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Effects of nimodipine on noise-induced hearing loss
Hearing Research 121 (1998) 139^146
E¡ects of nimodipine on noise-induced hearing loss Flint A. Boettcher *, Richard K. Caldwell, Michael Anne Gratton 1 , David R. White, Lesa R. Miles Department of Otolaryngology and Communicative Sciences, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2242, USA Received 4 December 1997; revised 13 April 1998; accepted 18 April 1998
Abstract The effects of nimodipine, a calcium channel blocker, on noise-induced hearing loss were examined in gerbils. Animals were implanted subcutaneously with a timed-release pellet containing either nimodipine (approximately 10 mg/kg/day) or placebo and exposed to either 102 or 107 dBA noise. Serum levels were tested in two subjects and were in the range known to protect humans from cerebral artery vasospasm and ischemia-related neurologic deficits. Nimodipine and control groups had similar amounts of noise-induced (a) permanent threshold shift ; (b) reductions in distortion product otoacoustic emissions ; (c) reductions in tuning and suppression of the compound action potential; and (d) loss of outer hair cells. The results suggest that nimodipine, at a dose which results in clinically relevant serum levels, does not provide protection from the effects of moderately intense noise exposures. z 1998 Published by Elsevier Science B.V. All rights reserved. Key words: Calcium channel blocker; Gerbil; Nimodipine; Noise-induced hearing loss
1. Introduction Noise-induced hearing loss (NIHL) is one of the most common occupational health hazards in industrial societies. The primary damage caused by excess noise exposure is to sensory hair cells in the organ of Corti, particularly outer hair cells (OHCs) (for a recent review, see Henderson and Hamernik, 1995). Noise exposure which causes permanent hearing loss typically results in bending, fusion, and fracture of hair cell stereocilia, vacuolization of hair cells, and ultimately hair cell death and degeneration. Hair cells are replaced by squamous epithelium which forms a scar in the place of the cell. The biological mechanisms underlying acoustic injury to the cochlea are not known. Calcium (Ca2 ) is a ubiquitous ion in mammalian cells (though at very low free intracellular concentra* Corresponding author. Tel.: +1 (803) 792-8291; Fax: +1 (803) 792-7736; E-mail: [email protected] 1 Present address: Boys Town National Research Hospital, 555 N. 30th St., Omaha, NE 68131, USA.
tions) and is critical in regulation of many cellular activities such as neurotransmitter release and cell movement. However, an excessive level of free intracellular Ca2 (resulting from entry through ion channels or release from intracellular stores) overactivates a series of enzymes including phospholipases, protein kinase C, proteases, endonucleases and depolymerases. The result is membrane breakdown, depolymerization of microtubules, and disruption of protein synthesis (Orrenius et al., 1992 ; Verity, 1992). Ca2 entry into cells can be controlled, in part, by drugs which block voltage-gated channels. Calcium channel blockers (CCBs) are drugs which restrict entry of Ca2 into cells. Nimodipine, a CCB of the dihydropyridine class, is lipophilic and readily crosses the blood-brain barrier (Allen et al., 1983). The drug blocks Ca2 channels of the L-type (large conductances with long-lasting e¡ects [Tsien, 1989; Hille, 1992]). Nimodipine is used to prevent cerebral artery spasms following subarachnoid hemorrhage (Allen et al., 1983; Mee et al., 1988) and to reduce neurologic de¢cits following ischemic stroke (Gelmers, 1985;
0378-5955 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 0 7 5 - 6
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Gelmers and Hennerici, 1990). One consequence of tissue ischemia is a large in£ux of Ca2 from extracellular £uid to the intracellular milieu during reperfusion of tissue. CCBs also have been shown experimentally to prevent or reduce Ca2 cytotoxicity following ischemia in the heart and kidney (for a review, see Nayler, 1988). The functions of Ca2 in hair cell (HC) activity are not completely understood, but several roles have been demonstrated or postulated, including regulation of: (a) slow motility of the OHCs (Dulon et al., 1991; Sridhar et al., 1997); (b) HC transduction (Hudspeth, 1986 ; see Fettiplace, 1992 for a recent review) and adaptation (Corey and Hudspeth, 1979; Ricci and Fettiplace, 1997 ; see Torre et al., 1995 for a recent review); (c) gating of ion channels (Hudspeth, 1986; Art et al., 1993); and (d) release of neurotransmitter (Siegel and Relkin, 1987; Guth et al., 1991). Calcium enters OHCs through several types of channels. Cation channels near stereocilia tips primarily allow entry of potassium, but Ca2 can also enter (Corey and Hudspeth, 1979; Lopez-Escamez and Schacht, 1995). L-type Ca2 channels have been demonstrated in OHCs (Santos-Sacchi and Dilger, 1988; Nakagawa et al., 1991) and 12 Ca2 channel subunits have been identi¢ed in the cochlea using the polymerase chain reaction (Green et al., 1996). Depolarizing the cell with potassium causes a large increase in intracellular Ca2 in hair cells, probably due to entry through voltage-sensitive Ca2 channels (Hudspeth, 1986; Fettiplace, 1992). Nifedipine, a CCB similar in structure and function to nimodipine, reduces Ca2 in£ux into hair cells due to cell depolarization (Ikeda et al., 1991). When nimodipine is administered directly to the cochlea, the compound action potential (CAP) threshold increases, CAP amplitude decreases, and CAP latency increases. In addition, the polarity of the negative component of the summating potential reverses (Bobbin et al., 1990). In vitro studies suggest that hair cell motility is reduced in the presence of nimodipine (Lin et al., 1995). The e¡ects of prolonged stimulation, such as with noise exposure, on Ca2 accumulation in OHCs has not been examined directly, but several groups have shown that mechanical stimulation of isolated hair cells results in increased intracellular Ca2 concentrations. Fridberger and Ulfendahl (1996) reported that mechanical stimulation of isolated OHCs by water jets resulted in increased intracellular Ca2 levels. The authors suggested that such increases in intracellular Ca2 could, over time, result in damage similar to that which occurs with noise exposure. Similarly, Lopez-Escamez and Schacht (1995) reported that vestibular hair cells show increased free Ca2 in the cells following mechanical stimulation with the bathing medium ; intact stereocilia tips were needed for the e¡ect suggesting that Ca2 may enter the cell via transduction channels. The purpose of this study was to determine if nimo-
dipine can reduce the amount of threshold shift caused by a noise exposure. Because one of the primary causes of permanent noise-induced hearing loss is damage to HCs, preventing Ca2 overload in HCs might reduce noise-induced hearing loss. Recently, Maurer et al. (1993) reported that diltiazem (another CCB) may reduce the hearing loss caused by noise exposure, but Boettcher (1996) did not ¢nd such protection by diltiazem. Similarly, Ison et al. (1997) reported that C57BL/ 6J mice receiving nimodipine did not show reductions in hearing loss caused by exposure to a wideband noise (1^100 kHz, 120 dB SPL, 2 min). 2. Materials and methods 2.1. Subjects Subjects were 46 young (3^7 months of age) gerbils (Meriones unguiculatus) raised at the Medical University of South Carolina in an acoustically treated environment. Four groups of 10 gerbils each were used : two experimental groups were given nimodipine and exposed to either 102 or 107 dBA noise and two control groups were given placebo and exposed to either noise. A ¢fth group of six subjects were given nimodipine but not exposed to noise. Hearing thresholds of each subject were determined with auditory brainstem response (ABR) prior to the experiment and one month following the o¡set of the noise exposure. Following the last ABR test, CAP tuning and suppression as well as amplitudes of distortion product otoacoustic emissions (DPOAEs) were measured. Cochleas were preserved in order to assess loss of hair cells. 2.2. Drug delivery Nimodipine was chosen because it is lipophilic, readily crosses the blood-brain barrier, and binds to neural membranes with very high a¤nity (less than 1 nM) (Scriabine et al., 1989). A stable serum level was desired for the duration of a noise exposure. Pellets containing either nimodipine or placebo were implanted at the nape of the neck in each subject. The pellet (Innovative Research Technologies, Toledo, OH) delivered 1 mg/ day, for a dose of approximately 10 mg/kg/day for 14 days (based on an average gerbil weight of 70 g). The administration of nimodipine via 24-h-delivery subcutaneous pellets has been shown to result in stable serum levels in the rat with minimal (less than 10%) reductions in mean arterial blood pressure (Perez-Trepichio and Jones, 1996). The dose is near that prescribed for humans (270^360 mg/day orally or approximately 3.9^5.1 mg/kg/day for a 70 kg person) to prevent cerebral artery spasm (Vinge et al., 1986; Kumana et al., 1993). Two subjects in the group given nimodipine but not
