NEUROTOXIC MECHANISMS IN PRIMARY CNS CELL CULTURES

原代中枢神经系统细胞培养中的神经毒性机制

• 批准号: 3251664
• 负责人: NEVILLE BROOKES
• 金额: $ 10.95万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-15 至 1992-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3251664
• 关键词: aminoacid metabolism; aminoacid transport; antidotes; astrocytes; blood brain barrier; brain metabolism; central nervous system; cryoscience; cytotoxicity; electron microscopy; electron probe spectrometry; environmental toxicology; fluorimetry; freeze etching; glucose metabolism; glucose transport; glutamates; high performance liquid chromatography; immunofluorescence technique; laboratory mouse; leucine; membrane permeability; mercury poisoning; methylmercury; mixed tissue /cell culture; neurotoxins; organ culture; paper chromatography; phase contrast microscopy; radiotracer; scintillation counter; spectrometry

Mercury, including its organic compounds, is a pervasive environmental and occupational neurotoxicant with multiple cellular targets of action. In addition to direct toxic effects on neurons, neurotoxicity results secondarily following impairment of other cell types that control the neuronal microenvironment, notably glia and the blood-brain barrier. Astroglia and cerebrovascular endothelial cells exhibit mutual trophisms in the expression of differentiated blood-brain barrier properties. These properties can now be reproduced in vitro in cell culture systems. Such in vitro systems offer an accessibility for quantitative analysis of cellular toxicity that is rarely afforded in situ. The ultimate objective of the proposed research is a detailed understanding of the effects of mercury on the selective permeability and transport properties of the blood-brain barrier and its cellular components, which would serve as a basis for new therapeutic interventions aimed at compensating or reversing such effects of mercury intoxication, or aimed at manipulating rates of mercury entry into or egress from the brain. A further long-term goal is the characterization and refinement of cell culture systems for their potential use in discriminating categories of toxicity to the blood-brain barrier. Specifically, the research will explore the selective impairment by mercuric chloride and methylmercury(II) chloride of characterized transport systems for anionic, neutral and cationic amino acids and glucose analogs in mouse cerebrovascular endothelial cells grown to confluence on porous membranes alone or 'enhanced' by co-culture with mouse cerebral astrocytes. Impairment of transport will be correlated with effects on the passive permeability of the endothelial cell layer, and with the cell content of mercury and its subcellular distribution. The role of serum constituents in modulating the migration of mercury into and across the endothelial cell layer will be explored. Radioactive tracers will be used to measure transport of amino acids and glucose analogs, and movement of mercury across the endothelial barrier. High-performance liquid chromatography will be used to measure endogenous free amino acid content in the cell cultures, and to validate the identify of radiolabeled species. Permeability of the cerebrovascular endothelial barrier will be assessed by measurement of electrical conductivity, and by spectrophotometric and fluorometric assays of molecular markers of varying bulk and charge. Scanning X-ray microanalysis of subcellular mercury distribution will be carried out using quick-freeze fixation and cryopreparative techniques.

汞，包括其有机化合物，是一种普遍存在的物质。 环境和职业神经毒物具有多种细胞 行动目标。 除了对神经元的直接毒性作用外， 神经毒性继发于其他功能损害 控制神经元微环境的细胞类型，特别是神经胶质细胞 和血脑屏障。 星形胶质细胞和脑血管 内皮细胞在表达中表现出相互营养性 差异化的血脑屏障特性。 这些属性 现在可以在细胞培养系统中进行体外复制。 这样在 体外系统提供了定量分析的可及性 细胞毒性很少在原位产生。 终极 拟议研究的目标是详细了解 汞对选择性渗透和传输的影响 血脑屏障及其细胞成分的特性， 这将作为新的治疗干预措施的基础 旨在补偿或扭转汞的此类影响 中毒，或旨在操纵汞进入的速率 或从大脑的出口。 进一步的长期目标是 细胞培养系统的表征和完善 在区分毒性类别方面的潜在用途 血脑屏障。 具体来说，该研究将探讨 氯化汞和甲基汞(II)的选择性损伤 阴离子、中性传输系统的氯化物 以及小鼠中的阳离子氨基酸和葡萄糖类似物 脑血管内皮细胞在多孔上生长至汇合 单独的膜或通过与小鼠大脑共培养来“增强” 星形胶质细胞。 交通损害将与 对内皮细胞层被动通透性的影响， 以及汞及其亚细胞的细胞含量 分配。 血清成分在调节中的作用 汞迁移进入并穿过内皮细胞层 将被探索。 放射性示踪剂将用于测量 氨基酸和葡萄糖类似物的运输以及 汞穿过内皮屏障。 高性能液体 色谱法将用于测量内源性游离氨基酸 细胞培养物中的含量，并验证 放射性标记物种。 脑血管的通透性 内皮屏障将通过测量电来评估 电导率，并通过分光光度和荧光测定 不同体积和电荷的分子标记。 扫描X射线 将进行亚细胞汞分布的微量分析 使用快速冷冻固定和冷冻制备技术。