LOW-LEVEL LEAD EFFECTS IN GLIAL/ENDOTHELIAL COCULTURES

胶质细胞/内皮细胞培养物中的低水平铅效应

• 批准号: 2018269
• 负责人: MARIE E LEGARE
• 金额: $ 8.44万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-21 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2018269
• 关键词: blood brain barrier; calcium flux; developmental neurobiology; enzyme activity; gap junctions; glia; glutamate ammonia ligase; infant animal; ion transport; laboratory rat; lead poisoning; membrane model; mitochondrial membrane; mixed tissue /cell culture; molecular pathology; vascular endothelium

The toxic effects of low lead upon the developing nervous system remain a major health issue in this country. Among the unsolved problems are mechanisms of cellular injury, as well as mechanisms of Pb uptake, storage, and tolerance in brain cells that accumulate Pb. The candidate's immediate career objective is to gain depth and experience in the field of neuroscience, specifically with astroglia and their interaction with the blood-brain barrier (BBB). We plans to thoroughly explore the multistep model of low-level lead (Pb) neurotoxicity presented in this application. The investigators propose to study the molecular changes that occur in astroglia and endothelial cells in culture, following exposure to low levels of Pb (0.01 to 1 microM). Parameters investigated will be Ca2+ homeostasis, mitochondrial membrane potential, and gap junction regulation. Reduction of glutamine synthetase activity in astroglia will also be measured as an indicator of Pb-induced toxicity. The investigators will also test the hypothesis that endothelial cells induce astroglial maturation and resistance to Pb2+ in co-culture. The candidate will exploit the powerful new technology of Interactive Laser Cytometry for probing the disruption of cell metabolism at discrete sites. the candidate's department is unique in that it has both state-of-the-art equipment and a highly trained support staff required for effective use of this technology. Interactive Laser Cytometry and biochemical assays will be combined to evaluate the molecular events transpiring in these cells after exposure to submicromolar Pb concentrations. In addition, an in vitro model of the blood-brain barrier (BBB), consisting of rat astroglial and brain capillary endothelial cells grown on opposite surfaces of a semi- permeable membrane, will be constructed and evaluated with respect to low-level Pb toxicity. Pb transport across the barrier formed by the cells will be measured, as well as Pb effects on barrier integrity. These experiments, which constitute Phase I of the research plan, will lead to her PhD degree. Finally, in Phase II, the candidate plans to specifically address the question of how lead enters cells (particularly astroglia and endothelial cells), and how it is stored. The candidate will be guided in this effort by a biochemist with extensive experience in protein-metal interactions, as well as transitional metal chemistry and metabolism. The PSA would relieve her of other responsibilities, particularly teaching, so she could commit 90% of her time to training and research. The candidate has two long-term career objectives, which she plans to attain as an academic scientist and teacher: first, to make significant strides in the development of a valid in vitro model of the BBB, and secondly to apply this model to study toxin-induced as well as infectious neuropathologies.

低铅对发育中的神经系统的毒性作用仍然存在 一个重大的健康问题 未解决的问题包括 细胞损伤机制，以及铅吸收机制， 储存和耐受性的脑细胞中积累铅。 的 候选人的直接职业目标是获得深度和经验 在神经科学领域，特别是星形胶质细胞及其 与血脑屏障（BBB）的相互作用。 我们计划彻底 探讨低水平铅神经毒性的多步骤模型 在这个应用程序中呈现。 研究人员建议研究 星形胶质细胞和内皮细胞的分子变化， 文化，暴露于低水平的铅（0.01至1微M）。 研究的参数将是Ca 2+稳态、线粒体膜 电位和间隙连接调节。 谷氨酰胺还原 还将测量星形胶质细胞中的合成酶活性作为指示剂 铅的毒性。 调查人员还将检验这一假设 内皮细胞诱导星形胶质细胞成熟， Pb 2+共培养。 候选人将利用强大的新 交互式激光细胞计数技术，用于探测 在离散的部位进行细胞代谢。 候选人的部门是独一无二的 因为它既拥有最先进的设备，又拥有训练有素的人员 有效利用这项技术所需的支助人员。 交互式激光细胞计数和生化分析将结合起来， 评估暴露后这些细胞中发生的分子事件 到亚微摩尔浓度的铅。 此外，体外模型， 血脑屏障（BBB），由大鼠星形胶质细胞和脑 毛细血管内皮细胞生长在半- 渗透膜将根据以下方面进行构建和评估 低铅毒性。 Pb迁移穿过由 电池将被测量，以及铅对屏障完整性的影响。 这些实验构成研究计划的第一阶段， 获得了博士学位 最后，在第二阶段，候选人计划 具体解决铅如何进入细胞的问题（特别是 星形胶质细胞和内皮细胞），以及它如何储存。 候选 将由一位经验丰富的生物化学家指导这项工作 在蛋白质-金属相互作用，以及过渡金属化学中， 和新陈代谢。 公共安全协会将解除她的其他责任， 特别是教学，所以她可以将90%的时间用于培训， 与研究 候选人有两个长期职业目标， 她计划成为一名学术科学家和教师：首先， 在开发有效的体外模型方面取得了重大进展， 第二，应用该模型研究毒素诱导的血脑屏障损伤， 传染性神经病理学

项目成果

Analysis of Pb2+ entry into cultured astroglia.

• DOI: 10.1006/toxs.1998.2492
• 发表时间: 1998-11
• 期刊: Toxicological sciences : an official journal of the Society of Toxicology
• 作者: Marie E. Legare;R. Barhoumi;E. Hébert;Gerald R. Bratton;Robert C. Burghardt;E. Tiffany‐Castiglioni

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters hippocampal astroglia-neuronal gap junctional communication.

• 发表时间: 2000-12
• 期刊: Neurotoxicology
• 影响因子: 3.4
• 作者: Marie E. Legare;W. Hanneman;R. Barhoumi;Robert C. Burghardt;E. Tiffany‐Castiglioni

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in primary rat astroglia: identification of biochemical and cellular targets.

• 发表时间: 1997
• 期刊: Neurotoxicology
• 影响因子: 3.4
• 作者: M. Légaré;W. Hanneman;R. Barhoumi;E. Tiffany‐Castiglioni