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exposed to noise were tested for serum levels. Blood was drawn one week following implantation and serum levels were determined using high-performance liquid chromatography (Qian and Gallo, 1992; Muck and Bode, 1994). Serum levels were 40 ng/ml in one subject and 76 ng/ml in the other. Comparisons of serum levels for various treatments are found in Section 4 below. 2.3. Noise exposure Gerbils were exposed to an octave band of noise centered at 2 kHz with an overall level of either 102 or 107 dBA. The exposure duration was 7 days and began 7 days after implantation of the drug. The noise was generated, ¢ltered, and attenuated with a TuckerDavis Technologies (TDT) WG-1 waveform generator, PF-1 programmable ¢lter, and PA-4 programmable attenuator, respectively. The noise was then routed to a power ampli¢er and presented through Klipsch loudspeakers located in a reverberant room. The animals remained in home cages throughout the exposures with ad lib food and water. The exposures were calibrated with a BpK sound level meter (levels were þ 2 dB throughout each cage). 2.4. Electrophysiological testing : ABR ABR testing was performed on anesthetized gerbils (ketamine 35 mg/kg and xylazine 8 mg/kg, i.m.) in a double-walled sound booth. Stimuli were Gaussian tone pips (1.8 ms duration, 0.75 ms rise-fall times, generated digitally) at octave intervals from 1 to 16 kHz, presented at levels of 10^80 dB SPL in 10 dB steps (200 presentations/level). Each stimulus was generated digitally with a TDT 16 bit D/A convertor, low-pass ¢ltered at 40 kHz with a TDT anti-aliasing ¢lter, routed to a TDT programmable attenuator, an HP manual attenuator, a mixer, and a headphone bu¡er. Stimuli were presented through a Beyer DT-48 headphone located approximately 3 mm from the pinna. The stimuli were calibrated with a customized probe consisting of a Knowles microphone, using a 1 kHz pure tone. Responses were recorded with subcutaneous wire electrodes placed at vertex (positive) and dorsal to each ear canal (negative). A bite bar served as the ground for the system. Activity was ampli¢ed (100 000U) and ¢ltered (0.3^3 kHz) with a Grass Instruments Model 12 Neurodata Acquisition System, then routed to a TDT A/D 16 bit convertor (25 kHz sampling rate). 2.5. Electrophysiological testing : CAP and DPOAEs Each subject was anesthetized with Nembutal (50 mg/kg, i.p.). Details of the surgery, system, and testing are described in Schmiedt (1986) and Boettcher and
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Schmiedt (1995). Brie£y, after surgical depth of anesthesia was obtained, one pinna was removed so that a customized sound source (consisting of a Beyer DT-48 headphone and a probe tube microphone (BpK 4134) mounted to a head holder) could be placed near the tympanic membrane. The auditory bulla was opened with a minimum of surgical noise and a silver-silver chloride electrode was placed on the bony niche surrounding the round window. Tuning and suppression of the CAP were determined using forward masking procedures (Dallos and Cheatham, 1976). Brie£y, maskers were 50 ms tones (masker o¡set preceded probe onset by 5 ms), swept in frequency at a series of levels. The frequency-intensity combinations at which the response to the probe was just masked were recorded in order to derive tuning curves. The masker was then set to the same frequency of the probe so that the response to the probe was just masked, and a third (suppressor) tone was presented in conjunction with the masker. The suppressor was swept in frequency at a series of levels to determine the suppression contours, i.e., the frequency-intensity combinations for which the probe was `unmasked'. DPOAEs were collected using the CUBe DISP program designed by J. Allen of ATpT Bell Labs. Stimuli were digitized and controlled with Ariel DSP boards located in a PC. The primaries (f1 and f2 ) were routed from the PC to a Crown ampli¢er, HP attenuators, and presented through the customized sound source. DPOAEs were recorded (4 s intervals) using the probe microphone equalized (Applied Research Technology, HD-31) to a £at response and routed to the PC for data storage and analysis). DPOAEs were collected over a frequency range of 0.5^20 kHz (f2 frequency) at eight points per octave, using primary levels of 60 and 50 dB SPL as well as 50 and 40 dB SPL (L1 and L2 , respectively). 2.6. Cochlear histology The cochleae of ¢ve animals from each group were analyzed for hair cell damage using the soft surface preparation technique (Gratton et al., 1990). Brie£y, a deeply anesthetized animal was killed by decapitation, temporal bones were quickly excised, the cochlea exposed and the stapes removed. The cochlea was perfused via a slit made in the round window membrane (4% paraformaldehyde in phosphate bu¡er [PB]) and immersed in ¢xative prior to post-¢xation and staining via perfusion of cold 1% OsO4 in 0.1 M PB. The cochlea was then drilled to thin the otic capsule and decalci¢ed, then microdissected and the organ of Corti was mounted in glycerin for microscopic study. Using di¡erential interference contrast (DIC) microscopy at 400U, the number of missing inner and OHCs was quanti¢ed by row from cochlear apex to base. The cells were con-
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sidered present if the cell body, cuticular plate and stereocilia were intact. Missing cells were identi¢ed by the presence of a phalangeal scar. Counts of the cells by type and condition were normalized and plotted in 2% intervals as a function of percent distance from the apex to obtain a cochleogram, using the frequency-place map for the gerbil described in Tarnowski et al. (1991). 3. Results 3.1. Threshold shifts Baseline thresholds for the control and experimental groups were not signi¢cantly di¡erent. Fig. 1 shows the mean permanent threshold shifts (PTS) ( þ 1 S.E.) recorded for animals exposed to the 102 dBA noise (upper panel) and 107 dBA noise (lower panel). For the 102 dB exposure, mean PTS at each frequency was not signi¢cantly di¡erent (P s 0.05) between the control and experimental groups. PTS was larger for the 107 dBA noise exposures, but there were no signi¢cant di¡erences between subject groups. Subjects exposed only to nimodipine had no PTS (not shown). 3.2. Distortion product otoacoustic emissions Fig. 2 shows mean DPOAE amplitudes plotted as a function of f2 frequency for animals exposed to the 102 dBA noise (upper panels) and 107 dBA noise (lower panels). Data are shown for stimulus levels of L1 = 50 dB SPL and L2 = 40 dB SPL (left panels) and levels of 60 and 50 dB SPL for L1 and L2 , respectively (right panels). Mean noise £oor levels are also shown for each group as are data for a large group of unexposed control animals (from Boettcher and Schmiedt, 1995). For both experiments, DPOAEs near the exposure band (2 kHz) were reduced similar degrees in both control and experimental groups. However, there was a trend for the nimodipine groups to have higher DPOAE amplitudes at approximately 6^10 kHz for each condition. Although a restricted range of the cochlea, it is possible that a small protective e¡ect of the drug occurred in this region. 3.3. Tuning curves and suppression areas Tuning curves of the compound action potential, de-
Fig. 1. Permanent threshold shifts (PTS) ( þ 1 S.E.) for gerbils exposed to 102 dBA (upper) or 107 dBA (lower) noise (octave band centered at 2 kHz). See key for treatment groups (n = 10 for each group).
