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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 神经茎和前体细胞代表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（即三个主要的CNS谱系）的增生细胞的池。 尽管关于多能神经细胞的特定功能存在争议，但确实存在重要的数据，这表明它们在受伤和老化的中枢神经系统的修复和维护中起着不可或缺的作用。 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，分别补充丢失和/或受损的神经元或神经胶质。 已经发现，神经发生的抑制与认知功能障碍的发作是暂时的，并且辐射诱导的神经茎和前体细胞的耗竭可能是患者接受颅放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，神经干和前体细胞也可能成为脑肿瘤干细胞。 共享的不成熟表达谱，可靠的增殖，与血管的关联以及相似的氧化还原特性是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。 神经茎和前体细胞在正常组织修复中具有双重功能，以及致癌的进展强调了它们在中枢神经系统中的重要性。 鉴于上述内容，我们的实验室对了解多能力神经细胞的氧化还原应激生物学感兴趣。 我们已经证明，在响应辐射时，这些细胞显示出可能持续多个月的氧化应激的剂量增加。 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，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，这些染料在某些活性氧（ROS）和氮（RNS）物种氧化后氧化后会变得荧光，产生可以通过荧光激活细胞分类（FACS）来量化的信号。 其他更定性的研究已使用活细胞制剂或通过共聚焦显微镜在各种应力​​后评估相似的终点。 这些技术的局限性围绕通过流动细胞传递单细胞悬浮液的必要性，或者无法分析通常在3维神经球中生长的神经干细胞的大生命聚集体，该神经球的大小可能在50-1500个细胞/球体的范围内。 我们提出的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（Beckman Laser Institute）中存在的技术提供了非侵入性的许多氧化还原相关终点的能力。使用两光子比率氧化还原荧光测定法可以可视化线粒体能量代谢。 这种方法已成功地看到了氧化胺还原腺苷二核苷酸（NADH）和氧化的黄素黄素腺嘌呤二核苷酸（FAD）之间的氧化还原夫妇的差异荧光性能。 我们想使用我们的神经细胞系统扩展这些类型的研究。 两光谱光谱的一个优点是能够在整个细胞（直径约150 nm）和完整的神经球内成像线粒体的氧化还原状态。 这消除了破坏这些球体的结构以通过流动池的构建。 这很重要，因为我们有数据表明，氧化还原过程更忠实地代表了体内的情况。 将进行实验，以确定是否可以使用两光子比率氧化还原型荧光法来定量辐射诱导的氧化应激，范围内剂量和辐射后时间。 可以通过同时成像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来实现结果的验证。 未来的工作将寻求对辐照细胞中的不同氧化还原夫妇进行成像，以确定能量代谢和氧化应激在完整的神经球中的变化。 一些可能的例子可能包括分析琥珀酸脱氢酶活性，膜结合的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，我们想在体内延伸两光谱光谱。 UCI的其他人成功完成了这项工作（Cahalan's Lab），我们想与Beckman Laser Institute的人们合作开发这项技术，以成像正常脑组织的氧化还原状态和在小鼠中植入的脑肿瘤的氧化还原状态。 已经开发了用于手术在啮齿动物颅骨中安装“光学窗口”的协议。 然后，这可以促进两光子光谱法监测多种代谢参数（线粒体活性，缺氧，氧气消耗）不仅遵循肿瘤进展，还遵循肿瘤和正常组织对各种介入疗法的反应。 总而言之，我们很高兴与贝克曼激光学院进行长期合作。 我们期待与研究所的众多才华横溢的人合作，以启动一系列研究，我们认为与理解正常和患病的中枢神经系统的压力反应至关重要并且相关。