Apoptotic and Necrotic Death of OHCs in NIHL

描述（由申请人提供）：我们以前的研究发现，在高水平的噪声暴露后，外毛细胞（OHC）通过凋亡和坏死死亡。细胞凋亡或坏死的发生率与噪声水平和暴露后耳蜗病变的进展有关;然而，负责确定噪声暴露后OHC死亡途径的细胞机制仍不清楚。本申请集中于一般假设，即OHC通过凋亡或坏死死亡的倾向是由垂死OHC的能量水平和/或氧化状态调节的。为了检验这一假设，龙猫将暴露于中心频率为4 kHz、声压级为110 dB的倍频程噪声中1小时。在实验的第一部分中，琥珀酸脱氢酶（SDH），一个重要的线粒体酶，参与ATP合成的电子传递链，将检查在噪声暴露后的凋亡和坏死的OHC的活性。在研究的第二部分中，将通过脑内应用3-硝基丙酸（3-NP）（一种不可逆的SDH抑制剂）来阻断OHC合成ATP。将检查细胞内ATP耗竭对噪声暴露后细胞凋亡和坏死产生的影响。在研究的最后部分，将检查耳蜗抗氧化能力的改变对死亡途径的影响。耳蜗的抗氧化能力将通过改变耳蜗谷胱甘肽（GSH）的水平来操纵，无论是用L-丁硫氨酸-[S，R]-亚砜亚胺，GSH合成抑制剂，或谷胱甘肽单乙酯，GSH的类似物。这些数据将有助于阐明耳蜗能量水平和抗氧化剂水平在确定噪声暴露后细胞凋亡或坏死的患病率中的作用。我们的长期目标是探索有效的治疗策略，以减少噪声引起的听力损失。由于坏死的OHC可能通过释放细胞内内容物对存活的OHC产生显著更高水平的毒性应激，因此防止细胞凋亡转化为坏死可以改善总体耳蜗损伤。负责调节细胞死亡途径的生物学机制的知识将为最终开发合理的保护策略提供基础。

DESCRIPTION (provided by applicant): Our previous studies have found that following a high level of noise exposure, outer hair cells (OHCs) die by both apoptosis and necrosis. The prevalence of apoptosis or necrosis is associated with the noise level and post-exposure progression of the cochlear lesion; however, the cellular mechanisms responsible for determining the pathways of OHC death following noise exposure are still not known. This application focuses on the general hypothesis that the propensity of OHCs to die by apoptosis or necrosis is regulated by the energy level and/or oxidative status of dying OHCs. To test this hypothesis, chinchillas will be exposed to an octave band noise centered at 4 kHz at 110 dB SPL for 1 hour. In the first part of the experiment, the activity of succinate dehydrogenase (SDH), an important mitochondrial enzyme that participates in ATP synthesis in the electron transport chain, will be examined in apoptotic and necrotic OHCs after the noise exposure. In the second part of the study, the synthesis of ATP by OHCs will be blocked by intracochlear application of 3-nitropropionic acid (3-NP), an irreversible inhibitor of SDH. The effect of intracellular ATP depletion on generation of apoptosis and necrosis following the noise exposure will be examined. In the last part of the study, the effect of the alteration of the cochlear antioxidant capacity on the death pathways will be examined. The cochlear antioxidant capacity will be manipulated by changing the level of cochlear glutathione (GSH) either with L-buthionine-[S, R]-sulfoximine, a GSH synthesis inhibitor, or with glutathione monoethyl ester, an analog of GSH. These data will help to elucidate the role of the cochlear energy level and the antioxidant level in determining the prevalence of either apoptosis or necrosis after noise exposure. Our long-term goal is to explore effective therapeutic strategies to reduce noise-induced hearing loss. Since necrotic OHCs potentially create a significantly greater level of toxic stress on surviving OHCs through release of intracellular contents, preventing the conversion of apoptosis to necrosis may ameliorate the overall cochlear damage. The knowledge of the biological mechanisms responsible for modulation of the cell death pathways will provide the basis for eventually developing a rational protective strategy.