Precise and efficient endocytosis is critical for sustained neurotransmission during continuous neuronal activity. Endocytosis is a prerequisite for maintaining the auditory function. However, the differences between the patterns of endocytosis in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) remain unclear. Both IHCs and OHCs were obtained from adult C57 mice. Patterns of endocytosis in cells were estimated by analyzing the uptake of FM1-43, a fluorescent. The observations were made using live confocal imaging, fluorescence intensities were calculated statistically. Results revealed the details about following phenomenon, i) sites of entry: the FM1-43 dye was found to enter IHC at the apical area initially, the additional sites of entry were then found at basolateral membrane of the cells, The entry of the dye into OHCs initially appeared to be occurring around whole apical membranes area, which then diffused towards the other membrane surface of the cells, ii) capacity of endocytosis: fluorescence intensity in IHCs showed significantly higher than that of OHCs (P<0.01). We have found different patterns of endocytosis between IHCs and OHCs, this indicated functional distinctions between them. Moreover, FM1-43 dye can be potentially used as an indicator of the functional loss or repair of cochlear hair cells.
- MeSH
- biologický transport fyziologie MeSH
- endocytóza fyziologie MeSH
- fluorescenční barviva analýza metabolismus MeSH
- kvartérní amoniové sloučeniny analýza metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- orgánové kultury - kultivační techniky MeSH
- pyridinové sloučeniny analýza metabolismus MeSH
- sluchové evokované potenciály fyziologie MeSH
- vnější vláskové buňky chemie metabolismus MeSH
- vnitřní vláskové buňky chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva MeSH
- FM1 43 MeSH Prohlížeč
- kvartérní amoniové sloučeniny MeSH
- pyridinové sloučeniny MeSH
The development, maturation, and maintenance of the inner ear are governed by temporal and spatial expression cascades of transcription factors that form a gene regulatory network. ISLET1 (ISL1) may be one of the major players in this cascade, and in order to study its role in the regulation of inner ear development, we produced a transgenic mouse overexpressing Isl1 under the Pax2 promoter. Pax2-regulated ISL1 overexpression increases the embryonic ISL1(+) domain and induces accelerated nerve fiber extension and branching in E12.5 embryos. Despite these gains in early development, the overexpression of ISL1 impairs the maintenance and function of hair cells of the organ of Corti. Mutant mice exhibit hyperactivity, circling behavior, and progressive age-related decline in hearing functions, which is reflected in reduced otoacoustic emissions (DPOAEs) followed by elevated hearing thresholds. The reduction of the amplitude of DPOAEs in transgenic mice was first detected at 1 month of age. By 6-9 months of age, DPOAEs completely disappeared, suggesting a functional inefficiency of outer hair cells (OHCs). The timing of DPOAE reduction coincides with the onset of the deterioration of cochlear efferent terminals. In contrast to these effects on efferents, we only found a moderate loss of OHCs and spiral ganglion neurons. For the first time, our results show that the genetic alteration of the medial olivocochlear (MOC) efferent system induces an early onset of age-related hearing loss. Thus, the neurodegeneration of the MOC system could be a contributing factor to the pathology of age-related hearing loss.
- Klíčová slova
- Age-related hearing loss, Islet1 transcription factor, Medial olivocochlear efferent system, Outer hair cells, Transgenic mouse,
- MeSH
- analýza přežití MeSH
- embryo savčí metabolismus patologie MeSH
- ganglion spirale patologie MeSH
- kochlea inervace patologie patofyziologie MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární motory metabolismus MeSH
- myši transgenní MeSH
- nedoslýchavost patologie patofyziologie MeSH
- neurony eferentní MeSH
- otoakustické emise spontánní MeSH
- počet buněk MeSH
- proteiny s homeodoménou LIM metabolismus MeSH
- sluchový práh MeSH
- stárnutí patologie MeSH
- transkripční faktor PAX2 metabolismus MeSH
- transkripční faktory metabolismus MeSH
- vnější vláskové buňky patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- insulin gene enhancer binding protein Isl-1 MeSH Prohlížeč
- messenger RNA MeSH
- molekulární motory MeSH
- Pax2 protein, mouse MeSH Prohlížeč
- Pres protein, mouse MeSH Prohlížeč
- proteiny s homeodoménou LIM MeSH
- transkripční faktor PAX2 MeSH
- transkripční faktory MeSH
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.
- MeSH
- apoptóza MeSH
- barvení a značení metody MeSH
- časové faktory 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
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- glukosa MeSH
- kyslík MeSH
An important mechanism underlying cochlear hair cell (HC) susceptibility to hypoxia/ischemia is the influx of Ca(2+). Two main ATP-dependent mechanisms contribute to maintaining low Ca(2+) levels: uptake of Ca(2+) into intracellular stores via smooth endoplasmic reticulum calcium ATPase (SERCA) and extrusion of Ca(2+) via plasma membrane calcium ATPase (PMCA). The effects of the SERCA inhibitors thapsigargin (10 nM-10 microM) and cyclopiazonic acid (CPA; 10-50 microM) and of the PMCA blockers eosin (1.5-10 microM) 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 microM and to OHCs at 10 microM. o-Vanadate had no effect on IHC and OHC counts in normoxia, but aggravated the ischemia-induced 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.
- MeSH
- ATPasy přenášející vápník přes plazmatickou membránu antagonisté a inhibitory MeSH
- Cortiho orgán cytologie fyziologie MeSH
- eosin farmakologie 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
- techniky in vitro 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
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ATPasy přenášející vápník přes plazmatickou membránu MeSH
- eosin MeSH
- inhibitory enzymů MeSH
- sarkoplazmatická Ca2+-ATPáza MeSH
- vanadáty MeSH
- vápník MeSH
