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   Abstract
   Introduction
   Methods
   Surface preparations
   Results
   Histology
   Discussion
   Acknowledgments
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Year : 2003  |  Volume : 5  |  Issue : 19  |  Page : 1-18
Is the aged rat ear more susceptible to noise or styrene damage than the young ear?

Institut National de Recherche et de Sécurité‚ Laboratoire Multinuisances, Vandoeuvre, France

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  Abstract 

Noise- and styrene-induced hearing and hair cell loss were studied in young (3 months) and aged (24-26 months) Long-Evans rats. The animals were exposed 6 h/d, 5 d/w for 4 weeks to (a) broadband noise centered at 8 kHz (92 or 97dB SPL), or (b) styrene (700 ppm). Auditory sensitivity was tested by recording evoked potentials from the inferior colliculus. Histological analyses of the organ of Corti, stria vascularis, and the spiral ganglions were also performed. Aged controls showed outer hair cell (OHC) loss at the basal and apical regions of the organ of Corti, and an increase in pigmentation concomitant to a decrease in vascularization of the stria vascularis, along with elevated thresholds relative to young controls. The 92-dB noise caused similar threshold shifts in both age groups, whereas the 97-dB noise caused more threshold shifts in the aged group compared to the young group. Recovery of the hearing thresholds depended both on the intensity of the noise and on the age of the animals. Aged rats had minimal hair cell loss as a result of styrene exposure, whereas young animals showed significant OHC loss, particularly in third row. Despite significant loss of OHCs, the young subjects showed styrene-induced threshold shifts only at high frequencies. In summary, the data show that : (a) there is an influence of age on both noise-induced and styrene-induced threshold shift and hair cell loss in rats and (b) the cochlea appear to have a redundancy in the number of OHCs, thus threshold shift does not necessarily occur with significant OHC loss.

Keywords: Acoustic trauma, styrene, solvent, age-related hearing loss, presbycusis

How to cite this article:
Campo P, Pouyatos B, Lataye R, Morel G. Is the aged rat ear more susceptible to noise or styrene damage than the young ear?. Noise Health 2003;5:1-18

How to cite this URL:
Campo P, Pouyatos B, Lataye R, Morel G. Is the aged rat ear more susceptible to noise or styrene damage than the young ear?. Noise Health [serial online] 2003 [cited 2019 Nov 15];5:1-18. Available from: http://www.noiseandhealth.org/text.asp?2003/5/19/1/31702

  Introduction Top


The number of aged people has increased appreciably over the last decade in developed countries and this trend is expected to continue through the new century. This demographic change is having profound effects on the social and health-care systems for elderly people. One of the most prevalent functional implications of ageing is the deterioration of hearing. Hearing loss directly related to ageing is called age­related hearing loss or presbycusis (Schuknecht and Gacek, 1993). Because aged workers are often considered as a population having a higher risk of work-related injuries, the effects of ageing on the peripheral and central auditory systems have been extensively studied in mice (Miller et al., 1998), chinchilla (McFadden et al., 1997, 1998), gerbils (Boettcher et al., 1995; Mills et al., 2001) and finally rats. The rat can be considered as a well-suited model in ageing research. It has been shown to exhibit (1) strial and spiral ganglion cell degenerations (Keithley et al., 1992), (2) anatomical changes in the afferent synaptic endings on neurons (Keithley and Croskrey, 1990) and (3) decreases in a-aminobutyric acid (GABA) in the inferior colliculus (Caspary et al., 1990). Furthermore, the rat and the human have similar metabolism of solvents (Kishi et al., 1988, Pelekis et al., 1997; Benignus et al. 1998; Davis et al., 2002), and thus it can be used to study both solvent ­induced hearing loss and noise-induced hearing loss (Lataye et al., 1997, 2000).

Several authors have studied the influence of age in rats (Borg 1982; Keithley et al., 1992; Palombi et al., 1996) and the interaction of age with noise exposure on hearing, although most of the studies on the effects of noise were performed with postnatal aged rats (Freeman et al., 1999, Rybalko and Syka, 2001). To the best of our knowledge, the interaction of age with solvent on hearing has never been studied, although aromatic solvents are widely used in industry and considered as significant contributors to occupational hearing disorders. Indeed, human studies have shown that chronic exposure to styrene, which is an organic solvent widely used in industry (Miller et al., 1994; Nylander-French et al., 1999) can cause auditory deficits in workers (Moller et al., 1990; Calabrese et al., 1996; Morata and Campo, 2001). Similarly, numerous animal experiments have shown that styrene can severely disrupt the auditory function (Yano et al., 1992; Crofton et al., 1994; Campo et al., 2001).

