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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.

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Representative cochleograms that show the percentage of missing IHCs and OHCs following ouabain treatment. No evidence of missing HCs was found ((a) and (b)). Panels (a) and (b) show the mean cochleograms (average of N = 5) at 24 h and 48 h following 50 μM ouabain treatment, respectively. However, approximately 20% of IHCs were missing at the basal turn of the cochlea (c), which shows the mean cochleogram at 72 h after treatment with 50 μM ouabain. (d), (e), and (f) show the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 500 μM ouabain, respectively. Note that the loss in HCs progresses from the base to the apex in a time-dependent manner. In addition, loss in IHCs was greater than OHCs. (g), (h), and (i) represent the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 1000 μM ouabain. At 24 h after 1000 μM ouabain treatment, approximately 85% of IHCs and 50% of OHCs were missing (g). When the culture period was extended to 48 h, most IHCs and OHCs were no longer present, with the notable exception of the tip of the apex (h). Treatment with 1000 μM ouabain for 72 h destroyed 100% of HCs (i).
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fig2: Representative cochleograms that show the percentage of missing IHCs and OHCs following ouabain treatment. No evidence of missing HCs was found ((a) and (b)). Panels (a) and (b) show the mean cochleograms (average of N = 5) at 24 h and 48 h following 50 μM ouabain treatment, respectively. However, approximately 20% of IHCs were missing at the basal turn of the cochlea (c), which shows the mean cochleogram at 72 h after treatment with 50 μM ouabain. (d), (e), and (f) show the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 500 μM ouabain, respectively. Note that the loss in HCs progresses from the base to the apex in a time-dependent manner. In addition, loss in IHCs was greater than OHCs. (g), (h), and (i) represent the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 1000 μM ouabain. At 24 h after 1000 μM ouabain treatment, approximately 85% of IHCs and 50% of OHCs were missing (g). When the culture period was extended to 48 h, most IHCs and OHCs were no longer present, with the notable exception of the tip of the apex (h). Treatment with 1000 μM ouabain for 72 h destroyed 100% of HCs (i).

Mentions: The cochlear HCs were treated with 50 μM ouabain for 24 h and 48 h presented as normal appearance (Figures 2(a) and 2(b)). However, 72 h after 50 μM ouabain treatment, about 20% of the IHCs were missing in the basal turn, but all of the OHCs were found to be intact (Figure 2(c)). Treatment with 500 μM ouabain for 24 h destroyed more than 50% of the IHCs and 20% of the OHCs. The loss of HCs typically began at the basal turn and migrated towards the apical turn of the cochlea (Figure 2(d)). When the duration of the culture was extended to 48 h, a dose of 500 μM ouabain destroyed 70% of the IHCs and 50% of the OHCs (Figure 2(e)). Extending the duration of the culture to 72 h, treatment with 500 μM ouabain destroyed 90% of the IHCs and 80% of the OHCs (Figure 2(f)). Increasing the concentration of ouabain to 1000 μM resulted in the destruction of more than 80% of the IHCs and about 50% of the OHCs 24 h after ouabain treatment (Figure 2(g)). By contrast, 90%–100% of cochlear HCs were missing 48 h and 72 h after 1000 μM ouabain treatment, respectively (Figures 2(h) and 2(i)). The graphic panels of cochleograms (Figure 2) clearly showed that ouabain-induced destruction of cochlear HCs expanded from the basal turn to the apical turn, and that destruction of IHCs was more severe and occurred earlier than that seen with OHCs.


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)

Representative cochleograms that show the percentage of missing IHCs and OHCs following ouabain treatment. No evidence of missing HCs was found ((a) and (b)). Panels (a) and (b) show the mean cochleograms (average of N = 5) at 24 h and 48 h following 50 μM ouabain treatment, respectively. However, approximately 20% of IHCs were missing at the basal turn of the cochlea (c), which shows the mean cochleogram at 72 h after treatment with 50 μM ouabain. (d), (e), and (f) show the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 500 μM ouabain, respectively. Note that the loss in HCs progresses from the base to the apex in a time-dependent manner. In addition, loss in IHCs was greater than OHCs. (g), (h), and (i) represent the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 1000 μM ouabain. At 24 h after 1000 μM ouabain treatment, approximately 85% of IHCs and 50% of OHCs were missing (g). When the culture period was extended to 48 h, most IHCs and OHCs were no longer present, with the notable exception of the tip of the apex (h). Treatment with 1000 μM ouabain for 72 h destroyed 100% of HCs (i).
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Related In: Results  -  Collection

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fig2: Representative cochleograms that show the percentage of missing IHCs and OHCs following ouabain treatment. No evidence of missing HCs was found ((a) and (b)). Panels (a) and (b) show the mean cochleograms (average of N = 5) at 24 h and 48 h following 50 μM ouabain treatment, respectively. However, approximately 20% of IHCs were missing at the basal turn of the cochlea (c), which shows the mean cochleogram at 72 h after treatment with 50 μM ouabain. (d), (e), and (f) show the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 500 μM ouabain, respectively. Note that the loss in HCs progresses from the base to the apex in a time-dependent manner. In addition, loss in IHCs was greater than OHCs. (g), (h), and (i) represent the mean cochleograms at 24 h, 48 h, and 72 h after treatment with 1000 μM ouabain. At 24 h after 1000 μM ouabain treatment, approximately 85% of IHCs and 50% of OHCs were missing (g). When the culture period was extended to 48 h, most IHCs and OHCs were no longer present, with the notable exception of the tip of the apex (h). Treatment with 1000 μM ouabain for 72 h destroyed 100% of HCs (i).
Mentions: The cochlear HCs were treated with 50 μM ouabain for 24 h and 48 h presented as normal appearance (Figures 2(a) and 2(b)). However, 72 h after 50 μM ouabain treatment, about 20% of the IHCs were missing in the basal turn, but all of the OHCs were found to be intact (Figure 2(c)). Treatment with 500 μM ouabain for 24 h destroyed more than 50% of the IHCs and 20% of the OHCs. The loss of HCs typically began at the basal turn and migrated towards the apical turn of the cochlea (Figure 2(d)). When the duration of the culture was extended to 48 h, a dose of 500 μM ouabain destroyed 70% of the IHCs and 50% of the OHCs (Figure 2(e)). Extending the duration of the culture to 72 h, treatment with 500 μM ouabain destroyed 90% of the IHCs and 80% of the OHCs (Figure 2(f)). Increasing the concentration of ouabain to 1000 μM resulted in the destruction of more than 80% of the IHCs and about 50% of the OHCs 24 h after ouabain treatment (Figure 2(g)). By contrast, 90%–100% of cochlear HCs were missing 48 h and 72 h after 1000 μM ouabain treatment, respectively (Figures 2(h) and 2(i)). The graphic panels of cochleograms (Figure 2) clearly showed that ouabain-induced destruction of cochlear HCs expanded from the basal turn to the apical turn, and that destruction of IHCs was more severe and occurred earlier than that seen with OHCs.

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