Evaluating anti-oxidant and autophagy compounds as potential therapies for mammalian aminoglycoside-induced hair cell loss

人口发展概述了老年人疾病的重要性日益增加，例如感音神经性听力损失（SNHL）（65岁至50%）。目前尚无法预防由药物、遗传或衰老等大多数原因引起的SNHL。噪音引起的听力损失可以用耳塞或护耳来预防，但在必要时往往不戴。SNHL很难治疗，因为它会造成感知声音的灵敏度丧失和失真。这部分是由于与外毛细胞（HC）损失相关的频率特异性的丧失，但也与内部HC上传入突触的丧失有关，这可能不会反映在听力敏感性的丧失中（Kujawa和Liberman， 2015）。助听器提高了灵敏度，但通常不能克服声音失真（Silverman, 1998）。预防和治疗SNHL的新方法迫在眉睫。先前使用体外干预的研究已经确定了一些导致HC损伤和丢失的途径（Battaglia, 2003; Brand, 2015）或影响HC再生的途径（Masuda, 2006）。这一知识库为保护hc的药物干预提供了机会。提交的项目涉及抗氧化和自噬化合物的评估，这些化合物可能预防和保护hc免受氨基糖苷（AG）诱导的毛细胞脱落。目的：短期目标是确定抗氧化剂在体内保护hc和听力免受损伤的相对潜力。II)自噬化合物在体外影响HC损伤的相对潜力。该项目的长期目标是增加对不同类型SNHL的预防和改善治疗方案，如ag诱导的HC损伤和噪声诱导的SNHL。方法：实验方案一：抗氧化剂的体内试验采用FVB小鼠，这些小鼠不会出现与年龄相关的听力损失或噪音敏感性增加。每只小鼠将暴露在105 dB SPL（声压级）的倍频噪声（12-24 kHz）中30分钟。在噪声后立即测量的暴露产生约65 dB的阈值位移，两周后测量的永久阈值位移为40 dB，主要是外部hc的损失。将比较在先前的体外筛选中确定最有效的前四种抗氧化剂分子的三种剂量的淋巴周围递送的效果。实验方案二：体外自噬筛选采用Pou4f3/GFP转基因小鼠，其中hc选择性表达绿色荧光蛋白（GFP）。Corti的器官将被显微解剖，对AGs相对不敏感的根尖匝将被丢弃。基部和中间的轮生将被分成短节或微外植体。通过靶向筛选，我们将评估screen - well Autophagy文库，使用与先前体外氧化还原筛选相同的检测方法，以识别具有影响ags诱导HC损伤能力的化合物。

Demographic development outlines an increasing importance for diseases of the elderly, such as sensorineural hearing loss (SNHL) (>65 years up to 50%). Prevention is not currently possible for SNHL induced by most causes such as drugs, genetics or aging. Noise-induced hearing loss can be prevented with ear plugs or protectors, but they are frequently not worn when necessary. SNHL is difficult to treat, since it creates both loss of sensitivity and distortion of perceived sounds. This is partly due to loss of frequency specifity associated with outer hair cell (HC) loss, but also to loss of afferent synapses on the inner HCs, that may not be reflected in loss of hearing sensitivity (Kujawa and Liberman, 2015). Hearing aids increase sensitivity, but typically do not overcome sound distortion (Silverman, 1998). New methods for SNHL prevention and treatment are urgently needed. Prior research using in vitro intervention has characterized a number of pathways that contribute to HC damage and loss (Battaglia, 2003; Brand, 2015) or influence HC regeneration (Masuda, 2006). This knowledge base provides opportunities for pharmacological intervention to protect HCs. The submitted project deals with the evaluation of anti-oxidant and autophagy compounds, which may prevent and protect HCs against aminoglycoside (AG)-induced hair cell loss. Aim:The short-term objective is to determineI) relative potential of antioxidants to protect HCs and hearing from damage in vivo.II) relative potential of autophagy compounds to influence HC damage in vitro.The long term goal of this project is to increase prevention and improve treatment options for different types of SNHL such as AG-induced HC damage and noise- induced SNHL.Methods:Experimental Plan I:For the in vivo test of anti-oxidants, FVB mice will be employed, which do not display age-related hearing loss or increased noise sensitivity. Each mouse will be exposed to octave-band noise (12-24 kHz) at 105 dB SPL (sound pressure level) for 30 minutes. The exposure produces approximately 65 dB of threshold shift measured immediately after noise, and 40 dB of permanent threshold shift measured two weeks later, and loss primarily of outer HCs. The effects of perilymph delivery of three dosages of the top four antioxidant molecules, identified as most effective in a previous in vitro screen, will be compared.Experimental Plan II:For the in vitro autophagy screen, Pou4f3/GFP transgenic mice will be used, in which HCs selectively express green fluorescent protein (GFP). The organs of Corti will be micro-dissected and the apical turns, relatively insensitive to AGs, will be discarded. The basal and middle turns will be divided into short segments, or micro-explants. By means of targeted screening we will assess the Screen-Well Autophagy Library, using an identical assay to that used for the prior in vitro redox screen, to identify compounds with the ability to influence AGS-induced HC damage.