Hair Cell Responses to Ototoxic Drugs

摘要 氨基糖苷类抗生素对于对抗危及生命的细菌性败血症至关重要。氨基糖苷 也会导致超过120，000人的永久性耳聋/平衡障碍和肾毒性 每年在美国，特别是在婴儿和早产儿中。这项研究的长期目标是 防止耳蜗摄取氨基糖苷类药物，从而防止耳毒性，保护听觉功能。 上一个项目期间的进展表明，细胞对氨基糖苷类的摄取可以是 在体外阻断，非选择性阳离子通道增强氨基糖苷类的清除。在 在这个提议中，我们将研究氨基糖苷类药物是如何从血管系统转运到血管系统的。 血迷路屏障进入耳蜗液体和组织在体内，并制定策略，这种运输 以防止氨基糖苷类药物诱导的内耳感觉毛细胞死亡。我们的工作假设是： 药理学试剂可以减少耳蜗中的氨基糖苷类摄取和毒性。 该项目的具体目标是： 首先，通过确定药理学药物如何调节氨基糖苷类药物的体外摄取， 改变模型细胞系和耳蜗外植体的细胞内环境，以增强或 抑制氨基糖苷类摄取和清除。我们将监测细胞内钙和pH值 水平，静息电位和膜电阻（目标1）。 第二，为了保护听觉功能和形态在体内使用抑制剂， 氨基糖苷类摄取我们将评估氨基糖苷类摄取抑制剂的疗效 应用听性脑干反应测听、共聚焦显微镜观察毛细胞 形态学，并构建细胞耳蜗图（目的2）。 第三，确定氨基糖苷类药物从血管到耳蜗的耳蜗内途径。 感觉毛细胞我们将使用耳蜗灌注技术， 以确定氨基糖苷类是否从内淋巴或外淋巴进入毛细胞。然后我们将 验证氨基糖苷类摄取抑制剂减少耳蜗内转运 通过一种或两种途径使用氨基糖苷类药物（目的3）。 确定减少氨基糖苷类药物转运进入耳蜗和耳蜗内的机制， 确定目前可用的药物是否可用作联合治疗剂以保护听觉功能 氨基糖苷类抗生素治疗期间发生的此外，确定细胞内机制（药物诱导的 或其它）的新的方法为临床医生筛选患者提供了新的见解 用于增加患者氨基糖苷类药物毒性风险的既存疾病和药物。叙事 了解氨基糖苷类抗生素如何穿过血液迷宫的机制 屏障进入内耳液是至关重要的，在防止氨基糖苷类诱导的耳毒性。 拟议中的研究将使我们能够制定战略，防止 氨基糖苷类药物进入内耳液，因此耳毒性后遗症，特别是终身 耳聋、耳鸣和前庭缺陷。

ABSTRACT Aminoglycoside antibiotics are essential for battling life-threatening bacterial sepsis. Aminoglycosides also cause permanent deafness/balance disorders and nephrotoxicity in more than 120,000 individuals each year in the US, particularly in infants and premature babies. The long-term goal of this research is to prevent cochlear uptake of aminoglycosides and thus ototoxicity, to preserve auditory function. Progress in the previous project period revealed that the cellular uptake of aminoglycosides can be blocked in vitro, and that non-selective cation channels enhance the clearance of aminoglycosides. In this proposal, we will investigate how aminoglycosides are transported from the vasculature, across the blood-labyrinth barrier into the cochlear fluids and tissues in vivo, and develop strategies to this transport to prevent aminoglycoside-induced inner ear sensory hair cell death. Our working hypothesis is that: pharmacological agents can reduce aminoglycoside uptake and toxicity in the cochlea. The specific aims of this project are: First, to regulate aminoglycoside uptake in vitro, by identifying how pharmacological agents change the intra-cellular milieu of model cell lines and cochlear explants to enhance or inhibit aminoglycoside uptake and clearance. We will monitor intracellular calcium and pH levels, the resting potential and membrane resistance (Aim 1). Second, to preserve auditory function and morphology in vivo using inhibitors of aminoglycoside uptake. We will assess the efficacy of aminoglycoside-uptake inhibitors using auditory brainstem response audiometry, confocal microscopy of hair cell morphology, and constructing cytocochleograms (Aim 2). And thirdly, to identify the intra-cochlear route of aminoglycosides from the vasculature to the sensory hair cells. We will use cochlear perfusion techniques, and sample cochlear fluids to determine if aminoglycosides enter hair cells from endolymph or perilymph. We will then verify that aminoglycoside uptake-inhibitors reduce the intra-cochlear transport of aminoglycosides by one or both routes (Aim 3). Identification of mechanisms that reduce aminoglycoside transport into, and within, the cochlea will determine if currently-available drugs can be used as co-therapeutics to preserve auditory function during life-saving aminoglycoside therapy. In addition, identifying intracellular mechanisms (drug-induced or otherwise) that enhance aminoglycoside uptake provides new insight for clinicians to screen patients for pre-existing conditions and medications that elevate the risk of aminoglycoside toxicity in patients. NARRATIVE Understanding mechanisms of how aminoglycosides antibiotics cross the blood-labyrinth barrier to enter the inner ear fluids is crucial in preventing aminoglycoside-induced ototoxicity. The proposed research will enable us to develop strategies to prevent the entry of aminoglycosides into the inner ear fluids and therefore ototoxic sequelae, particularly life-long deafness, tinnitus and vestibular deficits.