NONINVASIVE IMAGING OF NEURAL STEM AND PRECURSOR CELL FUNCTIONS

神经干和前体细胞功能的无创成像

• 批准号: 8362632
• 负责人: TATIANA B KRASIEVA
• 金额: $ 0.51万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362632
• 关键词: 3-Dimensional; Aging; Apoptosis; Architecture; Astrocytes; Binding; Biological Assay; Biology; Biotechnology; Blood Vessels; Brain Neoplasms; Caliber; Cell Cycle Checkpoint; Cell Proliferation; Cell physiology; Cells; Cephalic; Collaborations; Confocal Microscopy; Couples; Data; Disease; Dose; Dyes; Energy Metabolism; Flavin-Adenine Dinucleotide; Fluorescence-Activated Cell Sorting; Fluorometry; Funding; Future; Goals; Grant; Hypoxia; Image; Imagery; Imaging technology; Impaired cognition; Implant; Individual; Institutes; Lasers; Life; Link; Maintenance; Metabolic; Mitochondria; Molecular Profiling; Monitor; Mus; NADH; NADPH Oxidase; National Center for Research Resources; Neuroglia; Neurons; Nicotinamide adenine dinucleotide; Nitrogen; Normal tissue morphology; Oligodendroglia; Optics; Oxidation-Reduction; Oxidative Stress; Oxygen; Oxygen Consumption; Patients; Peroxidases; Play; Principal Investigator; Process; Property; Protocols documentation; Radiation; Radiation Tolerance; Radiation therapy; Research; Research Design; Research Infrastructure; Resources; Rodent; Role; Series; Signal Transduction; Source; Spectrum Analysis; Stress; Succinate Dehydrogenase; Suspension substance; Suspensions; System; Techniques; Technology; Time; Tumor Stem Cells; United States National Institutes of Health; Validation; Work; Wound Healing; biological adaptation to stress; brain tissue; cancer stem cell; cell preparation; cost; cranium; experience; gliogenesis; glutathione peroxidase; in vivo; injured; interest; irradiation; membrane activity; multipotent cell; nerve stem cell; neurogenesis; oxidation; precursor cell; ratiometric; repaired; research study; response; response to injury; tumor; tumor progression; two-photon

