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. Author manuscript; available in PMC: 2008 Apr 1.
Published in final edited form as: Hear Res. 2006 Dec 31;226(1-2):52–60. doi: 10.1016/j.heares.2006.12.011

Prophylactic and therapeutic functions of T-type calcium blockers against noise-induced hearing loss

Haiyan Shen a,b,*, Baoping Zhang a,*, June-Ho Shin a, Debin Lei a, Yafei Du a, Xiang Gao b, Qiuju Wang c, Kevin K Ohlemiller a, Jay Piccirillo a, Jianxin Bao a,**
PMCID: PMC1903349  NIHMSID: NIHMS21252  PMID: 17291698

Abstract

Cochlear noise injury is the second most frequent cause of sensorineural hearing loss, after aging. Because calcium dysregulation is a widely recognized contributor to noise injury, we examined the potential of calcium channel blockers to reduce noise-induced hearing loss (NIHL) in mice. We focused on two T-type calcium blockers, trimethadione and ethosuximide, which are anti-epileptics approved by the Food and Drug Administration. Young C57BL/6 mice of either gender were divided into three groups: a ‘prevention’ group receiving the blocker via drinking water before noise exposure; a ‘treatment’ group receiving the blocker via drinking water after noise exposure; and controls receiving noise alone. Trimethadione significantly reduced NIHL when applied before noise exposure, as determined by auditory brainstem recording. Both ethosuximide and trimethadione were effective in reducing NIHL when applied after noise exposure. Results were influenced by gender, with males generally receiving greater benefit than females. Quantitation of hair cell and neuronal density suggested that preservation of outer hair cells could account for the observed protection. Immunocytochemistry and RT-PCR suggested that this protection involves direct action of T-type blockers on α1 subunits comprising one or more Cav3 calcium channel types in the cochlea. Our findings provide a basis for clinical studies testing T-type calcium blockers both to prevent and treat NIHL.

Keywords: Noise-induced hearing loss, Mouse, T-type calcium channel, trimethadione, ethosuximide

1. Introduction

Excessive noise is the predominant cause of permanent sensorineural hearing loss. At least 30 million people in the United States encounter hazardous levels of noise at work, particularly in jobs such as construction, mining, agriculture, manufacturing, transportation, and in the military (LePrell et al, this volume; Bohnker et al., 2002; Henderson et al., 1998, 2003; Seixas et al., 2005). The incidence of noise-induced hearing loss (NIHL) continues to grow, moreover, partly due to growing popularity of portable music players with highly efficient headphones (Fligor and Cox, 2004; Serra et al., 2005). Although several promising approaches have been identified for reducing NIHL (Lefebvre et al., 2002; Niu and Canlon 2002; Kopke et al., 2005; Lynch and Kil 2005; Le Prell et al., this volume; Gagnon et al., this volume), there are currently no pharmacologic agents approved by the Food and Drug Administration (FDA) for this purpose.

Typically there are two phases of hearing loss after noise, a temporary threshold shift (TTS) that is most prominent in the first 24 hours, but may extend for 1–2 weeks, and permanent threshold shift (PTS) (Clark, 1991; Quaranta et al., 1998; Nordmann et al., 2000). Previous studies have suggested that TTS and PTS are two distinct phenomena with different cellular pathological changes. TTS may reflect reversible buckling of the pillar cell bodies (Nordmann et al., 2000), temporary strial edema and reduction of the endocochlear potential (Hirose and Liberman, 2003), or excitotoxic damage to afferent fibers (Pujol and Puel, 1999). Histopathologic correlates of PTS include permanent stereocilia damage, hair cell loss, and degeneration of afferent fibers in the organ of Corti (Slepecky, 1986; Saunders et al., 1991).

