and oligodendrocytes (square arrow). H&E×200; C: Optic nerve of a rat in MTM group. Severe destruction and edema (straight arrow) and decrease in number of astrocytes and oligodendrocytes (lined arrow). H&E×200; D: Optic nerve of a rat in MTM group. Dilated blood vessels with congestion (straight arrow).H&E×400; E: Optic nerve of a rat in MTME group. Mild hemorrhage (lined arrow), mild edema (squared arrow), mildly dilated blood vessels with congestion (straight arrow) H&E×200; F: Optic nerve of a rat in MTMR group. Optic nerve tissue resembling control group.H&E×200.

Experimental Design In all rabbits except those of the control group, MTX, diluted in sterile serum physiologic, 0.3 mg/kg per oral was applied for 7d by the aid of a tube. After this procedure to the rats of MTM, MTME and MTMR groups,20% methanol with a dose of 3 g/kg per oral was given by the aid of a tube. In MTME group, 4h after the application of methanol, 20% ethanol was applied by the same way with a dose of 0.5 g/kg. On the other hand, in MTMR group 4h after the application of methanol, Rutin (Solgar, USA), which was dissolved in distilled water, was applied by the same way with a dose of 50 mg/kg. Eight hours after the application of ethanol or rutin, all rats were sacri fi ced with high dose (50 mg/kg)thiopental (IE Ulagay, Turkey) anesthesia and optic nerves of all rats were dissected and obtained for the measurement of 8-OHdG levels, which is the end product of DNA damage,and for the histopathological evaluations; as defined before in literature before[10]. Tissue samples underwent routine processing, paraffin embedding, and sectioning. The tissues were stained with hematoxylin and eosin (H&E) and examined under light microscopy. Moreover, blood samples were also obtained from the animals in order to determine serum MDA,MOP, tGSH, SOD, IL-1β and TNF-α levels.

Statistical Analysis The statistical analyses were performed with the Software SPSS version 21.0 (SPSS for Windows software; SPSS Inc., Chicago, IL, USA). Numerical variables were expressed as mean±standard deviation. For the analysis of continuous variables, one way variance analysis (ANOVA)was performed. Subsequently, in determination of differentgroups, Duncan multiple comparison test was performed. For the analysis of quantified data of histopathological analyses,Kruskal-Wallis test was performed. Statistical significance was set at P＜0.05.

Methanol is metabolized in the liver by alcohol dehydrogenase enzyme to formaldehyde, and then by formaldehyde dehydrogenase to formic acid rapidly[20]. Formic acid is a toxic substance that inhibits aerobic metabolism and energy production inducing acidosis with high plasma anion gap. Due to the inhibition of cytochrome oxidase activity and prevention of mitochondrial oxygen production in optic nerve by formic acid, optic neuropathy takes place in methanol toxicity causing damaged axonal flow with mitochondrial edema and fragmentation of neuro fi laments[21-22]. Another important point in methanol metabolism is the requirement of foliate in the metabolism of formic acid to CO2 and H2O. Enhancing formic acid elimination may also improve the visual disturbances in methanol toxicity.

Optic nerve, retina, and basal ganglia are the main tissues that are at risk from methanol intoxication which may be associated with the high mitochondrial energy requirement in these tissues.Usually following a latent period of 12-36h after the ingestion time, most probably required for the methanol metabolism and formic acid accumulation, visual symptoms start which are generally painless, bilateral and severe[23]. Paasma et al[24]reported the 6y follow-up results after a methanol outbreak of 111 hospitalized patients and described that new neurological and visual complications were identified in more than one third of the patients. In order to avoid neurological and visual sequelae, prompt treatment of methanol ingestion including the correction of the systemic acidosis, foliate supplementation and inhibition of methanol metabolism are essential.

Prevention of formic acid formation by inhibiting liver alcohol dehydrogenase enzyme and restoration of normal pH to diminish the cellular formic acid entry by sodium bicarbonate or dialysis are the main steps in treatment[1]. Ethanol is a competitive inhibitor of liver alcohol dehydrogenase, which is one of the main treatment options in methanol intoxication[25].Fomepizole is another competitive inhibitor of aldehyde dehydrogenase that also prevents formic acid formation in methanol toxicity[26-27]. Some other treatment options, such as high dose prednisolone, erythropoietin and alpha-lipoic acid as an anti-oxidant were also reported to have promising effects in methanol-induced toxic optic neuropathy but prospective studies are required to determine the exact role of those treatment options[28-30].

Flavonoids are a group of natural poly-phenolic compounds playing significant roles in detoxification of free radicals[31].Rutin is a flavonoid with strong proton donating and free radical stabilizing properties; thus reducing generation of reactive oxygen species[32]. Rutin may also improve endothelial function by enhancing nitric oxide production in human endothelial cells which is also an anti-oxidant action[33].Recently, it was reported to inhibit glycation and creation of advanced glycation end products on eye lens protein[34].Rutin was also reported to prevent oxaliplatin induced painful peripheral neuropathy in an experimental model[9]. Moreover,the beneficial effects of Rutin on diabetic neuropathy were reported via attenuation of the oxidative stress and neuroinflammation[8]. To the best of our knowledge, Rutin was not studied before in treatment of methanol-induced optic neuropathy. In this study we also determined promising effects of Rutin in prevention of methanol induced optic neuropathy with its anti-inflammatory and anti-oxidant effects.

There are some limitations of this study that should be mentioned. First, as all experimental studies, it carries the bias associated with the differences between metabolisms of animals and human beings. Second is the short follow-up time in that study.

In conclusion, Rutin may prevent methanol-induced optic neuropathy via anti-inflammatory effects and decreasing the oxidative stress. New treatment options are warranted in this disease to avoid loss of vision in patients.

ACKNOWLEDGEMENTS

Conflicts of Interest: Taşli NG, None; Çimen FK, None;Karakurt Y, None; Uçak T, None; Mammadov R, None;Süleyman B, None; Kurt N, None; Süleyman H, None.

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