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Neural stem and precursor cells represent pools of proliferative cells that can migrate within the CNS and differentiate into neurons, astrocytes, and oligodendrocytes (i.e. the three main CNS lineages). While controversy exists regarding the specific functions of multipotent neural cells, significant data does exist suggesting they play integral roles in the repair and maintenance of the injured and aging CNS. In response to injury or disease, multipotent cells can undergo neurogenesis or gliogenesis to replenish lost and/or damaged neurons or glia respectively. Inhibition of neurogenesis has been found to be temporally coincident with the onset of cognitive dysfunction, and the radiation-induced depletion of neural stem and precursor cells may be one cause of the cognitive impairments experienced by patients subjected to cranial radiotherapy. Despite the protective role these cells have, recent evidence suggests that under certain circumstances, neural stem and precursor cells may also become brain tumor stem cells. The shared immature expression profiles, robust proliferation, association with blood vessels, and similar redox properties are some of the similarities suggesting a functional link between normal and cancer stem cells in the CNS. The possibility that neural stem and precursor cells have dual functions in normal tissue repair as well as carcinogenic progression underscores their importance in the CNS. Given the foregoing, our lab has been interested in understanding the redox stress biology of multipotent neural cells. We have demonstrated that in response to irradiation, these cells show a dose dependent increase in oxidative stress that can persist for many months. Oxidative stress found after biologically relevant doses (< 1Gy) impacts radiosensitivity, proliferation, cell fate, apoptosis, cell cycle checkpoints, adaptive responses and mitochondrial function. Many of our past studies have relied on the use of fluorogenic dyes in live cells that upon oxidation by certain reactive oxygen (ROS) and nitrogen (RNS) species become fluorescent, yielding a signal that can be quantified by fluorescence activated cell sorting (FACS). Other more qualitative studies have used living or fixed cell preparation to assess similar endpoints after a variety of stresses via confocal microscopy. Limitations of these technologies revolve around the necessity of passing single cell suspensions through a flow cell or the inability to assay large living aggregates of neural stem cells that typically grow in 3-dimensional neurospheres, that can range in size from 50-1500 cells/sphere. Our overall goal for this proposed collaboration is to extend our redox studies in multipotent neural cells using a variety of noninvasive spectroscopic techniques. The technologies present at the Beckman Laser Institute provide the capability to image many redox relevant endpoints non-invasively. The use of two-photon ratiometric redox fluorometry allows for the visualization of mitochondrial energy metabolism. This approach has successfully visualized the differential fluorescent properties of the redox couple between reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD). We would like to extend these types of studies using our neural cell system. One advantage two-photon spectroscopy provides is the ability to image the redox status of mitochondria throughout the cells within larger (~150 nm diameter) and intact neurospheres. This obviates the need to disrupt the architecture of these spheres to pass them through a flow cell. This is important since we have data suggesting that redox processes transpiring in intact spheres more faithfully represents the in vivo situation. Experiments would be conducted to determine whether two-photon ratiometric redox fluorometry could be used to quantify radiation-induced oxidative stress over a range of doses and post-irradiation times. Validation of results could be accomplished by simultaneously imaging a range of redox sensitive fluorogenic dyes our lab has used extensively in the past. Future work would seek to image different redox couples in irradiated cells to determine how energy metabolism and oxidative stress vary in intact neurospheres. Some possible examples might include analyzing succinate dehydrogenase activity, membrane bound NADPH oxidases, glutathione peroxidase as well as other cellular peroxidases. Many other possibilities and endpoints exist. Ultimately we would like to extend two-photon spectroscopy in vivo. Others at UCI have done this successfully (Cahalan's Lab) and we would like to work with the people at the Beckman Laser Institute to develop this technology for imaging the redox status of normal brain tissue and implanted brain tumors in mice. Protocols have been developed for surgically installing an "optical window" in the cranium of rodents. This may then facilitate the application of two-photon spectroscopy to monitor a variety of metabolic parameters (mitochondrial activity, hypoxia, oxygen consumption) to follow not only tumor progression but the response of tumors and normal tissue to various interventional therapies. In summary we are excited to initiate a long-term collaboration with the Beckman Laser Institute. We look forward to working with the many talented individuals at the Institute in our efforts to initiate a series of studies we believe will be important and relevant to understanding the stress response the normal and diseased CNS.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和子项目首席研究员的主要支持可能由其他来源提供，包括NIH的其他来源。子项目列出的总成本可能代表子项目使用的中心基础设施的估计数量，而不是由NCRR授予给子项目或子项目员工的直接资金。神经干细胞和前体细胞代表了可在中枢神经系统内迁移并分化为神经元、星形胶质细胞和少突胶质细胞（即中枢神经系统的三种主要谱系）的增殖细胞池。虽然关于多能神经细胞的具体功能存在争议，但确实有大量数据表明它们在损伤和老化的中枢神经系统的修复和维持中发挥着不可或缺的作用。在对损伤或疾病的反应中，多能细胞可以分别进行神经发生或胶质发生来补充丢失和/或受损的神经元或胶质细胞。神经发生的抑制已被发现与认知功能障碍的发作在时间上是一致的，辐射诱导的神经干和前体细胞的耗竭可能是颅脑放疗患者经历认知障碍的原因之一。尽管这些细胞具有保护作用，但最近的证据表明，在某些情况下，神经干细胞和前体细胞也可能成为脑肿瘤干细胞。共享的未成熟表达谱、强劲的增殖、与血管的关联以及相似的氧化还原特性是一些相似之处，表明中枢神经系统中正常干细胞和癌症干细胞之间存在功能联系。神经干细胞和前体细胞在正常组织修复和致癌进展中具有双重功能的可能性强调了它们在中枢神经系统中的重要性。