Molecular mechanisms underlying NIHL are only partly known, but probably include dysregulation of calcium (LeFebvre et al., 2002; LePrell et al, this volume; Henderson, 2006). Elevated intracellular calcium levels may impair mitochondrial function and compromise a host of cellular functions (Luer et al., 1996). Calcium elevation affects cell membranes by activation of phospholipase A2 and C, resulting in hydrolysis of membrane phospholipids, release of free fatty acids (Farooqui, et al., 2004) and lipid peroxidation (Sullivan et al., 2004). Disturbance in calcium homeostasis contributes to trauma-induced neuronal injury and age-related neuronal loss (Zipfel et al., 2000; Toescu et al., 2004). In the noise-exposed cochlea, calcium may participate in both hair cell and neuronal damage (Minami et al.., 2004; LeFebvre et al., 2002; Pujol and Puel, 1999). Calcium homeostasis in hair cells and spiral ganglion neurons (SGNs) is maintained by regulatory proteins such as calmodulin and calbindin (e.g., Hansen et al., 2003; Hackney et al., 2005), and by several types of calcium channels (Niedzielski et al., 1997; Morley et al., 1998; Parks, 2000; Lopez et al., 2003). Voltage-gated calcium channels (VGCCs) play a key role in calcium entry into neurons and control various calcium-dependent functions, including intracellular signaling and gene expression (Snutch and Reiner, 1992; Fuchs, 1996, Kochegarov, 2003; Errington et al., 2005). VGCCs can be divided into two groups: low-voltage activated calcium channels that respond to small (~10 mV) changes in the resting membrane potential, and high-voltage activated calcium channels that require stronger (~30 mV) depolarization to open (Perez-Reyes, 1998; Lacinova et al., 2000; Yunker and McEnery, 2003; Layton et al., 2005; Triggle, 2006). Diltiazem, a blocker of L-type calcium channels (high-voltage activated channels) was reported to attenuate NIHL (Heinrich et al., 1997). However, other studies (Boettcher, 1996; Ison et al., 1997; Boettcher et al., 1998) have not supported any protective effect of blocking L-type calcium channels. Recently, blockers of T-type calcium channels (low-voltage activated channels) were found to prevent cisplatin-induced death of cochlear cells in vitro (So et al., 2005). Whether these agents are able to prevent or treat NIHL has not been tested. Here we test the protective actions against NIHL of T-type calcium blockers trimethadione and ethosuximide, anti-epileptic drugs approved by the FDA, when administered in drinking water to inbred C57BL/6 mice. We show that both agents can reduce NIHL, whether applied before or after noise exposure, although efficacy may depend on gender. The primary action of T-type calcium antagonists may be to promote hair cell survival.

2. Methods

2.1 Animals

All animal procedures were approved by the Animal Studies Committee at Washington University in St. Louis. The study included 44 male and female C57BL/6J mice aged 2–3 months, purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed three to five per cage in a noise-controlled environment on a 12 hr light/dark cycle with light onset at 6:00 a.m.

2.2 Drug application

Animals were subject to one of two protocols, a ‘prevention’ protocol under which drugs were administered prior to a single noise exposure, and a ‘treatment’ protocol wherein drugs were administered only after noise. Trimethadione and ethosuximide were obtained from Sigma Chemical Co. (St. Louis, MO). Pilot experiments led us to select dosages of 200 mg/day/kg body weight for trimethadione and 1.5 g/day/kg body weight for ethosuximide. Dosages were kept constant throughout the experimental period by monitoring the amount of water uptake and body weight measured every three days. Drinking water containing the drug was kept in dark bottles and changed every three days. Animals in the prevention group received trimethadione in their drinking water for three weeks prior to noise. Animals in the therapy group received trimethadione or ethosuximide beginning immediately after noise exposure in drinking water for two weeks.

2.3 Noise Exposure

As described previously (e.g., Ohlemiller et al., 2000), noise exposures were performed in a foam-lined, double-walled soundproof room (Industrial Acoustics). The noise exposure apparatus consisted of a 21×21×11 cm wire cage mounted on a pedestal inserted into turntable. The cage was rotated at 1 revolution/80 s. A Motorola KSN1020A piezo ceramic speaker (four totals) was attached to each side of a metal frame surrounding the cage. Opposing speakers were driven by independent channels of a Crown D150A power amplifier. Noise was generated by two General Radio 1310 generators and filtered to 4.0–45.0 kHz by Krohn-Hite 3550 filters. The overall noise level was measured at the center of the cage using a B&K 4135 ¼ inch microphone in a combination with a B&K 2231 sound level meter set to broadband (0.2 Hz −70 kHz). Mice were exposed in pairs at 110 dB SPL for 30 min.

