Development of long-termed implants: In vivo and in vitro characterisation of the interactions of inner ear cells with platinum corrosion products in the context of cochlear implant stimulation

目前认为，植入人工耳蜗后电阻抗增加10 kΩ是由于电极的技术缺陷、诱发炎症过程和/或沿电极形成疤痕组织所致。然而，最近的研究报道，铂（Pt）电极接触表面的侵蚀是高阻抗的原因。此外，在电极-神经界面和内耳组织中可以证明Pt氧化物、Pt蛋白复合物和特定Pt的存在。虽然离子Pt的细胞毒性已被充分证明，但特定Pt的毒性潜能不仅取决于其颗粒大小、浓度和细胞类型，还取决于其内化后对溶酶体中水解酶氧化的敏感性。据推测，铂粒子的氧化产物可能与DNA形成络合物并随后诱导DNA链断裂。事实上，Pt腐蚀的长期影响，其腐蚀产物的组成以及它们与神经组织的相互作用尚不清楚。特别是，Pt腐蚀产物诱导细胞死亡的分子机制需要明确，以保护神经元组织免受氧化应激诱导的细胞损伤，并制造防止大量腐蚀产物释放的电极。目前的研究表明，顺铂介导的耳毒性不仅可以通过凋亡途径触发，还可以通过坏死和自噬途径触发。然而，Pt腐蚀产物尚未被确定为坏死坏死或自噬相关信号通路的诱导剂。因此，由Pt腐蚀产物诱导的细胞死亡相关信号通路的详细体外分析是本文所述项目的重点。为此，将建立大鼠原代螺旋神经节神经元、小鼠Corti细胞永生化器官HEI-OC1细胞系和大鼠Corti器官器官型培养的体外细胞培养模型，以表征和量化Pt纳米颗粒和Pt腐蚀产物给药后的细胞毒性作用。因此，电刺激Pt导线将成为腐蚀产物的来源。测定诱导50%细胞培养物细胞死亡的Pt浓度是研究Pt触发氧化应激和随后细胞死亡的分子机制的基础。获得的数据将允许检查凋亡/坏死/自噬信号交叉点的潜在抑制剂/诱导剂，以防止Pt暴露后严重的细胞损伤。因此，体外数据必须在体内以大鼠为动物模型进行评估。

So far, it was supposed that an increase of electrical impedance extending 10 kΩ following cochlear implant insertion was due to technical defects of the electrode, induction of inflammatory processes and/or formation of scar tissue along the electrode. Recent studies, however, reported eroded surfaces of the platinum (Pt) electrode contacts as the reason for high impedances. Furthermore, the presence of Pt oxides, Pt protein complexes and particular Pt could be demonstrated at the electrode-nerve-interface and in the inner ear tissues. Whereas the cytotoxicity of of ionic Pt is well documented, the toxic potential of particular Pt depends not only on the particle size, concentration and cell type, but also on its susceptibility to oxidation by hydrolytic enzymes in the lysosomes following internalization. It is supposed that the oxidation product of the Pt particles may form complexes with DNA and subsequently induce DNA strand breakings. In fact, long-term effects of Pt corrosion, compositions of its corrosion products as well as their interactions with neural tissues are not well understood. Especially, molecular mechanisms of cell death induced by Pt corrosion products need to be clarified to enable protection of neuronal tissues against oxidative stress induced cell damage and manufacturing of electrodes preventing extensive corrosion product release.Current studies demonstrate that cisplatin mediated ototoxicity is not only triggered by apoptotic pathways, but also via necroptosis and autophagy. However, Pt corrosion products have not been identified as inductors for necroptosis or autophagy related signal pathways. Thus, detailed in vitro-analysis of cell death related signal pathways induced by the Pt corrosion products are in the focus of the project described herein. For this, in vitro cell culture models of primary rat spiral ganglion neurons, HEI-OC1 cell line of the immortalized mouse organ of Corti cells and organotypic cultivation of the rat organ of Corti will be established to characterize and quantify cytotoxic effects following administration of Pt nanoparticles and Pt corrosion products. Hereby, electrical stimulation of Pt wires will be used as source of corrosion products. Determination of the Pt concentration inducing cell death of 50 % of the cell culture constitutes the basis of the examination of the molecular mechanisms of Pt triggred oxidative stress and subsequent cell death. The obtained data will allow the examination of potential inhibitors/inductors of the cross points in apoptosis/necroptosis/autophagy signaling to prevent severe cell damage following Pt exposure. Consequently, the in vitro data has to be evaluated in vivo using rats as animal model.