p75 Expression for Assessing Neurotoxicity

描述（由申请人提供）：神经毒性是许多化疗药物的常见剂量限制或显著副作用，强调在筛选新型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学终点。这些方法的成本和资源要求限制了可以评价的化学品的数量，这些评价的效用因其固有的敏感性和难以获得定量数据而降低。先前的工作已经证明了周围神经系统受到毒性损伤和物理损伤后，几种基因的调节。低亲和性神经生长因子受体（p75）已被广泛表征，并在损伤后表现出最大的增加和持续时间最长的升高表达。在结构变化开始之前检测mRNA升高的能力以及评估整个轴突变化的能力表明，p75的上调可能比形态学终点提供了更敏感的神经毒性指标。提出的项目是基于这样的假设，即p75在周围神经系统中的表达升高是对毒性损伤的一种可量化的一般反应，并且在形态改变发生之前。这一假设将通过将大鼠暴露于选择性靶向神经元、轴突或雪旺细胞的神经毒性药物中来验证，然后在光镜和电子显微镜水平上确定p75转录的升高是否先于结构变化的发生。采用实时RT-PCR检测周围神经中p75 mRNA的表达量，免疫组织化学检测神经中p75蛋白的表达水平。测定血浆和尿液中截断的p75在检测神经毒性方面的效用也将通过ELISA和等离子体共振来确定。形态学评估将用于确定p75表达与结构改变发生的时间关系。这些研究的意义在于开发更有效和敏感的方法来检测新的治疗药物的神经毒性，监测治疗期间患者的神经毒性，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.