2.4 Auditory brainstem recording (ABR)

ABR testing was performed prior to treatment, then 24 hours and 2–3 weeks after the noise exposure to estimate TTS and PTS, respectively. Thresholds were obtained as described previously (Ohlemiller et al., 2000; Bao et al., 2005). Prior to testing, all mice were anesthetized with 80 mg/kg ketamine and 15 mg/kg xylazine (i.p.). Otoscopic examination was performed to ensure that tympanic membranes were normal. Core temperature was maintained at 37.5±1.0 °C using a thermostatically-controlled heating pad in conjunction with a rectal probe (Yellow Springs Instruments Model 73A). Platinum needle electrodes (Grass) were inserted subcutaneously just behind the right ear, at the vertex, and in the back (ground). Electrodes were led to a Grass P15 differential amplifier (100–10,000Hz, x100), then to a custom amplifier providing another ×1,000 gain, and digitized at 30 kHz using a Cambridge Electronic Design Micro1401 in conjunction with SIGNAL™ and custom signal averaging software operating on a 120 MHz Pentium PC. Sinewave stimuli generated by a HP 3445 oscillator were shaped by a custom electronic switch to 5 ms total duration, including 1 ms rise/fall times. The stimulus was amplified by a Crown D150A amplifier and output to a KSN1020A piezo ceramic speaker. Toneburst stimuli at each frequency and level were presented 1,000 times at 20/sec. The minimum sound pressure level required for visual detection of wave I was determined at each frequency using a 5 dB minimum step size. To calibrate sound stimuli, a B&K 4135 ¼ inch microphone was placed where the ear would normally be located.

2.5 Tissue processing for histology

All animals were sacrificed immediately after the final ABR test. The methods for fixing, processing and analyzing mouse cochleae are described elsewhere (Ohlemiller et al., 2000). Briefly, mice were perfused transcardially with cold 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer. Both cochleae were isolated, the stapes removed, and immersed in the same fixative. Complete exposure of the cochlea to the fixative was ensured by making a small hole at the apex of the cochlear capsule, gently circulating the fixative over the cochlea using a transfer pipet. After decalcification in sodium EDTA, the cochleae were post-fixed in buffered 1% osmium tetroxide, dehydrated in an ascending acetone series, and embedded in Epon.

2.6. Morphometric analysis

Because the protective effects of T-type calcium antagonists might be exerted through hair cell survival, neuronal survival, or both, we conducted analyses designed to detect all three potential effects.

Hair cell counts

Estimates of hair cell survival were obtained for 4 drug-treated and 4 control mice, all male. To obtain counts, embedded cochleae were dissected into roughly half-turn segments and examined in mineral oil under Nomarski optics, using a calibrated ocular. Three separate inner hair cell (IHC) and outer hair cell (OHC) counts were obtained from non-overlapping 100 μm segments in the apex, upper base, lower base, and hook regions of the cochlea. In 2 of the animals, counts were made at a higher spatial resolution (Ou et al., 2000), and the percentage of missing IHCs and OHCs was determined by dividing the number of missing hair cells by the total number of hair cells (i.e. present plus absent) in the organ of Corti.

Neuronal counts

Spiral ganglion neuron counts were obtained for 14 male mice (8 drug-treated, 8 control). Cochleae were cut in the mid-modiolar plane at 4.0 μm m. In every second section though a total 50 sections per animal, the number of SGN nuclei in the apex, upper base, and lower base were counted under a 200x field using a computerized planimetry program (Image Pro Plus, Media Cybernetics). Because all sections were cut in the same orientation, it was possible to compare two groups by Mann-Whitney test.

