NONINVASIVE IMAGING OF NEURAL STEM AND PRECURSOR CELL FUNCTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 神经干细胞和前体细胞代表增殖细胞池，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(即三种主要的中枢神经系统谱系)。虽然关于多能神经细胞的具体功能存在争议，但有重要数据表明，它们在受损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。作为对损伤或疾病的反应，多能细胞可以通过神经发生或神经胶质发生来分别补充丢失和/或受损的神经元或神经胶质细胞。神经发生的抑制与认知功能障碍的发生在时间上是一致的，辐射引起的神经干细胞和前体细胞的枯竭可能是接受颅脑放射治疗的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瘤干细胞。共同的不成熟的表达谱，强劲的增殖，与血管的联系，以及相似的氧化还原特性是一些相似之处，表明在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，突显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，作为对辐射的反应，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。生物相关剂量(1Gy)后发现的氧化应激影响辐射敏感性、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，当某些活性氧(ROS)和氮(RNS)物种氧化时，这些染料会变成荧光，产生可以通过荧光激活细胞分类(FACS)进行量化的信号。其他更定性的研究使用活的或固定的细胞准备，通过共聚焦显微镜评估在各种压力下的相似终点。这些技术的局限性在于需要让单个细胞悬液通过流动细胞，或者无法检测通常生长在三维神经球中的大量活的神经干细胞聚集体，三维神经球的大小从50-1500个细胞/球不等。 我们提出的这项合作的总体目标是使用各种非侵入性光谱技术扩大我们在多潜能神经细胞中的氧化还原研究。贝克曼激光研究所现有的技术提供了非侵入性地对许多氧化还原相关终点进行成像的能力。使用双光子比率氧化还原荧光法可以可视化线粒体的能量代谢。这种方法成功地显示了还原的烟酰胺腺嘌呤二核苷酸(NADH)和氧化的黄素腺嘌呤二核苷酸(FAD)之间的氧化还原对的差异荧光性质。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势是能够在更大(~150 nm直径)和完整的神经球中成像整个细胞中线粒体的氧化还原状态。这就不需要破坏这些球体的结构来使它们通过流动细胞。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真实地代表了体内的情况。将进行实验，以确定是否可以使用双光子比率氧化还原荧光法在一定剂量和辐照后时间范围内对辐射引起的氧化应激进行量化。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受辐射的细胞中成像不同的氧化还原对，以确定完整神经球中能量代谢和氧化应激的变化。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。还存在许多其他可能性和端点。最终，我们希望在活体内扩展双光子光谱学。UCI的其他人已经成功地完成了这项工作(Cahalan‘s Lab)，我们希望与Beckman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瘤的氧化还原状态进行成像。已经开发出用于在啮齿类动物的头盖骨中外科安装“光学窗口”的方案。这可能有助于应用双光子光谱学来监测各种代谢参数(线粒体活动、缺氧、氧气消耗)，不仅可以跟踪肿瘤的进展，还可以跟踪肿瘤和正常组织对各种介入治疗的反应。 总而言之，我们很高兴能与贝克曼激光研究所展开长期合作。我们期待着与研究所的许多有才华的人合作，努力启动一系列研究，我们相信这对了解正常和患病的中枢神经系统的应激反应将是重要和相关的。

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.