Growth & Gene Expression in Primary Sensory Neurons

我的长期目标是成为一名独立的研究人员，并为我们对中枢神经系统（CNS）创伤的理解做出重大贡献，特别是脊髓创伤我的博士前工作主要集中在使用生化，分子和行为结果的挫伤脊髓损伤后的初始兴奋性毒性事件和慢性中枢疼痛的发展。为了成为脊髓损伤的独立研究者，我需要对中枢神经系统损伤后的神经再生有更多的了解。在这次研究中，我将通过评估损伤后生长相关基因的表达来研究为什么中枢神经系统不能启动新的轴突生长。这需要学习新的技术，如原位杂交，病毒载体递送，轴突生长的体外和体内测定、神经再生模型和脊髓损伤的新模型（背柱病变）在此奖学金期间接受的培训将使我获得成为独立赞助商所需的知识和技术技能19 NAMEANDDEGREE（S）Clifford J Woolf，MD，Phi）20职位/职级Richard J K.itz麻醉研究教授21研究兴趣/领域神经再生/疼痛神经生物学描述（请勿超过所提供的空间）中枢神经系统（CNS）损伤后的功能丧失是由于CNS内的轴突无法重新生长所致，这与周围神经系统（尽管经过多年的研究，对PNS再生能力的机制仍然知之甚少，但CNS再生失败的机制仍然知之甚少。最近，基因表达技术的进步使得使用高密度DNA微阵列同时筛选和鉴定数千个基因表达谱成为可能。但是，在此情况下，因此，需要进一步鉴定这些假定的生长相关基因（GAG）。本研究的目的是利用背根神经节（DRG）的独特性质，神经元以验证和表征通过微阵列分析鉴定的推定的GAG具有以下特征的推定的DRG GAG的表达谱将检查（i）在发育过程中上调，（ii）在成体中下调或不表达，（iii）在外周损伤后上调，和（iv）在中枢损伤后不表达或下调。(;将通过狭缝、北方和蛋白质印迹、免疫细胞化学、并通过原位杂交确定细胞定位。将在体外检测满足生长促进GAG标准的基因，并检测它们在CNS损伤后调节轴突生长和再生的能力。restdts将增加我们对中枢再生失败原因的理解，并可能为治疗中枢神经系统损伤提供新的治疗机会PHS 416-1（修订版12/98）表格第2页BB CC个人NRSA应用目录============================

18 GOALS FOR FELLOWSHIP TRAINING AND CAREER My long-term goal is to become an independent researcher and make significant contributions to our understanding of central nervous system (CNS) trauma, specifically trauma to the spinal cord My predoctoral work focused on the initial excitotoxic events and development of chronic central pain following contusion spinal cord injury using biochemical, molecular, and behavioral outcomes However, in order to become an independent investigator of spinal cord injury, I need a greater knowledge of nerve regeneration after CNS injury During this fellowship I will examine why the CNS fails to initiate new axonai growth by evaluating expression of growth associated genes after injury This requires learning new techniques, such as in situ hybridiazation, viral vector delivery, in vitro and in rive assays of axonal growth, models of nerve regeneration and a new model of spinal cord injury (dorsal column lesion) The training received during this fellowship will allow me to acquire the knowledge and technical skills I need to become independent SPONSOR 19 NAMEANDDEGREE(S) Clifford J Woolf, MD, Phi) 20 POSITION/RANK Richard J K.itz Professor of Anesthesia Research 21 RESEARCHINTERESTS/AREAS Neural Regeneration / Neurobiology of Pain DESCRIPTION (Do not exceed space provided) Loss of function following injury to the central nervous system (CNS) is due to a failure of axons within the CNS to re-grow This is in contrast to the peripheral nervous system (PNS) where re-growth occurs Despite years of study, the mechanisms resgonsible for the ability of the PNS, but failure oft.he CNS to regenerate remain poorly understood Recently, advances in gene expression technology have made it possible to screen and identify thousands of gene expression profiles simultaneously using high density DNA microarrays Using this new technology, genes that appear to regulate neuronal growth have been identified, however, identification by microarrays is inadequate evidence to draw conclusions about functional significance Further characterization of these putative growth associated genes (GAGs) is therefore reqnired The aim of this study is to use the unique properties of dorsal root ganglion (DRG) neurons to validate mad characterize putative GAGs identified by microarry analysis Expression profiles of putative DRG GAGs that have the following characteristics will be examined (i) upregulated during development, (it) down regulated or not expressed in adult, (iii) upregulated after peripheral injury, and (iv) not expressed or down regulated after a central lesion The expression profiles of putative DR(; GAGs will be validated by slot, Northern, and Western blots, immunocytochemistry, and by in situ hybridization to determine cellular localization Genes that fulfill the criteria for a growth promoting GAG will be examined in vitro and in rive for their ability to regulate axonal growth and regeneration after CNS injury These restdts will increase our understanding of the reasons for failure of central regeneration and may offer novel therapeutic opportunities to treat CNS injuries PHS 416-1 (Rev 12/98) Form Page 2 BB CC Individual NRSA Application Table of Contents ========================================Section End===========================================