Hair Cell Responses to Ototoxic Drugs

描述(申请人提供)：氨基糖苷类抗生素是与危及生命的细菌败血症作斗争所必需的。在美国，氨基糖苷类药物每年还会导致超过12万人永久性耳聋/平衡障碍和肾毒性，特别是对婴儿和早产儿。这项研究的长期目标是防止耳蜗胺糖苷类药物的摄取，从而防止耳毒性，以保护听觉功能。先前项目期间的进展显示，细胞对氨基糖苷类药物的摄取在体外可以被阻断，而非选择性阳离子通道可以增强氨基糖苷类药物的清除。在这个方案中，我们将研究氨基糖苷类药物是如何从血管系统，穿过血迷路屏障进入体内的耳蜗液和组织，并开发这种运输的策略，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：药物可以减少氨基糖苷类药物在耳蜗中的摄取和毒性。该项目的具体目标是：首先，通过确定药物如何改变模型细胞系和耳蜗外植体的细胞内环境来促进或抑制氨基糖苷的摄取和清除，来调节氨基糖苷在体外的摄取。我们将监测细胞内钙和pH水平、静息电位和膜电阻(目标1)。第二，使用氨基糖苷类摄取抑制剂在体内保存听觉功能和形态。我们将通过听性脑干反应测听、毛细胞形态的共聚焦显微镜和构建细胞耳蜗图来评估氨基糖苷类摄取抑制剂的有效性(目标2)。第三，确定氨基糖苷类药物在耳蜗内从血管到感觉毛细胞的途径。我们将使用耳蜗灌流技术，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摄取抑制剂通过一条或两条途径减少氨基糖苷类药物在耳蜗内的转运(目标3)。对减少氨基糖苷类药物进入耳蜗内和在耳蜗内转运的机制的识别将决定目前可用的药物是否可以在救命的氨基糖苷类药物治疗期间作为辅助疗法来保护听觉功能。此外，识别增加氨基糖苷摄取的细胞内机制(药物诱导或其他)为临床医生筛查患者先前存在的疾病和增加患者氨基糖苷中毒风险的药物提供了新的见解。了解氨基糖苷类抗生素如何通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引起的耳毒性至关重要。拟议的研究将使我们能够制定策略，防止氨基糖苷类药物进入内耳液，从而防止耳毒性后遗症，特别是终生耳聋、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.