rived with a forward-masking technique, were compared quantitatively using the Q10dB ¢lter measure, which represents the width (in frequency) of the curve 10 dB above the tip, divided by the probe frequency. Table 1 shows values and standard errors of the Q10dB for each frequency in each group. The Q10dB values were lower for the 107 dB exposure, but there were no signi¢cant di¡erences (t-tests) between experimental and control groups at either exposure level. Suppression of the compound action potential was examined qualitatively, in terms of whether it was present or absent at frequencies above the probe tone. Thus, suppression was considered present regardless of the size of the suppression contour. The suppression areas tended to be larger in the experimental group, but there were no di¡erences between groups in terms of presence or absence of suppression.
Table 1 Tuning curve Q10dB values (mean þ S.E.) Group
1 kHz
2 kHz
4 kHz
8 kHz
16 kHz
102-Placebo 102-Nimodipine 107-Placebo 107-Nimodipine
1.53 þ 0.29 1.16 þ 0.31 1.17 þ 0.20 1.14 þ 0.16
2.28 þ 0.38 1.75 þ 0.21 1.44 þ 0.23 1.05 þ 0.14
1.20 þ 0.19 1.01 þ 0.08 0.75 þ 0.13 0.90 þ 0.09
2.87 þ 0.46 4.12 þ 0.52 2.08 þ 0.49 3.10 þ 0.45
6.97 þ 1.50 6.93 þ 0.98 6.11 þ 0.65 7.16 þ 1.22
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Fig. 2. DPOAE amplitudes plotted as a function of f2 frequency for subjects exposed to 102 dBA (upper panels) or 107 dBA noise (lower panels). Data are shown for primary levels of 50 and 40 dB SPL (L1 and L2 , respectively; left panels) and 60 and 50 dB SPL (L1 and L2 , respectively; right panels). In each panel, DPOAEs are shown for unexposed controls, noise-placebo group, and noise-nimodipine group, as are noise £oors for the latter two groups.
3.4. Cochlear histology Virtually all inner hair cells remained intact in all groups. Approximately 20% of the OHC in the region corresponding to 6 kHz were found to be missing in the group exposed to the placebo and 102 dB noise (Fig. 3A). This sharp focal loss was evident primarily in the second and third OHC rows. A small focal region existed at 16 kHz and involved loss of 10% of third row OHC. In contrast, the OHC loss in the group exposed to the nimodipine and 102 dB noise (Fig. 3B) was slightly more widespread (6^9 kHz) and involved only 15% of the third row of OHC. The OHC loss shown in the low frequencies was noted in only one cochlea and was felt to be artifactual. The group exposed to the placebo and 107 dB loss (Fig. 3C) had a slightly greater OHC loss to all three HC rows (25%) than did the placebo/102 dB noise counterpart (Fig. 3A) in essentially the same frequency region (6^12 kHz). The OHC loss for subjects exposed to 107 dBA noise was approximately 15% over the frequency range of 4^10 kHz. The histological data thus suggest that little pro-
tection from the noise exposure is derived from treatment by nimodipine. 4. Discussion Nimodipine did not reduce the permanent hearing loss or hair cell loss caused by moderately intense noise exposure in the gerbil. Furthermore, nimodipine did not prevent the reductions in CAP tuning or suppression caused by noise exposure, and did not prevent noiseinduced reductions in DPOAE amplitudes except at the upper boundary of the hearing loss. The results are thus generally similar to those observed for diltiazem in this lab (Boettcher, 1996). Diltiazem (30 mg/kg/day) did not reduce PTS caused by exposure to noise (4 kHz, 90 dB SPL, 5 days), nor did it prevent or reduce the temporary threshold shift caused by exposure to 4 kHz tones (90 dB SPL, 20 min) in gerbils. Diltiazem had previously been reported to reduce threshold shift caused by noise exposure in guinea pigs (Maurer et al., 1993), but no such protection was observed in our lab. Maurer et
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but the drug regimen had an e¡ect on cochlear function, as the P1 latency of the ABR was increased in experimental subjects. The conclusions of Ison and colleagues are thus generally consistent with those of the present experiment. It is, however, worth considering several issues in experimental design which di¡er between Ison et al. (1997) and the present experiment. The C57 mouse loses hair cells and hearing early in life due to genetic factors and thus the baseline condition of the auditory system may have been changing over the course of the mouse experiments. Furthermore, the very intense noise exposure may have caused some mechanical damage to the cochlea, in a manner that di¡ers from more moderate, long-term noise exposures. Taken together, however, the results of Ison and colleagues and those of the present experiment suggest that nimodipine, at a dose which results in clinically relevant serum levels and at a dose which prolongs P1 latencies, does not reduce noise-induced hearing loss. 4.1. E¡ect for DPOAEs The single piece of evidence suggesting a protective e¡ect for nimodipine was that DPOAE amplitudes were less reduced in the nimodipine groups than the placebo groups. This e¡ect was limited to f2 frequencies of approximately 6^10 kHz, a region corresponding to the upper boundary of the PTS and the hair cell loss. This observation is interesting given that Boettcher and Schmiedt (1995) and Kim et al. (1992) have both reported increases in DPOAE amplitudes at the upper boundary of a hearing loss. However, given the lack of protection from noise in terms of threshold shift, CAP tuning and suppression, as well as hair cell loss, it is unlikely that the DPOAE observation is particularly relevant. 4.2. Why was nimodipine not e¡ective? Fig. 3. Mean percent of outer hair cells (OHCs) present is plotted against frequency for each group (n = 5 per group). Data for each row of OHCs are shown separately as indicated.