Because of the lack of data in the literature regarding age-styrene interactions, the main goal of the present investigation was to compare the noise effects with those of styrene on hearing in young adult and aged populations of Long-Evans rats. To achieve this, auditory function was tested by recording the near field auditory evoked potentials from the inferior colliculus and histopathological analysis was performed on the organ of Corti, stria vascularis, and spiral ganglion.


  Methods Top


Subjects

Subjects were 50 young (3 months old) and 52 aged (25-27 months old at the beginning of exposure) male Long-Evans rats obtained from Janvier Laboratories in France. Thirty six months can be considered as the end of the normal life span for this strain. The young animals were housed for one month before the start of the experiments in individual cages (350x180x184mm). Food (UAR C ie France, ref.: A04 10) and tap water were available ad libitum except during the exposure period. The aged animals were 7-9 months old when they arrived, then were kept for 17 months in the animal facility. Lighting (fluorescent lighting) was on from 7am to 7pm. The temperature in the animal quarters was 22±1°C and the relative humidity ranged from 50 to 55%. No significant difference in body weight was found between the exposed and control groups during the exposures. While conducting the research

described in this article, the investigators adhered to the Guide for Care and Use of Laboratory Animals, as mandated by the French Conseil d'Etat in Decret n°87-848 published in the French Journal Officiel on October, 20th, 1987, and the principles of the declaration of Helsinki.

Audiometry

To avoid repetition, only a cursory description of the method employed in this investigation is presented. Further details can be found in Campo et al. (1997). Anesthesia was induced with a mixture of ketamine (50 mg/kg) and xylazine (6 mg/kg). Rats were then placed on a stereotaxic table and a tungsten electrode was implanted into the right inferior colliculus, third relay of the afferent auditory pathways. A second electrode was implanted in the rostral cranium just below the dura mater to serve as the ground electrode. These two electrodes were then fastened to a transistor socket and fixed with dental cement to the skull. The animals were allowed to recover for four weeks before testing. One month after surgery, audiometric testing was performed in a soundproof booth on awake rats placed in a restraining device. The generation and the signal treatment were preformed with a Tucker-Davis Technologies apparatus equipped with Biosig software. The acoustic stimuli (two cycles for the rise/fall ramp, four cycles for the plateau) were gated sinusoidal stimuli at 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, 24 and 32 kHz, presented at a rate of 20 per second, with an analysis window of 30ms. The stimuli were transduced by a speaker (JBL, 2405) positioned 15 cm from the left pinna.

For each frequency, the acoustic calibration was carried out by measuring the sound pressure level emitted by the speaker, when it was driven by a continuous pure tone. The sound field was calibrated by positioning a microphone at a point normally occupied by the center of the animal's head. The targeted intensity is obtained by adjusting the level of the continuous pure tone.

The electrical signal from the implanted electrode was amplified (x2000) and filtered between 30 Hz and 3000 Hz. Averaged auditory evoked potentials were obtained from 260 presentations. An amplitude trough-to-peak (N1­P1) of 15 µv of the response was considered as the threshold value in our experimental conditions. For each animal, an audiogram was obtained prior to styrene exposure (T1), at the end of exposure (T2), and six weeks after exposure (T3). Compound and permanent threshold shifts were respectively defined as : CTS = T2 - T1 and PTS = T3 - T1.

Noise exposure

The animals were exposed to moderate-intensity noise. They were housed alone in individual cages with a speaker above the cages. The rats were exposed either to 92 ± 1.0 dB SPL (n = 8) or to 97 ± 1 dB SPL (n = 8) octave band noise centered at 8 kHz for 6 h/d, 5 d/w for 4 consecutive weeks. The exposure spectrum was chosen to cause hearing losses where auditory sensitivity is the highest. Neither food nor water was given to the animals during exposure. Eight age-matched controls were maintained in the same conditions without noise.

