p75 Expression for Assessing Neurotoxicity

描述(申请人提供)：神经毒性是许多化疗药物常见的剂量限制或显著副作用，强调在筛选新的化疗药物时评估神经毒性的重要性。目前筛选神经毒性的方法通常利用形态终点。这些方法的成本和资源要求限制了可以评估的化学品的数量，而且这些评估的效用因其固有的敏感性和难以获得定量数据而受到削弱。之前的研究已经证明，在有毒侮辱和身体损伤对周围神经系统造成伤害后，几个基因的表达上调。低亲和力神经生长因子受体(P75)的特征最为广泛，在损伤后表达上调幅度最大，持续时间最长。在结构变化开始之前检测到mRNA的升高的能力，以及评估整个轴突的变化的能力，表明p75的上调可能提供了比形态终点更敏感的神经毒性指标。拟议的项目基于这样一种假设，即周围神经系统中p75表达的升高是对毒性侮辱的一般反应，这种反应是可以量化的，并且先于形态变化的开始。这一假设将通过让大鼠暴露于选择性靶向神经元、轴突或雪旺细胞的神经毒剂，然后在光学和电子显微镜水平上确定p75转录升高是否先于结构变化来检验。采用实时定量RT-PCR方法检测周围神经中p75基因的表达，免疫组织化学方法检测神经中p75蛋白的表达水平。测定血浆和尿液中截短p75对检测神经毒性的作用也将通过酶联免疫吸附试验和血浆共振法确定。形态评估将被用来确定p75表达与结构变化开始的时间关系。这些研究的意义在于发展更有效和更灵敏的方法来检测新的治疗药物的神经毒性，监测患者在治疗过程中的神经毒性，确定神经毒性药物的生物激活途径，以及评估环境化合物的神经毒性。这些方法的好处可以通过促进新治疗剂的开发和在人类神经毒性发生之前识别神经毒性化学物质来实现。

DESCRIPTION (provided by applicant): Neurotoxicity is a common dose limiting or significant side effect of many chemotherapeutic agents emphasizing the importance of assessing neurotoxicity in the screening of novel chemotherapeutic agents. Current methods of screening for neurotoxicity typically utilize morphologic endpoints. The cost and resource requirements of these methods limits the number of chemicals that can be evaluated and the utility of these assessments is diminished by their inherent sensitivity and difficulty in obtaining quantitative data. Previous work has demonstrated up regulation of several genes following toxic insults and physical injury to the peripheral nervous system. The low affinity nerve growth factor receptor (p75) has been most extensively characterized and has exhibited the greatest increases and longest duration of elevated expression after injury. The ability to detect elevations in mRNA prior to the onset of structural changes together with the ability to assess changes along the entire axon suggest that up regulation of p75 may provide a more sensitive index of neurotoxicity than morphological endpoints. The proposed project is based upon the hypothesis that elevated p75 expression in the peripheral nervous system is a general response to toxic insult that is quantifiable and precedes the onset of morphological changes. This hypothesis will be tested through exposing rats to neurotoxic agents that selectively target the neuron, axon or Schwann cell and then determining if elevated p75 transcription precedes the onset of structural changes at the light and electron microscope level. The quantity of p75 mRNA will be determined in peripheral nerve using real time RT-PCR and the levels of p75 protein in nerve evaluated using immunohistochemistry. The utility of measuring truncated p75 in plasma and urine to I detect neurotoxtcity will also he determined by ELISA and plasmon resonance. Morphologic assessments will he used to determine the temporal relationship of p75 expression to the onset of structural changes. The significance of these investigations lies in the development of more efficient and sensitive methods for detecting neurotoxicity of new therapeutic agents, monitoring neurotoxicity in patients during therapy, defining bioactivation pathways of neurotoxic agents and assessing the neurotoxicity of environmental compounds. The benefits of these methods can be realized through facilitating development of new therapeutic agents and recognizing neurotoxic chemicals before the occurrence of human neurotoxicity.