al. (1995) also reported that patients given diltiazem prior to otologic surgery had less, but not signi¢cantly less, hearing loss as a result of surgery than patients given a placebo. There are con£icting data in the literature as to the ability of diltiazem to cross the bloodbrain barrier, whereas nimodipine readily crosses the barrier (Allen et al., 1983). Ison et al. (1997) reported that a daily oral dose of 30 mg/kg of nimodipine (mixed into a food supplement) did not reduce hearing loss in C57 mice caused by an intense wideband noise (1^100 kHz, 120 dB SPL, 2 min). Serum levels of nimodipine were not obtained,
Several possibilities may explain the negative results in this study : (a) that cochlear concentrations of nimodipine were too low or (b) that CCBs cannot reduce NIHL. Nimodipine was delivered at a rate of 1 mg/ day for 14 days, or approximately 10 mg/kg/day for 14 days (based on an average gerbil weight of 70 g). This resulted in serum levels of 40^76 ng/ml, although only two subjects were tested. The administration of nimodipine via 24-h-delivery subcutaneous pellets has been shown to result in stable serum levels in the rat (Perez-Trepichio and Jones, 1996). Therapeutic doses of nimodipine for the prevention of cerebral vasospasm following subarachnoid hemorrhage are typically 45^ 60 mg every 4 h by mouth (Vinge et al., 1986; Kumana et al., 1993). A 60 mg dose every 4 h results in average serum levels of 33.5 ng/ml (Kumana et al., 1993); however, serum levels may vary greatly after an oral dose as
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Vinge et al. (1986) reported serum levels of 7^96 ng/ml following oral doses of 45 mg, every 6 h. Peak serum levels occur approximately 0.7^1 h after administration (Ramsch et al., 1985). Thus, our delivery method, which resulted in serum levels of 40^76 ng/ml, were in the range commonly observed to have therapeutic effects in prevention of vasospasm following subarachnoid hemorrhage (Vinge et al., 1986; Mee et al., 1988). There remains the obvious question of what the levels of nimodipine were in the cochlear £uids. It has been estimated that the perilymph levels were likely to have been approximately 0.1^0.2 WM, given a molecular weight of 418 for nimodipine and assuming equal concentrations of nimodipine in serum and perilymph. Bobbin et al. (1990) examined the CAP, cochlear microphonic (CM) and summating potential (SP) before and after cochlear perfusion with various levels of nimodipine in arti¢cial perilymph. A concentration of 0.1 WM nimodipine did not a¡ect the physiologic measures. Increasing the dose to 0.33 WM caused a reduction in CAP amplitude, particularly at low stimulus levels, with a threshold shift of approximately 6 dB. The negative portion of the SP became slightly more positive. A higher dose (1 WM) resulted in a CAP threshold shift of approximately 30 dB and a polarity change in the negative SP. Although Bobbin et al. (1990) reported that physiologic changes occurring with the 0.33 WM concentration of nimodipine were not signi¢cant (and that those at 1 WM were signi¢cant), there is a trend in their data that suggests nimodipine had some e¡ect on hair cells at the 0.33 WM concentration. Furthermore, a threshold change of 30 dB is a fairly large change in cochlear function, again suggesting that lower concentrations may also a¡ect cochlear function. The concept that nimodipine has a large e¡ect at concentrations of 1 WM, but that the `threshold' of nimodipine e¡ects is much lower, is supported by in vitro work with hair cells. Lin et al. (1995) examined the response of OHCs to the bathing medium with Ca2 and a variety of CCBs. Nimodipine showed reduction of K currents at concentrations below 1 WM, but the half-inhibitory concentration (the concentration which decreased the K current by 50%) was 6 WM. In summary, the serum levels of nimodipine were similar to those observed in experimental and clinical studies of the ability of nimodipine to reduce neurologic injury and arterial spasm following subarachnoid hemorrhage. However, the levels of nimodipine in the cochlear £uids were not measured in this study. Data from in vivo and in vitro studies suggest that nimodipine a¡ects hair cells at very low concentrations, but signi¢cant e¡ects are not observed until concentrations are at or near 1 WM. This would suggest that a protective e¡ect by nimodipine against NIHL cannot be ruled out, but the dose level required would in itself cause at least a moderate temporary hearing loss.
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Acknowledgments The authors wish to thank Drs. Lynne Bianchi, Judy Dubno, John Mills, and Richard Schmiedt for comments on an earlier version of the manuscript. We also appreciate the estimate of nimodipine levels in cochlear £ues contributed by an anonymous reviewer. This study was supported by grants from the National Organization for Hearing Research and National Institutes of Health-NIDCD P50-DC-00422. Portions of this paper were presented at the Nineteenth Midwinter Meeting of the Association for Research in Otolaryngology, St. Petersburg Beach, FL, February, 1996.
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Hille, B., 1992. Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA. Hudspeth, A.J., 1986. The ionic channels of a vertebrate hair cell. Hear. Res. 2, 21^27. Ikeda, K., Saito, Y., Nishiyama, A., Takasaka, T., 1991. E¡ects of pH on intracellular calcium levels in isolated cochlear outer hair cells of guinea pigs. Am. J. Physiol. 261, C231^C236. Ison, J.R., Payman, G.H., Palmer, M.J., Walton, J.P., 1997. Nimodipine at a dose that slows ABR latencies does not protect the ear against noise. Hear. Res. 106, 179^183. Kim, D.O., Leonard, G., Smurzynski, J., Jung, M.D., 1992. Otoacoustic emissions and noise-induced hearing loss: Human studies. In: Dancer, A., Henderson, D., Salvi, R., Hamernik, R. (Eds.), Noise-Induced Hearing Loss, Mosby Yearbook, St. Louis, MO, pp. 98^105. Kumana, C.R., Kou, M., Yu, Y.L., Fong, K.Y., Fung, C.F., Chang, C.M., Muck, W., Lauder, I.J., 1993. Investigation of nimodipine pharmacokinetics in Chinese patients with acute subarachnoid hemorrhage. Eur. J. Clin. Pharmacol. 45, 363^366. Lin, X., Hume, R.I., Nuttall, A.L., 1995. Dihydropyridines and verapamil inhibit voltage-dependent K current in isolated outer hair cells of the guinea pig. Hear. Res. 88, 36^46. Lopez-Escamez, J.A., Schacht, J., 1995. Mechanically induced calcium increases in isolated vestibular hair cells of the guinea pig. Acta Otolaryngol. 115, 759^764. Maurer, J., Mann, W., Schneider, M., Strecker, G., 1993. Einsatz von Ca -Antagonisten bei Larmbelaëstung im Tierversuch. HNO 41, 192^197. Maurer, J., Riechelmann, H., Amedee, R.G., Mann, W.J., 1995. Diltiazem for prevention of acoustical trauma during otologic surgery. ORL, J. Oto-Rhino-Laryngol. Rel. Spec. 57, 319^324. Mee, E., Dorrance, D., Lowe, D., Neil-Dwyer, G., 1988. Controlled study of nimodipine in patients treated early after subarachnoid hemorrhage. Neurosurgery 22, 484^491. Muck, W., Bode, H., 1994. Bioanalytics of nimodipine ^ an overview of methods. Pharmazie 49, 130^139. Nakagawa, T., Kakehata, S., Akaike, N., Kommune, S., Takasaka, T., Uemura, T., 1991. Calcium channels in isolated outer hair cells of guinea pig cochlea. Neurosci. Lett. 125, 81^84. Nayler, W.G., 1988. Calcium Antagonists. Academic Press, San Diego, CA. Orrenius, S., McCabe, M.J., Jr., Nicotera, P., 1992. Ca2 -dependent mechanisms of cytotoxicity and programmed cell death. Toxicol. Lett. 64/65, 357^364.