2.7 Immunocytochemical staining

The cochleae were cryoprotected in 20% glycerol in PBS at −20 °C. The tissue was embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound (Sakura, USA) on dry ice. Frozen sections of 10 μmm were cut dorsal to ventral through the cochlea, in the sagittal plane relative to the head. Sections were rinsed in PBS and permeabilized in 0.3% Triton X-100 for 20 min. Tissues were blocked in 5% normal goat serum in PBS for 30 min and then incubated in the primary antibody for the α1G (Chemicon, USA), α1H (Santa Cruz Biotech. USA), or α1I (Chemicon, USA) at 1:100–1:1000 dilutions overnight at 4 °C. For diaminobenzidine (DAB) method, the tissue was processed using a Vectastain Elite avidin-biotin peroxidase kit (Vector Laboratories, USA) with DAB as the chromogen. Images were captured using a Nikon microscope or a confocal microscope with LaserSharp2000 program (Bio-Rad).

2.8 Real-time RT-PCR

Total RNA from cochlea at each animal was extracted using RNAqueous (Ambion). To avoid any DNA contamination during RNA extraction, at the final step of extraction, one microliter of DNase I was added to 49 μml elution buffer to extract total RNA from an RNA extraction column. The solution was incubated at 37 °C for 15 minutes and heated to 100 °C for five minutes to kill the DNase I. Then the RNA was quantified with RiboGreen RNA quantization reagent (Molecular Probes, Eugene, OR). Prior to reverse-transcription to generate cDNA, the quality of RNA was determined by checking ribosomal RNA integrity on a 3% denatured agarose gel. Ten microliters of total 50 μml RNA were reverse-transcribed in total 20 μml using random hexamers and Superscript II reverse transcriptase (Invitrogen, CA). Standard curves for GAPDH and three α1 subunits were made using their respective cDNA plasmids at five dilutions. Primers used for real-time RT-PCR of these mouse genes were:

GAPDH: 5-CCTGGCCAAGGTCATCCATGACAAC-3 and 5-TGTCATACCAGGAAATGAGCTTGAC-3;

α1G Subunit: 5-AATGGCAAGTCGGCTTCAGG-3 and 5-TGTCAGAGACCATGGACACCAG-3;

α1H Subunit: 5-ATGTTCCGGCCCTGTGAGGA-3 and 5-CCATGACGTAGTACATGATGTCC-3;

α1I Subunit: 5-ATCTGCTCCCTGTCGG-3 and 5-GAGAACTGGGTCGCTATG-3.

PCR protocols of the LightCycler System for GAPDH and each α1 subunit were optimized with the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche). Two microliters of each 20 μml cDNA were added in a 20 μml PCR reaction mixture containing 1 X PCR buffer, 0.5 μm M each primer, and Master Mix from the kit. The number of PCR cycles when the fluorescence intensity exceeds a predetermined threshold was measured automatically during PCR. Quantification of the initial amount of template molecules was achieved by calculating this number of PCR cycles with the number from the standard samples. The difference in the initial amount of total RNA among the samples was normalized in every assay using the house keeping gene, GAPDH. Besides using melting curve analysis in the program of the LightCycler System to ensure the right PCR product, we also examined each PCR product on a 3% agarose gel after each experiment.

3. Results

3.1 Post-exposure application of T-type blockers

No difference by treatment in pre-exposure ABR thresholds was noted for any test condition (see Figs. 1–3). Male mice treated with trimethadione after noise exposure showed modest (~10 dB) but significantly smaller TTS compared to controls (p=0.001 overall by 2-way ANOVA, no interactions) (‘24 h’ in Fig. 1A). Significant protection was not achieved in female mice (Fig. 1B). A similar pattern was found for the PTS, such that threshold shifts were ~10 dB less in male mice treated with trimethadione than in controls (p=0.003, no interactions) (‘2 w’ in Fig. 1A), while no difference was indicated for female mice (Fig. 1B). Male mice receiving ethosuximide after noise exposure showed protection similar to that afforded by trimethadione (Fig. 2). Protection from TTS (24 h) was suggested, but did not achieve significance. PTS, however, was 10–20 dB less than in controls (3 w) (p=0.001, no interactions).

