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. TUNELpositive 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.

• MeSH
• apoptóza MeSH
• barvení a značení metody   MeSH
• časové faktory MeSH
• financování organizované MeSH
• fragmentace DNA MeSH
• glukosa metabolismus   MeSH
• hyperglykemie metabolismus   patologie   MeSH
• hypoxie buňky MeSH
• koncové značení zlomů DNA in situ MeSH
• krysa rodu rattus MeSH
• kyslík metabolismus   MeSH
• novorozená zvířata MeSH
• orgánové kultury - kultivační techniky MeSH
• permeabilita buněčné membrány MeSH
• potkani Wistar MeSH
• vnější vláskové buňky metabolismus   patologie   MeSH
• vnitřní vláskové buňky metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• chemokin CXCL12 genetika   metabolismus   MeSH
• erythropoetin metabolismus   MeSH
• financování organizované MeSH
• hypoxie buňky MeSH
• krysa rodu rattus MeSH
• látky indukující angiogenezi metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• novorozená zvířata MeSH
• orgánové kultury - kultivační techniky MeSH
• počet buněk MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani Wistar MeSH
• receptory CXCR4 genetika   metabolismus   MeSH
• receptory erythropoetinu genetika   metabolismus   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• techniky in vitro MeSH
• vaskulární endoteliální růstový faktor A genetika   metabolismus   MeSH
• viabilita buněk MeSH
• vláskové buňky MeSH
• vnitřní ucho cytologie   metabolismus   zranění   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

An important mechanism underlying cochlear hair cell (HC) susceptibility to hypoxia/ischemia is the influx of Ca2+. Two main ATP-dependent mechanisms contribute to maintaining low Ca2+ levels: uptake of Ca2+ into intracellular stores via smooth endoplasmic reticulum calcium ATPase (SERCA) and extrusion of Ca2+ via plasma membrane calcium ATPase (PMCA). The effects of the SERCA inhibitors thapsigargin (10 nM-10 µM) and cyclopiazonic acid (CPA; 10-50 µM) and of the PMCA blockers eosin (1.5-10 µM) and o-vanadate (1-5 mM) on inner and outer hair cells (IHCs/OHCs) were examined in normoxia and ischemia using an in vitro model of the newborn rat cochlea. Exposure of the cultures to ischemia resulted in a significant loss of HCs. Thapsigargin and CPA had no effect. Eosin decreased the numbers of IHCs and OHCs by up to 25 % in normoxia and significantly aggravated the ischemia-induced damage to IHCs at 5 and 10 µM and to OHCs at 10 µM. o-Vanadate had no effect on IHC and OHC counts in normoxia, but aggravated the ischemiainduced HC loss in a dose-dependent manner. The effects of eosin and o-vanadate indicate that PMCA has an important role to play in protecting the HCs from ischemic cell death.

• Klíčová slova
• Calcium, Organ of Corti, Ischemia, PMCA, Rat,
• MeSH
• ATPasy přenášející vápník přes plazmatickou membránu antagonisté a inhibitory   MeSH
• Cortiho orgán cytologie   fyziologie   MeSH
• eosin farmakologie   MeSH
• financování organizované MeSH
• inhibitory enzymů farmakologie   MeSH
• ischemie enzymologie   MeSH
• krysa rodu rattus MeSH
• novorozená zvířata MeSH
• perilymfa fyziologie   MeSH
• potkani Wistar MeSH
• sarkoplazmatická Ca2+-ATPáza antagonisté a inhibitory   MeSH
• vanadáty farmakologie   MeSH
• vápník metabolismus   MeSH
• viabilita buněk fyziologie   MeSH
• vnější vláskové buňky účinky léků   MeSH
• vnitřní vláskové buňky účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH