Hair Cell Responses to Ototoxic Drugs

描述（由申请人提供）：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。在美国，氨基糖苷类药物每年也会导致超过120，000例永久性耳聋/平衡障碍和肾毒性，尤其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，氨基糖苷类药物的细胞摄取可以在体外被阻断，非选择性阳离子通道可以增强氨基糖苷类药物的清除。在这个提议中，我们将研究氨基糖苷类药物如何从血管系统中转运，穿过血迷路屏障进入体内耳蜗液和组织，并制定策略，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：药物可以减少耳蜗中氨基糖苷类药物的摄取和毒性。该项目的具体目标是：首先，通过确定药理学试剂如何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药物的摄取和清除，来调节氨基糖苷类药物的体外摄取。我们将监测细胞内钙和pH水平、静息电位和膜电阻（目的1）。第二，使用氨基糖苷类摄取抑制剂在体内保护听觉功能和形态。我们将使用听性脑干反应测听、毛细胞形态学共聚焦显微镜检查和细胞耳蜗图构建来评估氨基糖苷类摄取抑制剂的疗效（目的2）。第三，确定氨基糖苷类药物从血管系统到感觉毛细胞的耳蜗内途径。我们将使用耳蜗灌注技术，并采集耳蜗液样本，以确定氨基糖苷类药物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通过一种或两种途径减少氨基糖苷类的耳蜗内转运（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将确定目前可用的药物是否可用作辅助治疗剂，以在挽救生命的氨基糖苷类药物治疗期间保护听觉功能。此外，识别增强氨基糖苷类摄取的细胞内机制（药物诱导或其他）为临床医生提供了新的见解，以筛选患者的既存疾病和药物，提高患者的氨基糖苷类毒性风险。了解氨基糖苷类抗生素如何穿过血迷路屏障进入内耳液的机制对于预防氨基糖苷类抗生素诱导的耳毒性至关重要。拟议的研究将使我们能够制定战略，以防止氨基糖苷类药物进入内耳液，从而耳毒性后遗症，特别是终身耳聋，耳鸣和前庭功能障碍。

DESCRIPTION (provided by applicant): 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. 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.