Fig. 1.

Fig. 1

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Therapeutic function of trimethadione against NIHL. (A) ABR thresholds (Mean ± S.D) for male mice (n=3 in each group) at 3 months of age. Significant protection against both TTS (24 h) and PTS (2 w) was found in males receiving trimethadione in their drinking water after noise exposure (2-way ANOVA, no interactions). (B) ABR thresholds for female mice (n=4 for each group) at 3 months of age. Differences by treatment were not significant.

Fig. 3.

Fig. 3

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Prophylactic function of trimethadione. (A) ABR thresholds (Mean ± S.D) for male mice aged 3 months (n=6 for noise alone, n=7 for noise+drug). (B) ABR thresholds for female mice at 3 months old (n=3 for each group). Significant protection from PTS only (2 w) was found for both males and females receiving trimethadione two weeks before noise exposure (2-way ANOVA with p<.05 for differences at 20 kHz in males and 28.3 kHz in females by Tukey test*).

Fig. 2.

Fig. 2

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Therapeutic function of ethosuximide against NIHL. ABR thresholds (Mean ± S.D) are shown for male mice (n=5) aged 3 months. Significant protection from PTS only (3 w) was found in mice versus controls (2-way ANOVA, no interactions).

3.2 Pre-exposure application of trimethadione

We next tested whether trimethadione could reduce NIHL when applied prior to noise exposure. Analysis of variance showed no significant reduction in TTS versus controls in male female mice (‘24 h’ in Fig. 3A, B). In both males and females, however, trimethadione reduced PTS by 10–20 dB (‘2 w’ in Fig. 3A, B) (p=0.004 overall by 2-way ANOVA with p<.05 for differences at 20 kHz in males and 28.3 kHz in females by Tukey test). The efficacy of trimethadione in females when applied prior to noise therefore stands in apparent contrast to the lack of any effect when applied after noise.

3.3 Histological findings

Hair cell counts in male mice treated with trimethadione after noise exposure showed significantly better preservation of outer hair cells in the cochlear base in treated mice versus controls (p=0.024 overall by 2-way ANOVA with p<.05 for differences in the hook region by Tukey test) (Fig. 4A). No difference was found for inner hair cells. There was no significant difference by treatment in the number of SGNs between the control and trimethadione-treated mice (Fig. 4B), nor between controls and mice treated with ethosuximide (Fig. 5).

Fig. 4.

Fig. 4

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Quantitative comparisons of OHCs, IHCs, and SGNs between control and trimethadione-treated mice. (A) Mean (± S.D.) counts of IHC and OHC in the hook region (most basal 1 mm), lower base (LB), upper base (UB), and apex of the cochlea in 3-month-old male mice (n=3 for each group). There was a significant overall difference in OHC preservation by treatment (p=0.024 by 2-way ANOVA with p<.05 for differences in the hook by Tukey test*). (B). Counts of SGNs (± SD) in cochlear apex, middle, and base from the same mice as in A. SGN density was not significantly different by treatment (t-test).

Fig. 5.

Fig. 5

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Quantitative comparison of SGNs between control and ethosuximide-treated mice. Mean (± S.D.) SGNs in cochlear apex, middle, and base in 3-month-old male mice (n=4 for each group). SGN density was not significantly different by treatment (t-test).

With the exception of the hook region (most basal ~0.8 mm), neither drug-treated nor control animals typically showed a clear relation between ABR thresholds and the number of surviving hair cells after noise. Figure 6 illustrates this point using highly contrasting examples of a control mouse and a mouse given trimethadione prior to noise exposure. The control shows >25 dB of permanent ABR threshold shift at most frequencies, compared to <25 dB in the treated mouse. The control showed ~22% missing IHCs and 75% loss of OHCs in the deep basal region of the cochlea (32–64 kHz region), compared to ~2% and 69% OHCs, respectively, in the drug-treated mouse. These mice are consonant with an overall pattern of better hair cell preservation in drug-treated mice, and there is agreement between hair cell and ABR data in the extreme base. However, while lower frequency regions featured better threshold preservation in the treated mouse, hair cell losses were similar (<1% and 5% loss of IHCs and OHCs respectively in the control versus 2% and 4% loss of IHCs and OHCs in the drug-treated mouse).