Styrene exposure

The animals (n=13 young and n=14 aged) were exposed to 700 ppm styrene vapors (Sigma­Aldrich, 99%), for 6 h/d, 5 d/w for 4 consecutive weeks in inhalation chambers. The chambers contained eight animals housed in individual cages. Neither food nor water was given to the animals during exposure. Simultaneously, 13 young and 14 aged controls were housed in similar chambers ventilated with fresh air. Designed to sustain a dynamic and adjustable airflow (10-20 m 3 /h), the chambers (200 liters) were maintained at a negative pressure of no more than 3 mm H2O. The input air was filtered and conditioned to a temperature of 22-24°C and a relative humidity of 50-55%. The styrene was vaporized by bubbling an additional airflow through a flask containing the test compound. The solvent concentration in the chambers was measured by collecting atmosphere samples through glass tubes packed with activated charcoal. Styrene samples were desorbed with carbon disulfide and analyzed by a gas chromatograph (GC: Intersmat, 120FB I.G.C. model, France) using o-xylene as the internal standard. These analyses allowed daily calibration of another GC used for the continuous monitoring of exposure level; this GC was equipped with a flame ionization detector and an automatic gas sampling valve. Concentration measurements were performed at regular intervals (0.5 min). The spectrum of the noise inside the chamber was previously reported in Lataye and Campo (1997).

Histology

Six weeks post-exposure, the animals were deeply anesthetized with a heavy dose of ketamine (75 mg/kg), and then fixed by transcardiac perfusion with 300 ml of a tri­aldehyde fixative: 3% glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% DMSO in 0.08 M sodium cacodylate buffer, pH = 7.4 after flushing out red blood cells with buffer. The temporal bones were then removed, the tympanic bullae opened, and the cochleae fixed again by perilymphatic perfusion. Following the primary 24 h-fixation, the cochleae were post­fixed with 1% OsO 4 in 0.08 M cacodylate buffer (pH = 7.4) for 1 h and finally washed in a trihydrate solution of sodium cacodylate.


  Surface preparations Top


Cochleograms

The cochleae were dissected in 70% ethanol at room temperature. The organ of Corti was dissected and mounted in glycerin for counting of hair cells. Cells were counted as present when either the stereocilia, the cuticular plate or the cell nucleus could be visualized. No attempt was made to assess the degree of possible cellular damage to surviving cells. The frequency-place map established by MMuffler ller (1991) was used to superimpose the frequency coordinates on the length coordinates of the organ of Corti. A cochleogram showing the percentage of hair cell loss as a function of distance from the base of the cochlea was plotted for each animal. The results were averaged across each group of animals for comparison between groups.

Stria vascularis

Five young and five aged cochleae of different animals were fixed with 4% glutaraldehyde, then the bony capsule was removed, starting at the apex, to expose the stria vascularis and organ of Corti. The stria vascularis and spiral ligament were separated from the rest of the cochlea and stained with a 0.5% eosin solution for 5 min. Mounted in glycerin on glass microscope slides and cover slipped, strial tissue samples from young and aged cochleae were observed with a light microscope. Specimens were evaluated for qualitative differences in stria vascularis thickness, extent of vascularization, diameter of blood vessels, and accumulation of pigmented granules. No attempt was made to quantify the differences between young and aged specimens.

Spiral ganglion cells

The opposite cochleae which were not used for the surface preparations, were prepared for modiolar sections. These were entirely immersed at room temperature in 0.7 M ethylene diamine tetraacetic acid (EDTA, pH = 7.8) for eight hours, then dehydrated in ascending concentrations of ethanol up to 100%, and finally infiltrated with resin (Epon/Araldite).

Density

The fractionator, which is a quantitative stereological tool for sampling (Gundersen et al., 1988), was used to determine the density of the spiral ganglion cells (SGCs). The method can be described in the following way. Serial 2.5 µm­thick sections [t = 2.5 µm] were cut parallel to the mid-modiolar plane to obtain an averaged profile (A) of Rosenthal's canal of 0.032mm 2 for the middle spiral ganglion. The sections were serially collected onto slides and stained with cresyl violet. Every fourth (4) section was saved, and at least three sections were counted. Observations were made with a light microscope equipped with a Leica DC 100 camera. The entire section of Rosenthal's canal was readable on the computer screen at a magnification of 250. The image was captured on the computer, the Rosenthal's canal delineated and the surface calculated using custom imaging software. The counts of SGCs, type I and II indistinctively, were manual counts using a video-imaging system. They were directly based on the presence or not of the SGC nucleus. To minimize sampling errors, the nuclear diameters of the SGCs were first measured when their perimeters were clearly observable in the section. Because the nuclei are more or less spherical, the maximum diameter corresponded approximately to the section crossing the centre of the nucleus. The maximum diameter measured within the area of the canal was dmax = ~10µm, therefore four 2.5 µm-thick sections were sufficient for not counting twice the same nucleus [4 x t =10µm].

