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.

Molecular Biology Research Laboratory Department of Otorhinolaryngology Charité Universitätsmedizin Berlin Berlin Germany

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