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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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Organotypic cultures from the middle cochleae 72 h after ouabain treatment. The HCs were stained with Alexa Fluor 488-phalloidin. Auditory nerve fibers and SGNs were labeled with an antibody directed against neurofilament 200 and an Alexa Fluor 555 conjugated secondary antibody. (a) Cochlear organotypic culture from postnatal day 3 rat that was cultured for 72 h under normal conditions. (b) Rat cochlear cultures treated with 50 μM ouabain for 72 h. Note that all HCs were intact, whereas the density of the auditory nerve fibers was greatly reduced as compared with the normal control in (a). (c) Cochlear explants were treated with 500 μM ouabain for 72 h. Many SGNs and nerve fibers degenerated and significant percentage's of HCs were missing. (d) Cochlear cultures treated with 1 mM ouabain for 72 h resulted in complete destruction of both SGNs and HCs in the cochlea (Fluorescence microscope ×200).
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fig1: Organotypic cultures from the middle cochleae 72 h after ouabain treatment. The HCs were stained with Alexa Fluor 488-phalloidin. Auditory nerve fibers and SGNs were labeled with an antibody directed against neurofilament 200 and an Alexa Fluor 555 conjugated secondary antibody. (a) Cochlear organotypic culture from postnatal day 3 rat that was cultured for 72 h under normal conditions. (b) Rat cochlear cultures treated with 50 μM ouabain for 72 h. Note that all HCs were intact, whereas the density of the auditory nerve fibers was greatly reduced as compared with the normal control in (a). (c) Cochlear explants were treated with 500 μM ouabain for 72 h. Many SGNs and nerve fibers degenerated and significant percentage's of HCs were missing. (d) Cochlear cultures treated with 1 mM ouabain for 72 h resulted in complete destruction of both SGNs and HCs in the cochlea (Fluorescence microscope ×200).

Mentions: The photomicrograph (Figure 1(a)) showed the orderly arrangement of cochlear outer hair cells and inner hair cells with green fluorescent labeling and auditory nerve fibers with red fluorescent labeling. In addition, SGNs with red fluorescence in a normal cochlear explant cultured for 72 h are shown. This data indicated that the sensory HCs and auditory innervations had been regularly growing for at least three days in the cochlear organotypic primary culture system. Cochlear HCs were intact (Figure 1(b)), whereas the density of auditory nerve fibers was greatly reduced 72 h after 50 μM ouabain treatment. Adding 500 μM of ouabain to the culture medium for 72 h resulted in severe degeneration in SGNs and auditory nerve fibers with massive loss of cochlear HCs (Figure 1(c)). Increasing the ouabain dose to 1000 μM destroyed all of the SGNs, auditory nerve fibers, and cochlear HCs (Figure 1(d)).


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)

Organotypic cultures from the middle cochleae 72 h after ouabain treatment. The HCs were stained with Alexa Fluor 488-phalloidin. Auditory nerve fibers and SGNs were labeled with an antibody directed against neurofilament 200 and an Alexa Fluor 555 conjugated secondary antibody. (a) Cochlear organotypic culture from postnatal day 3 rat that was cultured for 72 h under normal conditions. (b) Rat cochlear cultures treated with 50 μM ouabain for 72 h. Note that all HCs were intact, whereas the density of the auditory nerve fibers was greatly reduced as compared with the normal control in (a). (c) Cochlear explants were treated with 500 μM ouabain for 72 h. Many SGNs and nerve fibers degenerated and significant percentage's of HCs were missing. (d) Cochlear cultures treated with 1 mM ouabain for 72 h resulted in complete destruction of both SGNs and HCs in the cochlea (Fluorescence microscope ×200).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Organotypic cultures from the middle cochleae 72 h after ouabain treatment. The HCs were stained with Alexa Fluor 488-phalloidin. Auditory nerve fibers and SGNs were labeled with an antibody directed against neurofilament 200 and an Alexa Fluor 555 conjugated secondary antibody. (a) Cochlear organotypic culture from postnatal day 3 rat that was cultured for 72 h under normal conditions. (b) Rat cochlear cultures treated with 50 μM ouabain for 72 h. Note that all HCs were intact, whereas the density of the auditory nerve fibers was greatly reduced as compared with the normal control in (a). (c) Cochlear explants were treated with 500 μM ouabain for 72 h. Many SGNs and nerve fibers degenerated and significant percentage's of HCs were missing. (d) Cochlear cultures treated with 1 mM ouabain for 72 h resulted in complete destruction of both SGNs and HCs in the cochlea (Fluorescence microscope ×200).
Mentions: The photomicrograph (Figure 1(a)) showed the orderly arrangement of cochlear outer hair cells and inner hair cells with green fluorescent labeling and auditory nerve fibers with red fluorescent labeling. In addition, SGNs with red fluorescence in a normal cochlear explant cultured for 72 h are shown. This data indicated that the sensory HCs and auditory innervations had been regularly growing for at least three days in the cochlear organotypic primary culture system. Cochlear HCs were intact (Figure 1(b)), whereas the density of auditory nerve fibers was greatly reduced 72 h after 50 μM ouabain treatment. Adding 500 μM of ouabain to the culture medium for 72 h resulted in severe degeneration in SGNs and auditory nerve fibers with massive loss of cochlear HCs (Figure 1(c)). Increasing the ouabain dose to 1000 μM destroyed all of the SGNs, auditory nerve fibers, and cochlear HCs (Figure 1(d)).

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