【作者】 郭秀明；

【导师】 唐荣华；

【作者基本信息】 华中科技大学 ， 神经病学， 2007， 博士

【摘要】 二硫化碳（Carbon disulfide,CS₂）是一种应用广泛的有机溶剂,为全身性毒物,可累及人体多系统多器官,神经系统是二硫化碳作用的主要靶器官,其毒作用机制尚不完全清楚。大量流行病学调查资料表明,长期接触一定浓度的CS₂可使记忆力受到不同程度的影响。动物实验业已证实CS₂能使染毒动物的行为发生改变。但目前对于CS₂对学习记忆影响机制方面缺乏研究,本研究通过观察不同浓度CS₂对大鼠学习记忆功能、海马组织超微结构、自由基代谢和NOS的影响以及VitE的干预作用,探讨CS₂对大鼠学习记忆功能的影响及其机制,为临床预防和治疗CS₂中毒提供理论依据。第一部分二硫化碳对大鼠学习记忆功能的影响目的:观察CS₂对大鼠学习记忆功能的影响。方法:将60只大鼠随机分为对照组和三组CS₂染毒组（分别为50mg/m³组、250mg/m³组、1250mg/m³组）,每组15只。采用动式吸入染毒,将大鼠置于容积为60L的有机玻璃染毒柜内,每天染毒4h,每周5d,对照组染毒装置内无CS₂,与染毒组放置时间相同。2个月后,Morris水迷宫及跳台实验检测实验大鼠的学习记忆功能。结果:与对照组相比,各染毒组Morris水迷宫测试显示染毒后大鼠平均逃逸潜伏期延长,在原平台象限的游泳时间缩短,差异具有显著性意义;而且随着CS₂染毒浓度的增加大鼠平均逃逸潜伏期有延长的趋势,在原平台象限的游泳时间有缩短的趋势;跳台实验结果显示CS₂染毒后大鼠训练期间的错误次数（SD₁）增加,250mg/m³组和1250mg/m³组与对照组比较差异有显著性意义;24h后大鼠第一次跳下平台的潜伏期（SDL）缩短,5min内错误总数（SD₂）增加,各染毒组与对照组比较均有显著性差异;而且SD₁和SD₂有随CS₂染毒浓度的增加而增加的趋势,SDL有随CS₂染毒浓度的增加而缩短的趋势。结论:低浓度CS₂亚急性中毒即可导致大鼠学习记忆能力障碍。大鼠CS₂亚急性中毒所致的学习记忆障碍有随CS₂浓度增高而严重的趋势。第二部分二硫化碳对大鼠海马组织氧自由基代谢的影响及VitE的干预作用目的:探讨CS₂对大鼠海马组织超微结构和氧自由基代谢的影响以及VitE对CS₂致脂质过氧化作用的影响。方法:以吸入染毒法制作不同浓度CS₂中毒大鼠模型;染毒2个月后,透射电镜观察海马组织超微结构;分别以TBA法、硝酸还原酶法、黄嘌呤氧化酶法和化学比色法检测各组大鼠海马的丙二醛（MDA）、一氧化氮（NO）含量以及超氧化物歧化酶（SOD）活性;观察染毒同时应用不同剂量维生素E（VitE）干预后上述自由基代谢变化情况。结果:电镜下主要表现为,神经元染色质聚集,核膜、胞膜皱缩或溶解,线粒体内出现空泡,线粒体脊结构破坏,50 mg/m³组神经元结构无明显改变,1250 mg/m³组损害最为严重。与对照组比较,各染毒组大鼠海马MDA含量增加,SOD活性降低,NO含量降低,经检验差异均有统计学意义。染毒同时给予不同剂量VitE干预后,各浓度CS₂染毒组MDA含量均有不同程度减少,当VitE量达到100mg/kg时,海马MDA含量各染毒组与无干预组比较差异有显著性意义;SOD活性有不同程度增加,当VitE量达到200mg/kg时,海马SOD活性各染毒组与无干预组比较差异有显著性意义;NO含量有不同程度增加,当VitE量达到200mg/kg时,50 mg/m³组海马NO含量与无干预组比较差异有显著性意义。结论:CS₂所致的大鼠学习记忆能力减退与氧自由基对海马组织的损伤及NO代谢障碍有关;应用VitE可干预CS₂的致脂质过氧化作用。第三部分二硫化碳对大鼠海马一氧化氮合酶活力和基因表达的影响目的:探讨CS₂对大鼠海马一氧化氮合酶活力和基因表达的影响。方法:以吸入染毒法制作不同浓度CS₂中毒大鼠模型;染毒2个月后,以NOS测定试剂盒测定大鼠海马结构型一氧化氮合酶（cNOS）和诱导型一氧化氮合酶（iNOS）活性;以半定量逆转录-聚合酶链式反应（RT-PCR）法测定海马nNOS-mRNA和iNOS-mRNA含量的变化;免疫组织化学染色,光镜下观察nNOS和iNOS在大鼠海马的表达情况。结果:与对照组比较,各染毒组大鼠海马cNOS活性降低,RT-PCR显示nNOS-mRNA含量减少,免疫组化染色显示nNOS表达减少,差异有显著性意义。各染毒组iNOS活性与对照组比较差异无显著性意义,iNOS-mRNA含量比对照组增加,免疫组化染色显示iNOS表达增加,250mg/m³组、1250mg/m³组与对照组比较差异有显著性。结论:CS₂致大鼠海马cNOS活力降低及nNOS-mRNA含量减少可能是CS₂干扰学习记忆功能的机制之一。第四部分大剂量VitE对二硫化碳染毒大鼠学习记忆能力的影响目的:探讨大剂量VitE对CS₂染毒大鼠学习记忆能力以及海马组织超微结构的影响。方法:以吸入染毒法制作不同浓度CS₂中毒大鼠模型,染毒同时给予200mg/kg VitE。染毒2个月后,应用Morris水迷宫和跳台实验检测大鼠学习记忆能力;透射电镜观察海马组织超微结构。结果:与无干预组比较,染毒同时给予200mg/kgVitE,大鼠平均逃逸潜伏期缩短,空间搜索实验显示大鼠经过7天寻找水下平台的学习后,在原平台象限的游泳时间延长,SD₂减少,SDL延长,50 mg/m³组差异有显著性意义。200mgVitE+250 mg/m³CS₂组神经元染色质聚集,核膜及胞膜皱缩,但程度较无VitE干预组减轻,200mgVitE+1250 mg/m³CS₂组改变不明显。结论:大剂量VitE可部分减轻CS₂导致的大鼠海马组织损伤;大剂量VitE可以预防低浓度CS₂对大鼠学习记忆功能的影响。更多还原

【Abstract】 Carbon disulfide （CS₂） is a kind of popular organic solvent. It is an intoxicant contributed to all over the body. Nervous system is the main target of CS₂ and the mechanism of toxic action is still unclear. Generous epidemiological investigation indicated that the memory ability could suffer different degree effect after long-term exposure to CS₂. Animal experiments also confirmed that CS₂ could change the behavior of toxic animals. However, at present, there is still no study involoved the mechanism of CS₂ on learning and memory ability. The goal of this study is to explore the effect and the mechanism of CS₂ on the learning