Perez-Trepichio, A.D., Jones, S.C., 1996. Evaluation of a novel nimodipine delivery system in conscious rats that allows sustained release for 24 h. J. Neurosci. Methods 68, 297^301. Qian, M., Gallo, J.M., 1992. High-performance liquid chromatographic determination of the calcium channel blocker nimodipine in monkey plasma. J. Chromatogr. 578, 316^320. Ramsch, K.D., Ahr, G., Tettenborn, D., Auer, L.M., 1985. Overview on pharmacokinetics of nimodipine in healthy volunteers and in patients with subarachnoid hemorrhage. Neurochiurgia 28 (Suppl. 1), 74^78. Ricci, A.J., Fettiplace, R., 1997. The e¡ects of calcium bu¡ering and cyclic AMP on mechano-electric transduction in turtle auditory hair cells. J. Physiol. 501, 111^124. Santos-Sacchi, J., Dilger, J.P., 1988. Whole cell currents and mechanical responses of isolated outer hair cells. Hear. Res. 35, 143^ 150. Schmiedt, R.A., 1986. Acoustic distortion in the ear canal. I. Cubic di¡erence tones: E¡ects on acute noise injury. J. Acoust. Soc. Am. 79, 1481^1490. Scriabine, A., Schuurman, T., Traber, J., 1989. Pharmacological basis for the use of nimodipine in central nervous system disorders. FASEB J. 3, 1799^1806. Siegel, J.H., Relkin, E.M., 1987. Antagonistic e¡ects of perilymphatic calcium and magnesium on the activity of single cochlear a¡erent neurons. Hear. Res. 28, 131^147. Sridhar, T.S., Brown, M.C., Sewell, W.F., 1997. Unique postsynaptic signaling at the hair cell e¡erent synapse permits calcium to evoke changes on two time scales. J. Neurosci. 17, 428^437. Tarnowski, B.I., Schmiedt, R.A., Hellstrom, L.I., Lee, F.S., Adams, J.C., 1991. Age-related changes in cochleas of Mongolian gerbils. Hear. Res. 54, 123^134. Torre, V., Ashmore, J.F., Lamb, T.D., Menini, A., 1995. Transduction and adaptation in sensory receptor cells. J. Neurosci. 15, 7757^7768. Tsien, R.Y., 1989. Fluorescent probes of cell signaling. Annu. Rev. Neurosci. 12, 227^253. Verity, M.A., 1992. Ca2 -dependent processes as mediators of neurotoxicity. Neurotoxicology 13, 139^148. Vinge, E., Andersson, K.E., Brandt, L., Ljunggren, B., Nilsson, L.G., Rosendal-Helgesen, S., 1986. Pharmacokinetics of nimodipine in patients with aneurysmal subarachnoid haemorrhage. Eur. J. Clin. Pharmacol. 30, 421^425.
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E¡ects of nimodipine on noise-induced hearing loss Flint A. Boettcher *, Richard K. Caldwell, Michael Anne Gratton 1 , David R. White, Lesa R. Miles Department of Otolaryngology and Communicative Sciences, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2242, USA Received 4 December 1997; revised 13 April 1998; accepted 18 April 1998
Abstract The effects of nimodipine, a calcium channel blocker, on noise-induced hearing loss were examined in gerbils. Animals were implanted subcutaneously with a timed-release pellet containing either nimodipine (approximately 10 mg/kg/day) or placebo and exposed to either 102 or 107 dBA noise. Serum levels were tested in two subjects and were in the range known to protect humans from cerebral artery vasospasm and ischemia-related neurologic deficits. Nimodipine and control groups had similar amounts of noise-induced (a) permanent threshold shift ; (b) reductions in distortion product otoacoustic emissions ; (c) reductions in tuning and suppression of the compound action potential; and (d) loss of outer hair cells. The results suggest that nimodipine, at a dose which results in clinically relevant serum levels, does not provide protection from the effects of moderately intense noise exposures. z 1998 Published by Elsevier Science B.V. All rights reserved. Key words: Calcium channel blocker; Gerbil; Nimodipine; Noise-induced hearing loss
1. Introduction Noise-induced hearing loss (NIHL) is one of the most common occupational health hazards in industrial societies. The primary damage caused by excess noise exposure is to sensory hair cells in the organ of Corti, particularly outer hair cells (OHCs) (for a recent review, see Henderson and Hamernik, 1995). Noise exposure which causes permanent hearing loss typically results in bending, fusion, and fracture of hair cell stereocilia, vacuolization of hair cells, and ultimately hair cell death and degeneration. Hair cells are replaced by squamous epithelium which forms a scar in the place of the cell. The biological mechanisms underlying acoustic injury to the cochlea are not known. Calcium (Ca2 ) is a ubiquitous ion in mammalian cells (though at very low free intracellular concentra* Corresponding author. Tel.: +1 (803) 792-8291; Fax: +1 (803) 792-7736; E-mail: [email protected] 1 Present address: Boys Town National Research Hospital, 555 N. 30th St., Omaha, NE 68131, USA.
