Limits...
Ouabain-induced apoptosis in cochlear hair cells and spiral ganglion neurons in vitro.

Fu Y, Ding D, Wei L, Jiang H, Salvi R - Biomed Res Int (2013)

Bottom Line: Little is known about the ototoxic effects in vitro.The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway.The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China ; Center for Hearing and Deafness, University at Buffalo, Buffalo, NY 14214, USA.

ABSTRACT
Ouabain is a common tool to explore the pathophysiological changes in adult mammalian cochlea in vivo. In prior studies, locally administering ouabain via round window membrane demonstrated that the ototoxic effects of ouabain in vivo varied among mammalian species. Little is known about the ototoxic effects in vitro. Thus, we prepared cochlear organotypic cultures from postnatal day-3 rats and treated these cultures with ouabain at 50, 500, and 1000 μM for different time to elucidate the ototoxic effects of ouabain in vitro and to provide insights that could explain the comparative ototoxic effects of ouabain in vivo. Degeneration of cochlear hair cells and spiral ganglion neurons was evaluated by hair-cell staining and neurofilament labeling, respectively. Annexin V staining was used to detect apoptotic cells. A quantitative RT-PCR apoptosis-focused gene array determined changes in apoptosis-related genes. The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway. The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.

Show MeSH

Related in: MedlinePlus

Apoptotic labeling in HCs and SGNs after ouabain treatment in cochlear organotypic cultures. (a) There was no evidence of positive Annexin V labeling in HCs 24 h after 50 μM ouabain treatment, although the stereocilia were disarrayed on the HCs. (b) In the same culture conditions as (a), apoptotic signals were detected in some SGNs (arrow). (c) Annexin V positive labeling was seen in HCs and the supporting cells in the region of the lateral sulcus 24 h after 500 μM ouabain treatment. (d) In the same culture conditions as (c), with 500 μM ouabain, most SGNs presented with positive Annexin V expression (arrows) showing apoptotic features (fluorescence microscope at a magnification of ×600). (e) At 24 h after 50 μM ouabain treatment, a quarter of the SGNs exhibited FITC-Annexin V positive labeling. (f) At 24 h after 500 μM ouabain treatment, the FITC-Annexin V positive labeling of SGNs was increased to 65%.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3818842&req=5

fig4: Apoptotic labeling in HCs and SGNs after ouabain treatment in cochlear organotypic cultures. (a) There was no evidence of positive Annexin V labeling in HCs 24 h after 50 μM ouabain treatment, although the stereocilia were disarrayed on the HCs. (b) In the same culture conditions as (a), apoptotic signals were detected in some SGNs (arrow). (c) Annexin V positive labeling was seen in HCs and the supporting cells in the region of the lateral sulcus 24 h after 500 μM ouabain treatment. (d) In the same culture conditions as (c), with 500 μM ouabain, most SGNs presented with positive Annexin V expression (arrows) showing apoptotic features (fluorescence microscope at a magnification of ×600). (e) At 24 h after 50 μM ouabain treatment, a quarter of the SGNs exhibited FITC-Annexin V positive labeling. (f) At 24 h after 500 μM ouabain treatment, the FITC-Annexin V positive labeling of SGNs was increased to 65%.

Mentions: Considering that cell shrinkage and condensation were detected by regular staining with neurofilament-200 immunolabeling in ouabain-treated SGNs, we used FITC-Annexin V techniques for the detection of apoptosis in cochlear HCs and SGNs. At 24 h after 50 μM ouabain treatment, the FITC-Annexin V labeling was negative in the cochlear HCs, despite disarrayed stereocilia on the HCs that were detected at this time (Figure 4(a)). However, the positive apoptotic signals (green fluorescence) were found in the condensed or fragmented SGNs (Figure 4(b)) 24 h after 50 μM ouabain treatment. At 24 h after 500 μM ouabain treatment, despite all HCs being present in the cochlea apical turn (Figure 2(d)), the F ITC-Annexin V labeled membranes had significantly increased in most HCs in the apical region and in some supporting cells in the outer sulcus region (Figure 4(c)). Most SGNs had greatly shrunk and showed obvious positive expression of Annexin V 24 h after 500 μM ouabain treatment (Figure 4(d)). By quantitative observations, 35 positive FITC-Annexin V labeled SGNs were detected from 140 SGNs 24 h after 50 μM ouaba in treatment, and the positive labeling was equivalent to 25% observed SGNs (Figure 4(e)). It is worthwhile to note that when the concentration of ouabain increased to 500 μM, 89 FITC-Annexin V positively labeled SGNs were found in 137 SGNs 24 h after ouabain treatment, an observation that equaled the 65% observed SGNs (see Figure 5F). By statistical analysis, a significant increase in the number of positively stained FITC-Annexin V labeled SGNs was detected 24 h after 50 μM or 500 μM ouabain treatment (One-way ANOVA, F = 301.6, P < 0.0001, and Newman-Keuls post hoc analysis, P < 0.05) as compared with normal controls. There was also a significant difference between the 50 μM ouabain treated group and the 500 μM ouabain treated group (Newman-Keuls post hoc analysis, P < 0.05). The Annexin V positive response in cochlear HCs and SGNs suggested that ouabain-induced cell destruction was involved in cell apoptosis. In addition, the apoptotic signals appeared in the SGNs earlier than in the cochlear HCs.


