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: 8169461
• 负责人: TATIANA B KRASIEVA
• 金额: $ 0.34万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169461
• 关键词: 3-Dimensional; Aging; Apoptosis; Architecture; Astrocytes; Binding; Biological Assay; Biology; Blood Vessels; Brain Neoplasms; Caliber; Cell Cycle Checkpoint; Cell Proliferation; Cell physiology; Cells; Cephalic; Collaborations; Computer Retrieval of Information on Scientific Projects Database; 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; Institution; Lasers; Life; Link; Maintenance; Metabolic; Mitochondria; Molecular Profiling; Monitor; Mus; NADH; NADPH Oxidase; Neuroglia; Neurons; Nicotinamide adenine dinucleotide; Nitrogen; Normal tissue morphology; Oligodendroglia; Optics; Oxidation-Reduction; Oxidative Stress; Oxygen; Oxygen Consumption; Patients; Peroxidases; Play; Process; Property; Protocols documentation; Radiation; Radiation Tolerance; Radiation therapy; Research; Research Design; Research Personnel; 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; cranium; experience; gliogenesis; glutathione peroxidase; in vivo; injured; interest; irradiation; membrane activity; multipotent cell; nerve stem cell; neurogenesis; oxidation; precursor cell; ratiometric; relating to nervous system; repaired; research study; response; response to injury; stem; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 神经干细胞和前体细胞代表增殖细胞池，它们可以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（即三个主要中枢神经系统谱系）。 虽然关于多能神经细胞的具体功能存在争议，但确实存在重要数据表明它们在修复和维护受损和衰老的中枢神经系统中发挥着不可或缺的作用。 为了响应损伤或疾病，多能细胞可以经历神经发生或神经胶质发生，以分别补充丢失和/或受损的神经元或神经胶质细胞。 人们发现，神经发生的抑制在时间上与认知功能障碍的发生同时发生，辐射引起的神经干细胞和前体细胞的消耗可能是接受颅脑放疗的患者出现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。 共同的未成熟表达谱、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。 鉴于上述情况，我们的实验室一直对了解多能神经细胞的氧化还原应激生物学感兴趣。 我们已经证明，为了响应辐射，这些细胞表现出剂量依赖性的氧化应激增加，这种增加可以持续数月。 生物学相关剂量（< 1Gy）后发现的氧化应激会影响放射敏感性、增殖、细胞命运、细胞凋亡、细胞周期检查点、适应性反应和线粒体功能。 我们过去的许多研究都依赖于在活细胞中使用荧光染料，这些染料在被某些活性氧 (ROS) 和氮 (RNS) 物质氧化后会发出荧光，产生可通过荧光激活细胞分选 (FACS) 进行量化的信号。 其他更定性的研究使用活细胞或固定细胞制剂，通过共聚焦显微镜评估各种应激后的类似终点。 这些技术的局限性在于需要使单细胞悬浮液通过流动池，或者无法分析通常在 3 维神经球中生长的大型活神经干细胞聚集体，其大小范围为 50-1500 个细胞/球。 我们拟议合作的总体目标是使用各种非侵入性光谱技术扩展我们在多能神经细胞中的氧化还原研究。 贝克曼激光研究所的技术提供了对许多氧化还原相关端点进行非侵入性成像的能力。使用双光子比率氧化还原荧光测定法可以实现线粒体能量代谢的可视化。 该方法成功地可视化了还原烟酰胺腺嘌呤二核苷酸 (NADH) 和氧化黄素腺嘌呤二核苷酸 (FAD) 之间氧化还原对的差异荧光特性。 我们希望利用我们的神经细胞系统来扩展这些类型的研究。 双光子光谱学的优点之一是能够对较大（直径约 150 nm）且完整的神经球内整个细胞中线粒体的氧化还原状态进行成像。 这样就无需破坏这些球体的结构以使它们通过流动池。 这很重要，因为我们有数据表明，在完整球体中发生的氧化还原过程更忠实地代表了体内情况。 将进行实验以确定双光子比率氧化还原荧光测定法是否可用于量化一定剂量和辐照后时间范围内辐射引起的氧化应激。 结果的验证可以通过同时对我们实验室过去广泛使用的一系列氧化还原敏感荧光染料进行成像来完成。 未来的工作将寻求对受辐射细胞中不同的氧化还原对进行成像，以确定完整神经球中能量代谢和氧化应激如何变化。 一些可能的例子可能包括分析琥珀酸脱氢酶活性、膜结合 NADPH 氧化酶、谷胱甘肽过氧化物酶以及其他细胞过氧化物酶。 存在许多其他可能性和终点。 最终我们希望在体内扩展双光子光谱。 UCI 的其他人（Cahalan 实验室）已经成功地做到了这一点，我们希望与贝克曼激光研究所的人员合作开发这项技术，用于对小鼠正常脑组织和植入性脑肿瘤的氧化还原状态进行成像。 已经制定了通过手术在啮齿动物的颅骨中安装“光学窗口”的方案。 这可能有助于应用双光子光谱来监测各种代谢参数（线粒体活性、缺氧、耗氧量），不仅可以跟踪肿瘤进展，还可以跟踪肿瘤和正常组织对各种介入治疗的反应。 总之，我们很高兴能够与贝克曼激光研究所建立长期合作。 我们期待与研究所的许多才华横溢的个人合作，努力启动一系列研究，我们相信这些研究对于理解正常和患病中枢神经系统的应激反应非常重要且相关。