and memory of rats by observing the learning and memory ability of intoxic rats, observing the ultrastructure of hippocampus, detecting metabolism of oxygen free radicals and measuring NOS of hippocampus. Fourthermore, the intervention effects of VitE on lipid peroxidation in rat hippocampus after long-term exposure to CS₂ was also observed. Part one Effects of carbon bisulfide on the learning and memory ability of ratsObjective To study the effect of carbon bisulfide （CS₂） on the learning and memory ability of rats. Methods Rats（n=60） were randomly divided into one control group and three CS₂-poisoned groups （50mg/m³ group, 250mg/m³ group and 1250mg/m³ group）. The toxic rat models were made by inhaling various doses CS₂ 4 hours every day, 5 days every week. After 2 months of inhaling intoxication, the learning and memory ability of all groups were examined by Morris water maze and step down test. Results Compared with the controls, Morris water maze showed that the average run away latent period of CS₂-poisoned groups elongated and the swimming time in the platform located quadrant shortened, and both of which were CS₂ concentration dependented. Step down test demonstrated that the error frequency maked by rats during training increased, the latence before rats stepped down the platform after 24h shortened and the total errors in 5min increased, and all of which were CS₂ concentration dependented. Conclusions CS₂ subacute intoxication include low concentration CS₂ subacute intoxication could induce disturbance of learning and memory ability of rats, which was CS₂ concentration dependented.Part Two Effects of carbon bisulfide on metabolism of oxygen- derived free radidicals and the intervention role of VitE in rat hippocampusObjective To explore the effects of CS₂ on ultrastructure of rat hippocampus and metabolism of oxygen-derived free radidicals. To study the effects of VitE on lipid peroxidation induced by CS₂ in rat hippocampus. Methods The toxic rat models were made by inhaling various doses CS₂. After 2 months of inhaling intoxication, the ultrastructure was observed by transmission electron microscope. The content of Maleic Dialdehyde （MDA） and Nitric Oxide （NO） and the activity of Superoxide Dismutase（SOD） in rat hippocampus were detected by Thibabituric Acid test, Nitrate reductase test, xanthine oxidase test and chemistry color match test respectively. Treated with different doses of vitamin E simultaneous, changes of above-mentioned markers were observed. Results The principal influence of CS₂ on rat hippocampus was Chromatin aggregation, nuclear envelope and cell membrane shrinkage or swell, vacuole appearance in mitochondria. There was no obvious change in 50mg/m³group, while the damage in 1250 mg/m³group was the most sever. Compared with the controls, CS₂ treatment significantly increased MDA levels, decreased SOD activity and NO contents. Treated with VitE simultaneous with intoxication, the content of MDA reduced, and when the dose of VitE reached 100mg/kg, the difference between all intoxic groups