In order to calculate the density of SGCs in a reconstructed volume (V = A x t x 4), the counts of the type I and II cells (N) were multiplied by 4 and divided by a correction factor F. This factor, modified from Hall et al., (1997), takes into account the nuclei sectioned by the knife and appearing in several adjacent sections. The density would be overestimated if a correction faction was not used. F corresponds to the number of successive sections in which one nucleus could appear several times. This therefore depends on the thickness of the section and on the maximum diameter of the nuclei (F = dmax / t). Finally, the density formula can be expressed as follows: D = (N x 4) / (F x V).

Transmission electron microscopy

Ultra-thin (< 1 µm) sections were used for the transmission electron microscopy (TEM). Ultra­thin sections were stained with uranyl acetate and lead citrate, and observed with a Zeiss EM910 electron microscope.

Statistical analysis

The Statgraphics® plus (version 5) software was used to run all the statistical analyses. A multifactor ANOVA, with "noise", "styrene" or "age" as a between-subject factor, and tone "frequency" as within subjects, was run. This ANOVA allowed evaluation of either the "noise", "styrene", "age" effect, or interactions such as "age x noise". When needed, the ANOVA test was completed by a post-hoc Bonferroni's test which allows to compare the means at each frequency. The statistical differences in spiral ganglion cell density between young and aged, and between exposed and unexposed animals were determined using the Student's t-test. Alpha levels of 0.05 (significant at 95%) were used for the significance of the tests.


  Results Top


Hearing testing

The mean auditory [brainstem (inferior colliculus)-evoked potentials] thresholds obtained from young (n = 50) and aged (n = 52) rats are plotted in [Figure - 1]. The best frequency sensitivity (12-15 dB SPL) ranged from 8 kHz to 12 kHz. The threshold differences between young and aged animals were significant F age (1, 100) = 10.2, p = 0.0019. The post hoc Bonferroni's test revealed that the differences were significant from 2 kHz to 4 kHz and at 10 kHz.

Noise-induced hearing loss

[Figure - 2]a shows significant CTS and PTS [PTS young: F noise (1, 14) = 23.42, p = 0.0003] within the 92 dB SPL noise-exposed group compared to the age-matched controls. The threshold shifts were located in a frequency range from 8 to 16 kHz (p < 0.05), with the peak occurring near 10 kHz. The maximum hearing loss occurred between 10-12 kHz which corresponds to approximately one-half octave (11.3 kHz) above 8 kHz: the centre frequency of the exposure. At this noise intensity, there were no differences between the young and aged groups in terms of CTS or PTS.

At 97 dB SPL, both the CTS and PTS [PTS young: F noise (1, 14) = 283.26, p < 0.0001] were significant from 8 kHz up to 32 kHz (p < 0.05) regardless of the age of the animals [Figure - 2]b. The difference in the CTS measured in the young and aged animals was not significant. In contrast, PTS was greater in the aged than in the young rats [F age (1,13)=10.90, p=0.0057].

[Figure - 2]c-d illustrate the recovery (CTS-PTS) in the young and aged rats exposed to either 92 or 97 dB noise. The amount of recovery was not significantly different between groups for 92 dB, but the aged rats had significantly less recovery than young rats for the 97 dB noise [F age (1, 6) = 36.61, p = 0.0009].

Styrene-induced hearing loss

[Figure - 3] shows CTS and PTS as a result of styrene exposure for young and aged rats. Only the young styrene-exposed rats show significant PTS [F styrene (1, 24) = 10.06, p = 0.0041]. A 15 dB styrene-induced hearing loss was located in the region of 16-20 kHz. Surprisingly, no difference was obtained between controls and aged styrene-treated rats. No significant recovery could be measured from the CTS induced by styrene at the end of the six-week post-exposure period.


