Protection of the cochlear hair cells in adult C57BL/6J mice by T-type calcium channel blockers

Abstract

The aim of the present study was to investigate the protective effect of T-type calcium channel blockers against presbycusis, using a C57BL/6J mice model. The expression of three T-type calcium channel receptor subunits in the cochlea of 6-8-week-old C57BL/6J mice was evaluated using reverse transcription-quantitative polymerase chain reaction. The results confirmed that the three subunits were expressed in the cochlea. In addition, the capacity of T-type calcium channel blockers to protect the cochlear hair cells of 24-26-week-old C57BL/6J mice was investigated in mice treated with mibefradil, benidipine or saline for 4 weeks. Differences in hearing threshold were detected using auditory brainstem recording (ABR), while differences in amplitudes were measured using a distortion product otoacoustic emission (DPOAE) test. The ABR test results showed that the hearing threshold significantly decreased at 24 kHz in the mibefradil-treated and benidipine-treated groups compared with the saline-treated group. The DPOAE amplitudes in the mibefradil-treated group were increased compared with those in the saline-treated group at the F2 frequencies of 11.3 and 13.4 kHz. Furthermore, the DPOAE amplitudes in the benidipine-treated group were increased compared with those in the saline-treated group at an F2 frequency of 13.4 kHz. The loss of outer hair cells (OHCs) was not evident in the mibefradil-treated group; however, the stereocilia of the inner hair cells (IHCs) were disorganised and sparse. In summary, these results indicate that the administration of a T-type calcium channel blocker for four consecutive weeks may improve the hearing at 24 kHz of 24-26-week-old C57BL/6J mice. The function and morphology of the OHCs of the C57BL/6J mice were significantly altered by the administration of a T-type calcium channel blocker; however, the IHCs were unaffected.

Keywords: T-type calcium channel; calcium channel blocker; hair cell; receptor.

Figure 1.

Quantified expression of three T-type channel subunits in the cochlea of 6–8-week-old C57BL/6J mice via reverse transcription-quantitative polymerase chain reaction revealed that the expression levels of the three subunits were low, and that α1H had the lowest level of expression.

Figure 2.

DPOAE amplitudes at the F2 frequency in 3 treatment groups of 24–26-week-old C57BL/6J mice. The DPOAE amplitudes in the mibefradil-treated group at F2 frequencies of 11.3 and 13.4 kHz were significantly increased compared with those in the saline-treated group. In addition, the DPOAE amplitudes in the benidipine-treated group at an F2 frequency 13.4 kHz were significantly increased compared with those in the saline-treated group. The DPOAE amplitudes at other F2 frequencies did not significantly differ between the mibefradil- or benidipine-treated and the saline-treated group. *P<0.05 between the saline- and mibefradil-treated groups. DPOAE, distortion product otoacoustic emission; SPL, sound pressure level.

Figure 3.

Morphological analysis of cochlea hair cells observed by scanning electron microscopy in the three treatment groups of 24–26-week-old C57BL/6J mice. (A) Morphology of hair cells in the saline-treated group indicated the rupture of the cuticular plate, lost outer hair cells (OHCs) and sparse stereocilia bundles on inner hair cells (IHCs). Spherical extrusions appeared on the outside of the stereocilia of inner hair cells. (B) Morphology of hair cells in the mibefradil-treated group showed no obvious loss of OHCs; however, the stereocilia of the IHCs were disorganised and sparse. (C) Morphology of hair cells in the benidipine-treated group indicated a small quantity of missing OHCs, and disorganised or sparse hair cells. White arrows indicate spherical extrusion (scale bar, 10 µm).