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.
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