Ouabain-induced apoptosis in cochlear hair cells and spiral ganglion neurons in vitro.

Fu Y, Ding D, Wei L, Jiang H, Salvi R - Biomed Res Int (2013)

Apoptotic labeling in HCs and SGNs after ouabain treatment in cochlear organotypic cultures. (a) There was no evidence of positive Annexin V labeling in HCs 24 h after 50 μM ouabain treatment, although the stereocilia were disarrayed on the HCs. (b) In the same culture conditions as (a), apoptotic signals were detected in some SGNs (arrow). (c) Annexin V positive labeling was seen in HCs and the supporting cells in the region of the lateral sulcus 24 h after 500 μM ouabain treatment. (d) In the same culture conditions as (c), with 500 μM ouabain, most SGNs presented with positive Annexin V expression (arrows) showing apoptotic features (fluorescence microscope at a magnification of ×600). (e) At 24 h after 50 μM ouabain treatment, a quarter of the SGNs exhibited FITC-Annexin V positive labeling. (f) At 24 h after 500 μM ouabain treatment, the FITC-Annexin V positive labeling of SGNs was increased to 65%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818842&req=5

fig4: Apoptotic labeling in HCs and SGNs after ouabain treatment in cochlear organotypic cultures. (a) There was no evidence of positive Annexin V labeling in HCs 24 h after 50 μM ouabain treatment, although the stereocilia were disarrayed on the HCs. (b) In the same culture conditions as (a), apoptotic signals were detected in some SGNs (arrow). (c) Annexin V positive labeling was seen in HCs and the supporting cells in the region of the lateral sulcus 24 h after 500 μM ouabain treatment. (d) In the same culture conditions as (c), with 500 μM ouabain, most SGNs presented with positive Annexin V expression (arrows) showing apoptotic features (fluorescence microscope at a magnification of ×600). (e) At 24 h after 50 μM ouabain treatment, a quarter of the SGNs exhibited FITC-Annexin V positive labeling. (f) At 24 h after 500 μM ouabain treatment, the FITC-Annexin V positive labeling of SGNs was increased to 65%.
Mentions: Considering that cell shrinkage and condensation were detected by regular staining with neurofilament-200 immunolabeling in ouabain-treated SGNs, we used FITC-Annexin V techniques for the detection of apoptosis in cochlear HCs and SGNs. At 24 h after 50 μM ouabain treatment, the FITC-Annexin V labeling was negative in the cochlear HCs, despite disarrayed stereocilia on the HCs that were detected at this time (Figure 4(a)). However, the positive apoptotic signals (green fluorescence) were found in the condensed or fragmented SGNs (Figure 4(b)) 24 h after 50 μM ouabain treatment. At 24 h after 500 μM ouabain treatment, despite all HCs being present in the cochlea apical turn (Figure 2(d)), the F ITC-Annexin V labeled membranes had significantly increased in most HCs in the apical region and in some supporting cells in the outer sulcus region (Figure 4(c)). Most SGNs had greatly shrunk and showed obvious positive expression of Annexin V 24 h after 500 μM ouabain treatment (Figure 4(d)). By quantitative observations, 35 positive FITC-Annexin V labeled SGNs were detected from 140 SGNs 24 h after 50 μM ouaba in treatment, and the positive labeling was equivalent to 25% observed SGNs (Figure 4(e)). It is worthwhile to note that when the concentration of ouabain increased to 500 μM, 89 FITC-Annexin V positively labeled SGNs were found in 137 SGNs 24 h after ouabain treatment, an observation that equaled the 65% observed SGNs (see Figure 5F). By statistical analysis, a significant increase in the number of positively stained FITC-Annexin V labeled SGNs was detected 24 h after 50 μM or 500 μM ouabain treatment (One-way ANOVA, F = 301.6, P < 0.0001, and Newman-Keuls post hoc analysis, P < 0.05) as compared with normal controls. There was also a significant difference between the 50 μM ouabain treated group and the 500 μM ouabain treated group (Newman-Keuls post hoc analysis, P < 0.05). The Annexin V positive response in cochlear HCs and SGNs suggested that ouabain-induced cell destruction was involved in cell apoptosis. In addition, the apoptotic signals appeared in the SGNs earlier than in the cochlear HCs.

Bottom Line: Little is known about the ototoxic effects in vitro.The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway.The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China ; Center for Hearing and Deafness, University at Buffalo, Buffalo, NY 14214, USA.

ABSTRACT
Ouabain is a common tool to explore the pathophysiological changes in adult mammalian cochlea in vivo. In prior studies, locally administering ouabain via round window membrane demonstrated that the ototoxic effects of ouabain in vivo varied among mammalian species. Little is known about the ototoxic effects in vitro. Thus, we prepared cochlear organotypic cultures from postnatal day-3 rats and treated these cultures with ouabain at 50, 500, and 1000 μM for different time to elucidate the ototoxic effects of ouabain in vitro and to provide insights that could explain the comparative ototoxic effects of ouabain in vivo. Degeneration of cochlear hair cells and spiral ganglion neurons was evaluated by hair-cell staining and neurofilament labeling, respectively. Annexin V staining was used to detect apoptotic cells. A quantitative RT-PCR apoptosis-focused gene array determined changes in apoptosis-related genes. The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway. The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.

Show MeSH
Related in: MedlinePlus