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: 7954802
• 负责人: TATIANA B KRASIEVA
• 金额: $ 0.3万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954802
• 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; 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 神经干细胞和前体细胞代表可在CNS内迁移并分化成神经元、星形胶质细胞和少突胶质细胞（即三种主要CNS谱系）的增殖细胞库。 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受损和老化CNS的修复和维护中发挥不可或缺的作用。 在对损伤或疾病的反应中，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。 已发现神经发生的抑制与认知功能障碍的发作在时间上一致，并且辐射诱导的神经干细胞和前体细胞的耗竭可能是接受颅脑放射治疗的患者所经历的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、稳健的增殖、与血管的关联以及相似的氧化还原性质是暗示CNS中正常干细胞和癌症干细胞之间的功能联系的一些相似性。 神经干细胞和前体细胞在正常组织修复以及致癌进展中具有双重功能的可能性强调了它们在CNS中的重要性。 鉴于上述情况，我们实验室一直有兴趣了解多能神经细胞的氧化还原应激生物学。 我们已经证明，在响应辐射，这些细胞显示出剂量依赖性的氧化应激增加，可以持续数月。 生物学相关剂量（< 1Gy）后发现的氧化应激影响放射敏感性、增殖、细胞命运、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，这些荧光染料在被某些活性氧（ROS）和氮（RNS）物质氧化后变得发荧光，产生可以通过荧光激活细胞分选（FACS）定量的信号。 其他更多的定性研究使用活细胞或固定细胞制备物，通过共聚焦显微镜评估各种应力后的类似终点。 这些技术的局限性围绕着使单细胞悬浮液通过流动池的必要性或不能测定通常在三维神经球中生长的神经干细胞的大的活聚集体，所述三维神经球的尺寸范围可以为50-1500个细胞/球。 我们的总体目标，这项拟议的合作是扩大我们的氧化还原研究多能神经细胞使用各种非侵入性光谱技术。 贝克曼激光研究所目前的技术提供了非侵入性成像许多氧化还原相关端点的能力。双光子比率氧化还原荧光法的使用允许线粒体能量代谢的可视化。 这种方法已经成功地可视化还原型烟酰胺腺嘌呤二核苷酸（NADH）和氧化型黄素腺嘌呤二核苷酸（FAD）之间的氧化还原对的差异荧光特性。 我们希望使用我们的神经细胞系统来扩展这些类型的研究。 双光子光谱学提供的一个优点是能够在较大（~150 nm直径）和完整的神经球内对整个细胞中线粒体的氧化还原状态进行成像。 这消除了破坏这些球体的结构以使它们通过流动池的需要。 这一点很重要，因为我们有数据表明，在完整的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法是否可以用来量化辐射诱导的氧化应激在一定范围内的剂量和照射后的时间。 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的荧光染料进行成像来完成。 未来的工作将寻求在辐照细胞中成像不同的氧化还原对，以确定能量代谢和氧化应激在完整的神经球中如何变化。 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学。 UCI的其他人已经成功地做到了这一点（Cahalan的实验室），我们希望与贝克曼激光研究所的人合作开发这项技术，用于成像小鼠正常脑组织和植入脑肿瘤的氧化还原状态。 在啮齿动物的颅骨上手术安装“光学窗口”的方案已经开发出来。 然后，这可以促进双光子光谱学的应用，以监测各种代谢参数（线粒体活性、缺氧、耗氧量），从而不仅跟踪肿瘤进展，而且跟踪肿瘤和正常组织对各种介入治疗的反应。 总之，我们很高兴能与贝克曼激光研究所开展长期合作。 我们期待着与研究所的许多有才华的人合作，努力开展一系列研究，我们相信这对了解正常和患病的CNS的应激反应是重要的和相关的。