Growth & Gene Expression in Primary Sensory Neurons

18团契培训和职业生涯我的长期目标是成为一名独立的研究员，并对我们了解中枢神经系统(CNS)创伤，特别是脊髓创伤做出重大贡献我的博士前工作重点是脊髓挫伤后最初的兴奋毒性事件和慢性中枢性疼痛的发展，使用生化、分子和行为结果。然而，为了成为脊髓损伤的独立调查者，在这次团契期间，我需要更多关于CNS损伤后神经再生的知识。我将研究为什么CNS未能通过评估损伤后生长相关基因的表达来启动新的轴突生长。这需要学习新的技术，如原位杂交，病毒载体传递，体外和河流轴突生长分析，神经再生模型和新的脊髓损伤模型(背柱损伤)在这次团契期间接受的培训将使我获得成为独立赞助商19 NAMEANDDEGREE(S)Clifford J Woolf，MD，20职位/职级麻醉研究教授Richard J K.itz 21研究神经再生/神经生物学疼痛描述(不超过所提供的空间)中枢神经系统(CNS)损伤后的功能丧失是由于中枢神经系统(CNS)内的轴突未能重新生长这与周围神经系统(PNS)形成对比的是，尽管经过多年的研究，PNS的能力是可以解释的机制，但CNS再生的失败最近仍然知之甚少，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。使用这项新技术，似乎调节神经元生长的基因已经被鉴定出来，然而，用微阵列鉴定并不足以得出关于功能意义的结论。因此，需要对这些假定的生长相关基因(GAG)进行进一步的表征。本研究的目的是利用背根节(DRG)神经元的独特特性来验证通过微阵列分析识别的具有以下特征的假定的GAG的特征：(I)在发育过程中上调，(I)下调或不在成人中表达，(Iii)外周损伤后上调，以及(Iv)中枢损伤后不表达或下调，推测DR(；GAG将通过Slot、Northern和Western blotts、免疫细胞化学和原位杂交来确定细胞定位，以确定细胞定位。符合促生长GAG标准的基因将在体外和体内进行检测，以了解它们调节中枢神经系统损伤后轴突生长和再生的能力。这些研究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(Rev 12/98)来自第2页BB CC个体NRSA应用目录========================================Section End===========================================

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===========================================