Fig. 6.

Fig. 6

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Example ABR threshold shifts and cytocochleograms for a pair of mice testing the prophylactic function of trimethadione. (A) and (B) ABR threshold shifts and cytocochleogram for the control mouse (male, 3 months old). (C) and (D) ABR threshold shifts and cytocochleogram for trimethadione-treated mouse (male, 3 months old). The mice were exposed to the noise in the same cage at the same time. The treated mouse shows better hearing and better preservation of hair cell function in the extreme base. Overall, hair cell survival was not a good predictor of threshold sensitivity in either animal.

3.4 Presence of T-type calcium channels in the cochlea

The molecular target for the protection of NIHL by T-type calcium blockers is expected to be T-type calcium channels in the cochlea. The family of T-type calcium channels is composed of three members (Cav3.1, Cav3.2, and Cav3.3) based on their respective main pore-forming alpha subunits: α1G, α1H, or α1I (Perez-Reyes, 1998; Lacinova et al., 2000; Yunker and McEnery et al., 2003). So et al. (2005) showed the presence of α1G mRNA for the Cav3.1 channel in the P2 rat organ of Corti. It was not determined in that study whether α subunits for all three difference T-type calcium channels were present in the mature cochlea. We performed immunostaining on cochlear sections from 2 month old C57BL/6J mice using antibodies against each subunit. SGNs showed robust immunoreactivity for the α1H subunit, while weak staining was found for both α1G and α1I subunits (Fig. 7A). By contrast, weak expression of the α1G and α1I subunits but not the α1H subunit (data not shown) was observed in the organ of Corti. The expression of the α1G subunit was found in IHCs, pillar cells, deiters cells, and OHCs, and the expression of the the α1I subunit was found in OHCs and deiters’ cells (Fig. 7B). Using real-time RT-PCR, we also examined the expression level of mRNAs encoding the three α subunits in whole cochlea of 2 month old mice (Fig. 7C). Consistent with immunostaining results, the expression level of the α1H subunit in the cochlea appeared the highest of the three subunits, with mRNA levels more than 2-fold higher than that of the α1G subunit and ~100-fold higher than that of the α1I subunit. The ratio between each α1 subunit and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 3.49×10−3 (SD±4.1×10−3) for the α1G subunit, 8.18×10−3(± 17.6×10−3) for the α1H subunit, and 4.29×10−5 (± 8.58×10−5) for the α1I subunit. No significant difference was found in the expression level of each subunit between male and female mice (t-test; n=7 for each group; p=0.976, 0.174, and 0.09 for the α1G, α1H, and α1I respectively) (Fig. 7C). The type and placement of T-type calcium channels in the cochlea appear consistent with an effect of T-type blockers that is directed at cochlea, and specific to the organ of Corti and/or spiral ganglion.

Fig. 7.

Fig. 7

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Presence of alpha subunits for T-type calcium channels in the mature cochlea. (A) Immunocytochemistry showing all three alpha subunits in SGNs. The scale bar is 25 μm m. (B) Immunocytochemistry showing presence of α1G and α1I subunits in the organ of Corti. The scale bar is 25 μm m. (C) The expression level of each alpha subunit in the cochlea. Mean (+SD) ratio between each alpha subunit and GAPDH in female and male groups was shown (n=6 for each group; t-test; p=0.976, 0.174, and 0.09 for the α1G, α1H, and α1I respectively).

4. Discussion

Our data indicate that blockers of T-type calcium channels can reduce both temporary and permanent threshold shifts caused by noise exposure in C57BL/6J mice. Since these blockers are anti-convulsant drugs already approved by the FDA, they may be appropriate for testing in a clinical setting for protection against permanent NIHL in humans. Importantly, protection could be observed for drug administration after noise exposure. Recent evidence suggests that noise injury represents an ongoing process, beginning during exposure, but extending for days beyond (see Le Prell et al., this volume). Agents that can be effectively administered after noise exposure probably interfere with delayed, perhaps reversible, steps in the injury cascade. Such agents also offer added practicality, since potentially injurious exposure will often be recognized only after it has occurred.