  Histology Top


Cochleograms

Age effect

The average cochleogram obtained with the young controls (n = 10) revealed that small amounts of hair cell loss were scattered along the organ of Corti, but they did not exceed 1 % of all cells for any turn. Since the cochleogram was flat whatever the row of hair cells considered, it is not shown. [Figure - 4]a shows the cochleogram obtained with 9 aged controls. Large losses of OHC can be observed at the apical part of the cochleae (OHC 3 : ~ 50% ; OHC2: ~ 28.5% ; OHC 1 : ~ 22% at about 0.5 kHz) and to a lesser extent at the basal part of the cochleae (OHC 3 : ~ 26% ; OHC 2 : ~ 9.5% ; OHC1: ~ 2% at about 55 kHz). The loss of the third row of outer hair cells (~7.5% ) was greater than those of the second (~2.5%) or first rows (~3.2%) between 8 and 30 kHz. The inner hair cells were well preserved, except at the extreme apical part of the cochlea.

Noise effect

The cochleograms obtained with the group exposed to 92 dB were not different from those obtained with the controls regardless of the age of the animals. For this reason, they are not shown. In contrast, the cochleograms differed for aged and young rats exposed to 97 dB SPL. In [Figure - 4]b, the cochleogram obtained from young rats shows that the cell losses of the first row (~ 9.3%) were greater than those of the second (~ 3.8%) and third (~ 2%) rows between 12 and 34 kHz.

The hair cell losses induced by the 97 dB exposure were greater in the aged rats [Figure - 4]c than in the young rats [Figure - 4]b. Moreover, the losses in the aged group were located in a higher frequency range: from 23 kHz to 37 kHz.

Styrene effect

[Figure - 4]d illustrates the cochleogram (n = 5) obtained for the styrene-exposed young group. The losses were massive in the third row where 60.5% of the cells are missing from 1 to 28 kHz. The losses are not as large in the second row (28%) as in the third row, but larger than that in the first row (13%). Frequencies above 30 kHz seem to be well preserved for each row of OHCs. Similarly, IHC were well preserved. [Figure - 4]e corresponds to the styrene-exposed aged group.

There is a clear difference compared to the young group. The losses are less large regardless of the row of OHC. Over a frequency range from 2 kHz to 30 kHz, the percentages were 29.6% at the level of the OHC 1 , 6.7 % at OHC 2 and 5.1% at OHC 3 .

For the sake of clarity, all the percentages reported in this section have been summarized in [Table - 1].

Stria vascularis

In the stria vascularis of aged animals, the most obvious difference from young animals was the increased size and incidence of pigmented granules of melanin, as illustrated in [Figure - 5]. This phenomenon is true regardless of the turn of the cochlea. Other qualitative differences between young and aged rats included reduced vascularization (a vs. b), decreased diameter of blood vessels (a vs. b), and thinning of the strial tissue (a vs. b). The ageing effects are particularly clear at the apical turn of the cochlea. No evident difference was observed in animals exposed either to noise or to styrene. At the noise intensity or at the styrene concentration tested in this investigation, we have not seen any influence of these two noxious factors on the stria vascularis.

Spiral ganglion cells

[Figure - 6]a-b shows cochleae from young and aged controls respectively, cut in the mid­modiolar plane. There was a large decrease in the density of spiral ganglion cells and afferent fibres, especially evident in the apical part of the spiral ganglion.

[Figure - 7] illustrates the density of spiral ganglion cells calculated in unexposed and exposed young and aged rats. Obviously, there was a significant difference between unexposed young and aged groups, the largest difference being calculated at the level of the apical ganglion (p = 0.002). There was therefore a degeneration of spiral ganglions in the aged animals. As expected from the literature, there was no noise effect on the spiral ganglions, so we did not plot these negative results for sake of clarity. As far as the styrene exposure was concerned, the differences between controls and exposed groups were not significant. However, a decrease in the perikaryon number can be observed within the median ganglion between the unexposed and the styrene-exposed young animals. Finally, the aged rats did not show any difference between the controls and the styrene-exposed animals.

From a morphological point of view, large lipofuscin accumulations can be seen in the cytoplasm of the spiral ganglion cells in the aged animals [Figure - 8]. The young animals were free of lipofucsin accumulation. Only type I spiral ganglion neurons showed these accumulations.