tions) and is critical in regulation of many cellular activities such as neurotransmitter release and cell movement. However, an excessive level of free intracellular Ca2 (resulting from entry through ion channels or release from intracellular stores) overactivates a series of enzymes including phospholipases, protein kinase C, proteases, endonucleases and depolymerases. The result is membrane breakdown, depolymerization of microtubules, and disruption of protein synthesis (Orrenius et al., 1992 ; Verity, 1992). Ca2 entry into cells can be controlled, in part, by drugs which block voltage-gated channels. Calcium channel blockers (CCBs) are drugs which restrict entry of Ca2 into cells. Nimodipine, a CCB of the dihydropyridine class, is lipophilic and readily crosses the blood-brain barrier (Allen et al., 1983). The drug blocks Ca2 channels of the L-type (large conductances with long-lasting e¡ects [Tsien, 1989; Hille, 1992]). Nimodipine is used to prevent cerebral artery spasms following subarachnoid hemorrhage (Allen et al., 1983; Mee et al., 1988) and to reduce neurologic de¢cits following ischemic stroke (Gelmers, 1985;
0378-5955 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 0 7 5 - 6
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Gelmers and Hennerici, 1990). One consequence of tissue ischemia is a large in£ux of Ca2 from extracellular £uid to the intracellular milieu during reperfusion of tissue. CCBs also have been shown experimentally to prevent or reduce Ca2 cytotoxicity following ischemia in the heart and kidney (for a review, see Nayler, 1988). The functions of Ca2 in hair cell (HC) activity are not completely understood, but several roles have been demonstrated or postulated, including regulation of: (a) slow motility of the OHCs (Dulon et al., 1991; Sridhar et al., 1997); (b) HC transduction (Hudspeth, 1986 ; see Fettiplace, 1992 for a recent review) and adaptation (Corey and Hudspeth, 1979; Ricci and Fettiplace, 1997 ; see Torre et al., 1995 for a recent review); (c) gating of ion channels (Hudspeth, 1986; Art et al., 1993); and (d) release of neurotransmitter (Siegel and Relkin, 1987; Guth et al., 1991). Calcium enters OHCs through several types of channels. Cation channels near stereocilia tips primarily allow entry of potassium, but Ca2 can also enter (Corey and Hudspeth, 1979; Lopez-Escamez and Schacht, 1995). L-type Ca2 channels have been demonstrated in OHCs (Santos-Sacchi and Dilger, 1988; Nakagawa et al., 1991) and 12 Ca2 channel subunits have been identi¢ed in the cochlea using the polymerase chain reaction (Green et al., 1996). Depolarizing the cell with potassium causes a large increase in intracellular Ca2 in hair cells, probably due to entry through voltage-sensitive Ca2 channels (Hudspeth, 1986; Fettiplace, 1992). Nifedipine, a CCB similar in structure and function to nimodipine, reduces Ca2 in£ux into hair cells due to cell depolarization (Ikeda et al., 1991). When nimodipine is administered directly to the cochlea, the compound action potential (CAP) threshold increases, CAP amplitude decreases, and CAP latency increases. In addition, the polarity of the negative component of the summating potential reverses (Bobbin et al., 1990). In vitro studies suggest that hair cell motility is reduced in the presence of nimodipine (Lin et al., 1995). The e¡ects of prolonged stimulation, such as with noise exposure, on Ca2 accumulation in OHCs has not been examined directly, but several groups have shown that mechanical stimulation of isolated hair cells results in increased intracellular Ca2 concentrations. Fridberger and Ulfendahl (1996) reported that mechanical stimulation of isolated OHCs by water jets resulted in increased intracellular Ca2 levels. The authors suggested that such increases in intracellular Ca2 could, over time, result in damage similar to that which occurs with noise exposure. Similarly, Lopez-Escamez and Schacht (1995) reported that vestibular hair cells show increased free Ca2 in the cells following mechanical stimulation with the bathing medium ; intact stereocilia tips were needed for the e¡ect suggesting that Ca2 may enter the cell via transduction channels. The purpose of this study was to determine if nimo-
dipine can reduce the amount of threshold shift caused by a noise exposure. Because one of the primary causes of permanent noise-induced hearing loss is damage to HCs, preventing Ca2 overload in HCs might reduce noise-induced hearing loss. Recently, Maurer et al. (1993) reported that diltiazem (another CCB) may reduce the hearing loss caused by noise exposure, but Boettcher (1996) did not ¢nd such protection by diltiazem. Similarly, Ison et al. (1997) reported that C57BL/ 6J mice receiving nimodipine did not show reductions in hearing loss caused by exposure to a wideband noise (1^100 kHz, 120 dB SPL, 2 min). 2. Materials and methods 2.1. Subjects Subjects were 46 young (3^7 months of age) gerbils (Meriones unguiculatus) raised at the Medical University of South Carolina in an acoustically treated environment. Four groups of 10 gerbils each were used : two experimental groups were given nimodipine and exposed to either 102 or 107 dBA noise and two control groups were given placebo and exposed to either noise. A ¢fth group of six subjects were given nimodipine but not exposed to noise. Hearing thresholds of each subject were determined with auditory brainstem response (ABR) prior to the experiment and one month following the o¡set of the noise exposure. Following the last ABR test, CAP tuning and suppression as well as amplitudes of distortion product otoacoustic emissions (DPOAEs) were measured. Cochleas were preserved in order to assess loss of hair cells. 2.2. Drug delivery Nimodipine was chosen because it is lipophilic, readily crosses the blood-brain barrier, and binds to neural membranes with very high a¤nity (less than 1 nM) (Scriabine et al., 1989). A stable serum level was desired for the duration of a noise exposure. Pellets containing either nimodipine or placebo were implanted at the nape of the neck in each subject. The pellet (Innovative Research Technologies, Toledo, OH) delivered 1 mg/ day, for a dose of approximately 10 mg/kg/day for 14 days (based on an average gerbil weight of 70 g). The administration of nimodipine via 24-h-delivery subcutaneous pellets has been shown to result in stable serum levels in the rat with minimal (less than 10%) reductions in mean arterial blood pressure (Perez-Trepichio and Jones, 1996). The dose is near that prescribed for humans (270^360 mg/day orally or approximately 3.9^5.1 mg/kg/day for a 70 kg person) to prevent cerebral artery spasm (Vinge et al., 1986; Kumana et al., 1993). Two subjects in the group given nimodipine but not
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exposed to noise were tested for serum levels. Blood was drawn one week following implantation and serum levels were determined using high-performance liquid chromatography (Qian and Gallo, 1992; Muck and Bode, 1994). Serum levels were 40 ng/ml in one subject and 76 ng/ml in the other. Comparisons of serum levels for various treatments are found in Section 4 below. 2.3. Noise exposure Gerbils were exposed to an octave band of noise centered at 2 kHz with an overall level of either 102 or 107 dBA. The exposure duration was 7 days and began 7 days after implantation of the drug. The noise was generated, ¢ltered, and attenuated with a TuckerDavis Technologies (TDT) WG-1 waveform generator, PF-1 programmable ¢lter, and PA-4 programmable attenuator, respectively. The noise was then routed to a power ampli¢er and presented through Klipsch loudspeakers located in a reverberant room. The animals remained in home cages throughout the exposures with ad lib food and water. The exposures were calibrated with a BpK sound level meter (levels were þ 2 dB throughout each cage). 