4.1 Consideration of the animal model

We chose B6 mice for study because these mice share the background of knockout and transgenic models we hope to test to support the present findings. The use of B6 mice, which carry the Cdh23ahl allele, may have impacted our results, insofar as this allele increases the extent of noise injury, and Cdh23 may bind calcium (Davis et al., 2001, 2003). It is possible that our results will prove specific to B6 mice. Recent studies have highlighted differences completely unrelated to Cdh23 in the response to noise of B6 and other strains such as CBA/J (Ohlemiller, 2006; Gagnon et al., this volume).

4.2 Cellular targets of noise injury and protection by T-type blockers

The principal cochlear targets of most permanent noise injury are hair cells and afferent neurons, although most neuronal loss may be secondary to hair cell loss (Slepecky, 1986; Saunders et al, 1991; Robertson, 1983; Pujol and Puel, 1999). Outer hair cells, especially, may be either lost or non-lethally damaged by noise (e.g., Hunter-Duvar et al., 1972; Liberman and Kiang, 1978; Ou et al, 2000). Quantitation of both hair cells and neurons indicated significant protection by trimethadione and ethosuximide only for outer hair cells, and primarily in the cochlear base. We therefore propose that the cellular basis of protection by T-type calcium blockers on NIHL is preservation of OHC function and survival. Consistent with this, hair cells and supporting cells appeared to express both α1G and α1I calcium channel subunits. Flunarizine, another T-type calcium channel blocker, was shown previously to inhibit cisplatin-induced death of cultured auditory cells (So et al., 2005). In that study, however, the mechanism was proposed to be inhibition of lipid peroxidation and mitochondrial permeability transition, not blockage of T-type calcium channels. Experiments combining pharmacological and transgenic approaches may help narrow the molecular targets for trimethadione and ethosuximide and other T-type blockers.

4.3 T-type calcium channel blockers and gender difference

Gender differences we observed in the protection against NIHL afforded by trimethadione and ethosuximide could be an artifact of small sample sizes by gender, or may be due to different cochlear expression levels of the α1H channel subunit between male and female mice. RT-PCR revealed a nearly significant difference in the expression level of α1I between male and female mice at two months of age. Notably, a previous study also yielded gender differences in the efficacy of calcium blockers. An apparent delay of presbycusis in patients taking calcium blockers for other illnesses was found only in females (Mills et al., 1999). We observed the α1I subunit only in SGNs, which showed no enhanced survival in treated animals, and are not expected to account for most permanent NIHL. It is therefore not clear that differences in the expression of the α1I subunit can explain gender-specific aspects of the present or previous results.

4.4 Conclusions

Blockers of T-type calcium channels reduce permanent NIHL in C57BL/6J mice. Molecular mechanisms underlying this functional benefit remain unclear, but may involve direct action of blockers on the α1G and/or α1I subunits of one or more Cav3 calcium channels in hair cells or supporting cells. Since blockers for T-type calcium channels are anti-convulsant drugs approved by the FDA to treat epilepsy, they may be appropriate for tests in the clinical setting to treat and prevent NIHL in humans.

Acknowledgments

A preliminary report was presented at the 2005 International Symposium on “Pharmacologic Strategies for Prevention and Treatment of Hearing Loss and Tinnitus.” J.B. formed the original idea and experimental design. We thank Dr. Richard Chole for his valuable suggestions. We thank Drs. Barbara Bohne and Gary Harding for thoughtful suggestions and analyzing data for Figure 6. We also thank the technical assistance provided by Patricia M. Gagnon and Jaclyn M. Lett. The project was supported by grants to J.B. from the National Institute of Health (AG001016 and AG024250), and the National Organization for Hearing Research Foundation.

Footnotes

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