  Discussion Top


Noise effects

The strong causal relationship between noise exposure and hearing loss is well documented (Saunders et al., 1985; Henderson et al., 1995; Borg et al., 1995), and has been conclusively demonstrated in this study. Indeed, the electro­physiological data showed that 92 and 97 dB SPL octave band noises centered at 8 kHz clearly caused a permanent hearing deficit [Figure - 2]a-b whose maximum amplitude was positioned half an octave (10-12 kHz) above the exposure frequency. This half-octave shift is well known. Briefly, if the active displacement of the basilar membrane is located at the stimulation frequency (8 kHz), the peak of the traveling wave envelope migrates basalward with increasing intensity. Noise-induced hearing loss is located near the peak of the envelope (10-12 kHz) at 92 and 97 dB SPL. The extension of threshold elevation up to 32 kHz with the 97 dB SPL noise was due to the greater amplitude of the travelling wave envelope in the basal cochlea. For this reason, the cochlear trauma is located at and above 10­12 kHz. Likewise the half-octave shift, the extension of the noise effects to the basal cochlea with increasing intensity are well known and detailed by McFadden (1986).

For the 97 dB SPL exposure [Figure - 2]b, there were no differences in CTS between groups, but aged animals had greater PTS than young animals. Therefore, the recovery (CTS - PTS) capability depended on the age of the animals [Figure - 2]c-d. Recovery depended on the intensity of the noise (92 vs. 97 dB) and the age (young vs. old) of the subjects. In summary, aged rats are by and large equally vulnerable to moderate-level noise (92 dB SPL), but may be more vulnerable to moderate-high level noise (97 dB SPL) than young rats, specifically in the high frequency region. Such a statement has already been made in a previous work with chinchillas (McFadden et al., 1998).

As far as the histological findings are concerned, the hair cell losses obtained in the young [Figure - 4]b and aged rats [Figure - 4]c exposed to 97 dB were low relating to the electrophysiological data. In fact, such results were expected since the noise effects concerned essentially the stereocilia of the hair cells. Fusion and splayed stereocilia can be easily observed at this noise intensity (Lataye et al., 2000). So, by only counting the hair cells, the cochlear trauma is probably under­estimated. Although the order of the hair cell losses (OHC 1 >OHC 2 >OHC 3 ) was similar in young and aged animals, the locations of the losses were different. For example, the cell losses were centred at 20-22 kHz place [Figure - 4]b in the young subjects, whereas they were located in a higher frequency range, near the 30 kHz place [Figure - 4]c, in the aged animals. Such a difference in tonotopicity of the trauma could be due to changes in the physical features of the organ of Corti depending on the age of the animals. Indeed, the organs of Corti in aged control rats were already characterized by numerous cell losses, specifically in the third row of OHCs at the apex and at the base of the cochlea [Figure - 4]a. Such morphological data have been previously reported by Keithley et al. (1992) and may be sufficient to modify the mechanical properties of the basilar membrane, and thereby the location of the trauma.

The young rats showed a better capability of recovering the auditory sensitivity than the aged rats [Figure - 2]d. One possibility to account for the recovery discrepancy between aged and young rats is that threshold sensitivity could be affected by other degenerative changes in the cochlea, such a strial pathology [Figure - 5] and neural degeneration [Figure - 6],[Figure - 7]. Gratton et al. (1996) had already observed a strial pathology in the apical and basal cochlear turns, but the authors had not checked the neural degeneration in the gerbil. The functional recovery could be also explained by a certain plasticity of the central nervous system. Willot and Lu, (1982), and Salvi and coworkers, (1996) have already hypothesized that a cochlear trauma could be counterbalanced by a central adaptation of the inhibitory influences within the inferior colliculus (IC). Briefly, a release of the inhibition within the IC could then counterbalance the decrease in the inputs and thereby ensure a sufficient excitability of the afferent neurons. Such a hypothesis could explain the difference in recovery between the aged and young rats. In fact, the aged rats could already have used this plasticity power to counterbalance the ageing effect and more specifically, the loss of afferent neurons [Figure - 6]b. To verify this hypothesis, a GABA dosage will be performed in the IC of aged and young animals to evaluate the inhibition power of the two age groups.