2.4. Electrophysiological testing : ABR ABR testing was performed on anesthetized gerbils (ketamine 35 mg/kg and xylazine 8 mg/kg, i.m.) in a double-walled sound booth. Stimuli were Gaussian tone pips (1.8 ms duration, 0.75 ms rise-fall times, generated digitally) at octave intervals from 1 to 16 kHz, presented at levels of 10^80 dB SPL in 10 dB steps (200 presentations/level). Each stimulus was generated digitally with a TDT 16 bit D/A convertor, low-pass ¢ltered at 40 kHz with a TDT anti-aliasing ¢lter, routed to a TDT programmable attenuator, an HP manual attenuator, a mixer, and a headphone bu¡er. Stimuli were presented through a Beyer DT-48 headphone located approximately 3 mm from the pinna. The stimuli were calibrated with a customized probe consisting of a Knowles microphone, using a 1 kHz pure tone. Responses were recorded with subcutaneous wire electrodes placed at vertex (positive) and dorsal to each ear canal (negative). A bite bar served as the ground for the system. Activity was ampli¢ed (100 000U) and ¢ltered (0.3^3 kHz) with a Grass Instruments Model 12 Neurodata Acquisition System, then routed to a TDT A/D 16 bit convertor (25 kHz sampling rate). 2.5. Electrophysiological testing : CAP and DPOAEs Each subject was anesthetized with Nembutal (50 mg/kg, i.p.). Details of the surgery, system, and testing are described in Schmiedt (1986) and Boettcher and
141
Schmiedt (1995). Brie£y, after surgical depth of anesthesia was obtained, one pinna was removed so that a customized sound source (consisting of a Beyer DT-48 headphone and a probe tube microphone (BpK 4134) mounted to a head holder) could be placed near the tympanic membrane. The auditory bulla was opened with a minimum of surgical noise and a silver-silver chloride electrode was placed on the bony niche surrounding the round window. Tuning and suppression of the CAP were determined using forward masking procedures (Dallos and Cheatham, 1976). Brie£y, maskers were 50 ms tones (masker o¡set preceded probe onset by 5 ms), swept in frequency at a series of levels. The frequency-intensity combinations at which the response to the probe was just masked were recorded in order to derive tuning curves. The masker was then set to the same frequency of the probe so that the response to the probe was just masked, and a third (suppressor) tone was presented in conjunction with the masker. The suppressor was swept in frequency at a series of levels to determine the suppression contours, i.e., the frequency-intensity combinations for which the probe was `unmasked'. DPOAEs were collected using the CUBe DISP program designed by J. Allen of ATpT Bell Labs. Stimuli were digitized and controlled with Ariel DSP boards located in a PC. The primaries (f1 and f2 ) were routed from the PC to a Crown ampli¢er, HP attenuators, and presented through the customized sound source. DPOAEs were recorded (4 s intervals) using the probe microphone equalized (Applied Research Technology, HD-31) to a £at response and routed to the PC for data storage and analysis). DPOAEs were collected over a frequency range of 0.5^20 kHz (f2 frequency) at eight points per octave, using primary levels of 60 and 50 dB SPL as well as 50 and 40 dB SPL (L1 and L2 , respectively). 2.6. Cochlear histology The cochleae of ¢ve animals from each group were analyzed for hair cell damage using the soft surface preparation technique (Gratton et al., 1990). Brie£y, a deeply anesthetized animal was killed by decapitation, temporal bones were quickly excised, the cochlea exposed and the stapes removed. The cochlea was perfused via a slit made in the round window membrane (4% paraformaldehyde in phosphate bu¡er [PB]) and immersed in ¢xative prior to post-¢xation and staining via perfusion of cold 1% OsO4 in 0.1 M PB. The cochlea was then drilled to thin the otic capsule and decalci¢ed, then microdissected and the organ of Corti was mounted in glycerin for microscopic study. Using di¡erential interference contrast (DIC) microscopy at 400U, the number of missing inner and OHCs was quanti¢ed by row from cochlear apex to base. The cells were con-
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sidered present if the cell body, cuticular plate and stereocilia were intact. Missing cells were identi¢ed by the presence of a phalangeal scar. Counts of the cells by type and condition were normalized and plotted in 2% intervals as a function of percent distance from the apex to obtain a cochleogram, using the frequency-place map for the gerbil described in Tarnowski et al. (1991). 3. Results 3.1. Threshold shifts Baseline thresholds for the control and experimental groups were not signi¢cantly di¡erent. Fig. 1 shows the mean permanent threshold shifts (PTS) ( þ 1 S.E.) recorded for animals exposed to the 102 dBA noise (upper panel) and 107 dBA noise (lower panel). For the 102 dB exposure, mean PTS at each frequency was not signi¢cantly di¡erent (P s 0.05) between the control and experimental groups. PTS was larger for the 107 dBA noise exposures, but there were no signi¢cant di¡erences between subject groups. Subjects exposed only to nimodipine had no PTS (not shown). 3.2. Distortion product otoacoustic emissions Fig. 2 shows mean DPOAE amplitudes plotted as a function of f2 frequency for animals exposed to the 102 dBA noise (upper panels) and 107 dBA noise (lower panels). Data are shown for stimulus levels of L1 = 50 dB SPL and L2 = 40 dB SPL (left panels) and levels of 60 and 50 dB SPL for L1 and L2 , respectively (right panels). Mean noise £oor levels are also shown for each group as are data for a large group of unexposed control animals (from Boettcher and Schmiedt, 1995). For both experiments, DPOAEs near the exposure band (2 kHz) were reduced similar degrees in both control and experimental groups. However, there was a trend for the nimodipine groups to have higher DPOAE amplitudes at approximately 6^10 kHz for each condition. Although a restricted range of the cochlea, it is possible that a small protective e¡ect of the drug occurred in this region. 3.3. Tuning curves and suppression areas Tuning curves of the compound action potential, de-
Fig. 1. Permanent threshold shifts (PTS) ( þ 1 S.E.) for gerbils exposed to 102 dBA (upper) or 107 dBA (lower) noise (octave band centered at 2 kHz). See key for treatment groups (n = 10 for each group).
rived with a forward-masking technique, were compared quantitatively using the Q10dB ¢lter measure, which represents the width (in frequency) of the curve 10 dB above the tip, divided by the probe frequency. Table 1 shows values and standard errors of the Q10dB for each frequency in each group. The Q10dB values were lower for the 107 dB exposure, but there were no signi¢cant di¡erences (t-tests) between experimental and control groups at either exposure level. Suppression of the compound action potential was examined qualitatively, in terms of whether it was present or absent at frequencies above the probe tone. Thus, suppression was considered present regardless of the size of the suppression contour. The suppression areas tended to be larger in the experimental group, but there were no di¡erences between groups in terms of presence or absence of suppression.
Table 1 Tuning curve Q10dB values (mean þ S.E.) Group
1 kHz
2 kHz
4 kHz
8 kHz
16 kHz
102-Placebo 102-Nimodipine 107-Placebo 107-Nimodipine
1.53 þ 0.29 1.16 þ 0.31 1.17 þ 0.20 1.14 þ 0.16
2.28 þ 0.38 1.75 þ 0.21 1.44 þ 0.23 1.05 þ 0.14
1.20 þ 0.19 1.01 þ 0.08 0.75 þ 0.13 0.90 þ 0.09
2.87 þ 0.46 4.12 þ 0.52 2.08 þ 0.49 3.10 þ 0.45
6.97 þ 1.50 6.93 þ 0.98 6.11 þ 0.65 7.16 þ 1.22
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Fig. 2. DPOAE amplitudes plotted as a function of f2 frequency for subjects exposed to 102 dBA (upper panels) or 107 dBA noise (lower panels). Data are shown for primary levels of 50 and 40 dB SPL (L1 and L2 , respectively; left panels) and 60 and 50 dB SPL (L1 and L2 , respectively; right panels). In each panel, DPOAEs are shown for unexposed controls, noise-placebo group, and noise-nimodipine group, as are noise £oors for the latter two groups.