and the group without intervention is significant; The activity of SOD increased,and when the dose of VitE reached 200mg/kg, the difference between all intoxic groups and the group without intervention is significant; The content of NO increased, and when the dose of VitE reached 200mg/kg, the difference between 50mg/m³ group and the group without intervention is significant. Conclusions The decline of learning and memory ability of CS₂ poisoned rats is related to the impairment of hippocampus induced by oxygen-derived free radidicals and the metabolism disturbance of NO. VitE could interfere neurotoxicity of CS₂.Part Three Effects of carbon bisulfide on the activity and gene expression of nitricoxide synthase in rat hippocampus Objective To explore the effects of CS₂ on the NOS activity and expression of NOS-mRNA of rat hippocampus. Methods The toxic rat models were made by inhaling various doses CS₂. After 2 months of inhaling intoxication, the activity of cNOS and iNOS in hippocampus was detected by NOS Test Kit. The content of nNOS-mRNA and iNOS-mRNA was measured by semi-quantitative reverse transcription polymerase chain reaction（RT-PCR） method. The expression of nNOS and iNOS in rats hippocampus was observed by light microscope after immunohistochemical stain. Results Compared with the controls, the activity of cNOS significantly decreased. The activity of iNOS changed slightly. CS₂ treatment significantly decreased nNOS-mRNA content. The content of iNOS-mRNA in 250mg/kg CS₂ group and 1250mg/kg group increased significantly. Conclusions These results suggested that the activity of cNOS and the expression of nNOS might be related to the effect of CS₂ on learning and memory ability.Part Four Effects of large dose of VitE on the learning and memory ability of CS₂–poisoned ratsObjective To study the influence of large dose of VitE on ultrastructure of hippocampus and the learning and memory ability of CS₂ -poisoned rats. Methods The toxic rat models were made by inhaling various doses CS₂, meanwhile the poisoned rats were treated with 200mg/kg VitE. After 2 months of inhaling intoxication, the learning and memory ability was detected and the ultrastructure was observed by transmission electron microscope. Results Compared with the group without intervention, treated with 200mg/kg VitE simultaneous with intoxication, Morris water maze showed that the average run away latent period of CS₂-poisoned groups shortened and the swimming time in the platform located quadrant elongated. Step down test demonstrated that the error frequency maked by rats during training decreased. The latence before rats stepped down the platform after 24h elongated and the total errors in 5min decreased. The change of ultrastructure of hippocampus in 200mg/kg VitE +250mg/m³ group was obvious, while 200mg/kg VitE +1250 mg/m³ group changed slightly. Conclusions Application of large dose of VitE could partly reduce the damage of rat hippocampus induced by CS₂ and preserve the effect of low concentration CS₂ on the learning and memory ability.更多还原