Gene Expression in Rat Cochlea Following Loss of Hair Cells

描述(申请人提供)：人类听力损失最常归因于耳蜗毛细胞的丧失。毛细胞具有听觉感受器的功能，是螺旋神经节神经元的唯一传入细胞。毛细胞还为SGN提供促进存活的神经营养因子。遗传疾病、噪音创伤、衰老和耳毒性药物都可能导致毛细胞死亡。这种毛细胞的丧失导致SGN的去传入，最终导致外周轴突的丧失和神经元的死亡。SGN在一段时间内逐渐死亡，从大鼠的几个月到人类的几十年不等。目前尚不清楚在此期间是什么为SGN提供营养支持，也不知道为什么它最终不足以防止SGN死亡。回答这些问题可能会提出在耳聋后无限期维持SGN存活的方法。也不知道为什么一些SGN在毛细胞丧失后不久就死亡，而另一些则持续存在。了解这些SGN群体之间的差异可能会导致治疗以维持它们的生存。拟议研究的目标包括定位神经营养支持的替代来源，并确定所提供的神经营养因子。此外，我希望了解毛细胞缺失后仍存活数周的神经节与最初的神经节有何不同。神经营养支持的替代来源包括Corti器官的支持细胞、耳蜗核和螺旋神经节雪旺细胞。通过定量聚合酶链式反应和微阵列形式的基因表达分析，将完成对这些来源以及螺旋神经节的检查。从出生后第8天(P8)到P16，每天注射卡那霉素使大鼠耳聋，并在23、32、60和88天与年龄匹配的听力对照组一起实施安乐死。这些时间点是根据SGN的死亡过程选择的：P23相当于SGN死亡的开始，到P32已经发生了大量的SGN变性，P60处于退化期的中段，在P90时几乎没有SGN残留。RNA将被分离并转化为cDNA用于基因表达分析。在分析过程中，重点将放在神经营养因子、凋亡和促生存因子，以及与神经生存和生理学相关的基因上。

DESCRIPTION (provided by applicant): Human hearing loss is most often attributed to the loss of hair cells in the cochlea. Hair cells function as auditory receptors and are the sole afferent input to the spiral ganglion neurons (SGNs). Hair cells also provide survival-promoting neurotrophic factors for the SGNs. Genetic disorders, noise trauma, aging, and ototoxic drugs may all cause death of the hair cells. This loss of hair cells results in deafferentation of the SGNs, eventually causing loss of the peripheral axon and death of the neuron. SGNs die gradually over a period of time, ranging from months in rats, to decades in humans. It is not yet known what provides trophic support for the SGNs during this period nor why it is ultimately insufficient in preventing SGN death. Answering these questions might suggest means of maintaining SGN survival indefinitely after deafening. It is also unknown why some SGNs die shortly after loss of hair cells while others persist. Knowing the differences between these populations of SGNs may result in treatments to maintain their survival. Goals of the proposed study include localization of an alternative source for neurotrophic support and identification of the neurotrophic factors provided. Additionally, I wish to understand what distinguishes SGNs still surviving many weeks after hair cell loss from the initial population of SGNs. Alternative sources for neurotrophic support include supporting cells of the organ of Corti, the cochlear nucleus, and spiral ganglion Schwann cells. Examination of these sources, along with the spiral ganglion, through gene expression analysis in the forms of quantitative PCR and microarrays will be completed. Rats will be deafened with daily injections of kanamycin from postnatal days 8 (P8) through P16 and euthanized at 23, 32, 60, and 88 along with age-matched hearing controls. These time points were chosen based on the course of SGN death: P23 corresponds to the onset of SGN death, a statistically significant amount of SGN degeneration has occurred by P32, P60 is midway through the period of degeneration, and at P90 few SGNs remain. RNA will be isolated and converted to cDNA to be used for gene expression analysis. Emphasis during analysis will be on neurotrophic factors, apoptotic and pro-survival factors, and genes associated with neural survival and physiology.