3.4. Cochlear histology Virtually all inner hair cells remained intact in all groups. Approximately 20% of the OHC in the region corresponding to 6 kHz were found to be missing in the group exposed to the placebo and 102 dB noise (Fig. 3A). This sharp focal loss was evident primarily in the second and third OHC rows. A small focal region existed at 16 kHz and involved loss of 10% of third row OHC. In contrast, the OHC loss in the group exposed to the nimodipine and 102 dB noise (Fig. 3B) was slightly more widespread (6^9 kHz) and involved only 15% of the third row of OHC. The OHC loss shown in the low frequencies was noted in only one cochlea and was felt to be artifactual. The group exposed to the placebo and 107 dB loss (Fig. 3C) had a slightly greater OHC loss to all three HC rows (25%) than did the placebo/102 dB noise counterpart (Fig. 3A) in essentially the same frequency region (6^12 kHz). The OHC loss for subjects exposed to 107 dBA noise was approximately 15% over the frequency range of 4^10 kHz. The histological data thus suggest that little pro-
tection from the noise exposure is derived from treatment by nimodipine. 4. Discussion Nimodipine did not reduce the permanent hearing loss or hair cell loss caused by moderately intense noise exposure in the gerbil. Furthermore, nimodipine did not prevent the reductions in CAP tuning or suppression caused by noise exposure, and did not prevent noiseinduced reductions in DPOAE amplitudes except at the upper boundary of the hearing loss. The results are thus generally similar to those observed for diltiazem in this lab (Boettcher, 1996). Diltiazem (30 mg/kg/day) did not reduce PTS caused by exposure to noise (4 kHz, 90 dB SPL, 5 days), nor did it prevent or reduce the temporary threshold shift caused by exposure to 4 kHz tones (90 dB SPL, 20 min) in gerbils. Diltiazem had previously been reported to reduce threshold shift caused by noise exposure in guinea pigs (Maurer et al., 1993), but no such protection was observed in our lab. Maurer et
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but the drug regimen had an e¡ect on cochlear function, as the P1 latency of the ABR was increased in experimental subjects. The conclusions of Ison and colleagues are thus generally consistent with those of the present experiment. It is, however, worth considering several issues in experimental design which di¡er between Ison et al. (1997) and the present experiment. The C57 mouse loses hair cells and hearing early in life due to genetic factors and thus the baseline condition of the auditory system may have been changing over the course of the mouse experiments. Furthermore, the very intense noise exposure may have caused some mechanical damage to the cochlea, in a manner that di¡ers from more moderate, long-term noise exposures. Taken together, however, the results of Ison and colleagues and those of the present experiment suggest that nimodipine, at a dose which results in clinically relevant serum levels and at a dose which prolongs P1 latencies, does not reduce noise-induced hearing loss. 4.1. E¡ect for DPOAEs The single piece of evidence suggesting a protective e¡ect for nimodipine was that DPOAE amplitudes were less reduced in the nimodipine groups than the placebo groups. This e¡ect was limited to f2 frequencies of approximately 6^10 kHz, a region corresponding to the upper boundary of the PTS and the hair cell loss. This observation is interesting given that Boettcher and Schmiedt (1995) and Kim et al. (1992) have both reported increases in DPOAE amplitudes at the upper boundary of a hearing loss. However, given the lack of protection from noise in terms of threshold shift, CAP tuning and suppression, as well as hair cell loss, it is unlikely that the DPOAE observation is particularly relevant. 4.2. Why was nimodipine not e¡ective? Fig. 3. Mean percent of outer hair cells (OHCs) present is plotted against frequency for each group (n = 5 per group). Data for each row of OHCs are shown separately as indicated.
al. (1995) also reported that patients given diltiazem prior to otologic surgery had less, but not signi¢cantly less, hearing loss as a result of surgery than patients given a placebo. There are con£icting data in the literature as to the ability of diltiazem to cross the bloodbrain barrier, whereas nimodipine readily crosses the barrier (Allen et al., 1983). Ison et al. (1997) reported that a daily oral dose of 30 mg/kg of nimodipine (mixed into a food supplement) did not reduce hearing loss in C57 mice caused by an intense wideband noise (1^100 kHz, 120 dB SPL, 2 min). Serum levels of nimodipine were not obtained,
Several possibilities may explain the negative results in this study : (a) that cochlear concentrations of nimodipine were too low or (b) that CCBs cannot reduce NIHL. Nimodipine was delivered at a rate of 1 mg/ day for 14 days, or approximately 10 mg/kg/day for 14 days (based on an average gerbil weight of 70 g). This resulted in serum levels of 40^76 ng/ml, although only two subjects were tested. The administration of nimodipine via 24-h-delivery subcutaneous pellets has been shown to result in stable serum levels in the rat (Perez-Trepichio and Jones, 1996). Therapeutic doses of nimodipine for the prevention of cerebral vasospasm following subarachnoid hemorrhage are typically 45^ 60 mg every 4 h by mouth (Vinge et al., 1986; Kumana et al., 1993). A 60 mg dose every 4 h results in average serum levels of 33.5 ng/ml (Kumana et al., 1993); however, serum levels may vary greatly after an oral dose as
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Vinge et al. (1986) reported serum levels of 7^96 ng/ml following oral doses of 45 mg, every 6 h. Peak serum levels occur approximately 0.7^1 h after administration (Ramsch et al., 1985). Thus, our delivery method, which resulted in serum levels of 40^76 ng/ml, were in the range commonly observed to have therapeutic effects in prevention of vasospasm following subarachnoid hemorrhage (Vinge et al., 1986; Mee et al., 1988). There remains the obvious question of what the levels of nimodipine were in the cochlear £uids. It has been estimated that the perilymph levels were likely to have been approximately 0.1^0.2 WM, given a molecular weight of 418 for nimodipine and assuming equal concentrations of nimodipine in serum and perilymph. Bobbin et al. (1990) examined the CAP, cochlear microphonic (CM) and summating potential (SP) before and after cochlear perfusion with various levels of nimodipine in arti¢cial perilymph. A concentration of 0.1 WM nimodipine did not a¡ect the physiologic measures. Increasing the dose to 0.33 WM caused a reduction in CAP amplitude, particularly at low stimulus levels, with a threshold shift of approximately 6 dB. The negative portion of the SP became slightly more positive. A higher dose (1 WM) resulted in a CAP threshold shift of approximately 30 dB and a polarity change in the negative SP. Although Bobbin et al. (1990) reported that physiologic changes occurring with the 0.33 WM concentration of nimodipine were not signi¢cant (and that those at 1 WM were signi¢cant), there is a trend in their data that suggests nimodipine had some e¡ect on hair cells at the 0.33 WM concentration. Furthermore, a threshold change of 30 dB is a fairly large change in cochlear function, again suggesting that lower concentrations may also a¡ect cochlear function. The concept that nimodipine has a large e¡ect at concentrations of 1 WM, but that the `threshold' of nimodipine e¡ects is much lower, is supported by in vitro work with hair cells. Lin et al. (1995) examined the response of OHCs to the bathing medium with Ca2 and a variety of CCBs. Nimodipine showed reduction of K currents at concentrations below 1 WM, but the half-inhibitory concentration (the concentration which decreased the K current by 50%) was 6 WM. In summary, the serum levels of nimodipine were similar to those observed in experimental and clinical studies of the ability of nimodipine to reduce neurologic injury and arterial spasm following subarachnoid hemorrhage. However, the levels of nimodipine in the cochlear £uids were not measured in this study. Data from in vivo and in vitro studies suggest that nimodipine a¡ects hair cells at very low concentrations, but signi¢cant e¡ects are not observed until concentrations are at or near 1 WM. This would suggest that a protective e¡ect by nimodipine against NIHL cannot be ruled out, but the dose level required would in itself cause at least a moderate temporary hearing loss.
145
Acknowledgments The authors wish to thank Drs. Lynne Bianchi, Judy Dubno, John Mills, and Richard Schmiedt for comments on an earlier version of the manuscript. We also appreciate the estimate of nimodipine levels in cochlear £ues contributed by an anonymous reviewer. This study was supported by grants from the National Organization for Hearing Research and National Institutes of Health-NIDCD P50-DC-00422. Portions of this paper were presented at the Nineteenth Midwinter Meeting of the Association for Research in Otolaryngology, St. Petersburg Beach, FL, February, 1996.
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