Molecular Biology Research Laboratory, Department of Otorhinolaryngology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Physiol Res. 2009;58(6):895-902. doi: 10.33549/physiolres.931466. Epub 2008 Dec 17.
Ischemia can contribute to the inner ear pathology and hearing loss. To determine the susceptibility of inner and outer hair cells (IHCs/OHCs) to ischemic and post-ischemic period, we used organotypic cultures of the organ of Corti isolated from P3 rats as an in vitro model of inner ear ischemia (oxygen-glucose deprivation, OGD). We identified the hair cells (HCs) by phalloidin staining. The cells with damaged cellular membrane integrity were identified by propidium iodide (PI)-exclusion assay. The cells with fragmented chromosomal DNA were detected by TUNEL assay. Organotypic cultures were subjected to a mild (3 h duration) or severe (4 h duration) OGD, followed by a recovery period of 21 h and 20 h, respectively. Mild OGD induced a loss of 10-20% HCs, whereas severe OGD induced loss of 35% HCs. We confirmed that OHCs are less vulnerable to OGD than IHCs. Of all missing OHCs, 80-90% was lost during the OGD period and 10-20% during the recovery period. In contrast, the loss of IHCs was equal during both experimental periods. The OGD period was mainly associated with PI-positive nuclei. TUNEL-positive nuclei were a minor fraction during the OGD period and increased during the recovery period, indicating the progression of DNA fragmentation. Our results implicate a differential susceptibility of IHCs and OHCs during and after ischemia-like insult, which may be of therapeutic consequence.
缺血可导致内耳病理学和听力损失。为了确定内毛细胞 (IHC) 和外毛细胞 (OHC) 对内耳缺血和缺血后时期的易感性，我们使用从 P3 大鼠分离的耳蜗器官型培养物作为内耳缺血 (氧葡萄糖剥夺，OGD) 的体外模型。我们通过鬼笔环肽染色鉴定毛细胞 (HC)。通过碘化丙啶 (PI) 排除测定鉴定细胞膜完整性受损的细胞。通过 TUNEL 测定检测碎片化的染色体 DNA。将器官型培养物进行轻度 (3 小时持续时间) 或重度 (4 小时持续时间) OGD，然后分别进行 21 小时和 20 小时的恢复期。轻度 OGD 导致 10-20%的 HC 丧失，而重度 OGD 导致 35%的 HC 丧失。我们证实 OHC 比 IHC 对 OGD 的敏感性较低。在所有缺失的 OHC 中，80-90%在 OGD 期间丢失，10-20%在恢复期丢失。相比之下，IHC 的丢失在两个实验期间相等。OGD 期间主要与 PI 阳性核相关。TUNEL 阳性核在 OGD 期间是一个较小的部分，在恢复期增加，表明 DNA 片段化的进展。我们的结果表明，在内耳缺血样损伤期间和之后，IHC 和 OHC 的易感性存在差异，这可能具有治疗意义。
