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. 2002 Feb 15;22(4):1218-27.
doi: 10.1523/JNEUROSCI.22-04-01218.2002.

Inhibition of caspases prevents ototoxic and ongoing hair cell death

Jonathan I Matsui  1 , Judith M Ogilvie, Mark E Warchol
Collaborators, Affiliations

Inhibition of caspases prevents ototoxic and ongoing hair cell death

Jonathan I Matsui et al. J Neurosci. .

Abstract

Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

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Figures

Fig. 1.
Fig. 1.
Hair cells labeled for immunoreactivity to calretinin. Utricles from control animals were fixed and processed for immunohistochemistry with the use of an antibody directed against calretinin. Stereocilia bundles and the cuticular plate are labeled. Scale bar, 20 μm.
Fig. 2.
Fig. 2.
Neomycin induces nuclear chromatin changes in hair cells. Utricles were cultured for 24 hr with 1 mm neomycin (A) or control medium (B), and then they were fixed and stained with bisbenzimide. Dying cells in neomycin-treated cultures were shaped irregularly and had pyknotic nuclei, whereas cells in control cultures were stained uniformly. Pyknotic cells were quantified in 25,000 μm2regions throughout the organ (8 regions per organ; C). Results are the mean ± SEM for three experiments from 13 organs. Data point may obscure error bar. Scale bar, 1 μm.
Fig. 3.
Fig. 3.
Ultrastructural changes in hair cells after 24 hr of neomycin treatment. Shown are transmission electron microscopy micrographs of hair cells from utricles cultured in 1 mmneomycin (A) or control medium (B). Hair cells in neomycin-treated cultures had condensed and frag- mented nuclear chromatin (*). Vacuoles, intracellular membrane whorls (arrows), and lipid inclusions (arrowheads) were observed also. In control cultures, the hair cells appeared normal, with no significant morphological changes. Mitochondria were intact in both control and degenerating cells. Scale bar, 2 μm.
Fig. 4.
Fig. 4.
Inhibition of macromolecule synthesis promotes hair cell survival in the presence of neomycin. Utricles were cultured for 24 hr with 1 mm neomycin and 1 μg/ml cycloheximide or control medium. Calretinin-labeled cells were quantified in 10,000 μm2 regions of both the extrastriolar (6 regions per organ) and striolar (4 regions per organ) areas. Results are the mean ± SEM for three experiments from 10–12 organs. Data point may obscure the error bar.
Fig. 5.
Fig. 5.
Caspase inhibitors prevent hair cell death after neomycin treatment. Utricles were cultured for 24 hr with 1 mm neomycin and 0.1% DMSO (carrier) or 50 μmBAF. Cultures were fixed, and the nuclei were stained with Hoechst (A) or were processed for TUNEL labeling (B). Pyknotic cells were quantified in 25,000 μm2 regions throughout the organ (8 regions per organ). TUNEL+ cells were quantified in 25,000 μm2 regions throughout the organ (6 regions per organ). Results are the mean ± SEM for three experiments from 10–13 organs. Data point may obscure the error bar.
Fig. 6.
Fig. 6.
Photomicrograph of hair cells that were treated with neomycin and with or without BAF. Utricles were cultured for 24 hr in 1 mm neomycin and 0.1% DMSO (A, B) and 50 μm BAF (A). Enhanced numbers of hair cells were present in BAF-treated cultures compared with cultures that contained neomycin alone. Scale bar, 10 μm.
Fig. 7.
Fig. 7.
Caspase inhibitors promote hair cell survival after neomycin treatment. In short-term experiments the utricles were cultured for 24 hr with 1 mm neomycin and 0.1% DMSO (A, C), 50 μm BAF (A), or 25 μm zVAD (C). In long-term experiments (B) the utricles were cultured for 24 hr with 0.1% DMSO, 1 mm neomycin, or 1 mm neomycin and 50 μm BAF; they were washed and then cultured for 5 d in control medium. Calretinin+ cells were quantified in 10,000 μm2 regions of both the extrastriolar (6 regions per organ) and striolar (4 regions per organ) areas. In the long-term cultures the calretinin+cells were quantified in the extrastriolar region only. Similar levels of hair cell survival were observed in BAF/neomycin-treated cultures and control cultures after 5 DIV. Results are the mean ± SEM for three experiments from 10–13 organs.
Fig. 8.
Fig. 8.
BAF reduces ongoing cell death in the avian inner ear. Utricles (A, B) or isolated sensory epithelia (C) were cultured for 24 hr with 0.1% DMSO (controls) or 10, 50, or 100 μm BAF. Fixed specimens then were processed for TUNEL labeling (A) or BrdU immunohistochemistry (B, C). TUNEL+or BrdU+ cells were quantified in the extrastriolar regions in each organ and then were normalized to 25,000 μm2. Results are the mean ± SEM for three experiments from seven to nine organs. Significantly fewer TUNEL+ cells were present in BAF-treated specimens (A). In addition, correspondingly fewer BrdU+ cells were present in BAF-treated cultures compared with controls (B). C, Plot of proliferation index (BrdU+ cells/total cells) in a 10,000 μm2 region, demonstrating that BAF does not affect supporting cell proliferation directly. Combined results from these experiments suggest a causative relationship between ongoing hair cell death and ongoing supporting cell proliferation.
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