Styrene effects

The electrophysiological data showed that exposure to 700 ppm styrene caused threshold shift [Figure - 3], whereas the histological data showed hair cell loss [Figure - 4]. Styrene-induced hearing loss has already been reported in rats (Yano, 1992; Crofton, 1994, Campo, 2001, Pouyatos et al., 2002). But, to the best of our knowledge, no data have been published on styrene effects as a function of age of the exposed subjects. Based on the electrophysiolog­ical results reported in the present study, aged rats were insensitive to 700 ppm styrene compared to young animals contrary to all expectations [Figure - 3].

As has previously been reported in the rat, styrene-induced hearing loss was located in the mid-frequency range, namely in the vicinity of the 16-20 kHz area. No significant recovery was observed 6 weeks post-exposure. For young animals, a 15-dB PTS was observed in conjunction with large hair cell losses (OHC 3 : 60.5% > OHC 2 : 28% > OHC 1 : 13%). The hair cell losses were in reverse order with regard to that observed following the noise exposure. Moreover, the amplitude of the PTS values were modest ~15 dB [Figure - 3] with regard to the hair cell losses observed in the cochleogram [Figure - 4]c. As far as the inner hair cells were concerned, they appeared to be much less sensitive to styrene-induced injury than OHCs.

The aged animals showed no significant threshold shift due to the styrene exposure although hair cell losses (29.6 - 7.3 = 22.3%) occurred in the third row of OHCs [Figure - 4]a,e.

Such a loss of OHCs was modest with regard to that stated in the young rats, and apparently insufficient to modify the functional results [Figure - 3]. This statement associated with others in previous experiments (Campo et al., 1997; Loquet et al., 1999) in which the third row of OHCs was damaged without causing any threshold shift. This suggests that the third row of OHC does not seem to influence auditory sensitivity in the rat. The redundancy of OHCs has been reported by others (Loquet et al., 1999) and appears to be a particular trait of the rat. Furthermore, the apical part of the organ of Corti does not play a major role in the audiometric low-frequency responses. By way of proof, Prosen et al. (1990) showed that 50% outer hair cell loss in the apical 30% of the cochlea does not cause hearing loss at any frequency. Based on that, it was not surprising that the lack of OHC observed in the aged controls [Figure - 4]a at the apex and throughout the third row of OHC were without major consequence on auditory threshold on the rat.

Noise and styrene effects: a comparison

Based on the histological data, the noise caused less hair cell losses than the styrene, although the magnitude of PTSs were greater following the noise exposure than following the styrene exposure. In fact, these results can easily be explained by the respective mechanisms responsible for the noise- or styrene-induced hearing losses. Noise-induced hearing loss could be summarized by a stereocilia pathology (Lataye and Campo, 1997; Lataye et al., 2000), whereas styrene-induced hearing loss would be caused by a poisoning of the organ of Corti which would disorganize the membranous structures of the outer hair cells (Campo et al., 2001). The effect of styrene was therefore different from that of noise and was mainly caused by a chemical process.

The difference of vulnerability between young and aged subjects

The concept of reduced metabolic activity is often evoked to explain the differences in sensitivity between aged and young subjects (Miller et al, 1998). If this decrease in the metabolic activity would be the reason of the difference of vulnerability between young and aged subjects, the aged ear should be more sensitive to trauma from environmental insults, including noise and drug stress. In fact, if the concept of reduced metabolic activity can explain the noise effects, it could not explain the styrene effects since the aged ears were less sensitive to styrene than the young ear. The difference in sensitivity to styrene between age groups could be due to either a weight difference between animals, or to a maturity difference. Indeed, the aged rats weighed more than the young rats (500 g vs. 300 g) at the beginning of exposure. Such a difference in weight could constitute a stock reserve for the solvent before metabolizing it. By controlling the weight difference between two different groups of the same age, we should be able to test the weight factor on the effects of styrene intoxication. An experiment will be carried out to test this issue. The maturity of the cochlea could also play a major role in the sensitivity between the age groups. The existence of a critical period for styrene-induced hearing and hair cell loss will also be examined in future studies.


  Acknowledgments Top


The authors wish to thank Dr. E. Keithley from the University of California for her help in reading the pictures and Christian Barthelemy for his technical assistance. This study was supported by European grant QLK4-2000-00293 and by Institut National de Recherche et de Securite.[42]

 
  References Top

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