Age-related changes in cochleas of mongolian gerbils

123

Hearing Research, 54 (1991) 123-134 0 1991 Elsevier Science Publishers B.V. 037%5955/91/$03.50

HEARES

01584

Age-related

changes in cochleas of mongolian gerbils

B.I. Tarnowski, R.A. Schmiedt, L.I. Hellstrom, F.S. Lee and J.C. Adams Department of Otolaryngology and Communicative Sciences, Medical University of South Carolina, Charleston, South Carolina, U.S.A. (Received

10 August

1990; accepted

27 January

1991)

The effects of aging on the gerbil cochlea were studied in 16 animals raised in a quiet environment. Animals were tested at ages ranging from 33 to 36 months, the approximate average lifespan of gerbils in our colony. Hearing sensitivity was assessed by measures of whole-nerve compound action potential (CAP) thresholds and surface preparations of the organ of Corti were subsequently examined by light microscopy for losses of sensory hair cells. These quiet-aged animals showed a wide range of hair-cell losses and threshold shifts. Outer hair cells often showed significant losses while inner hair cells were rarely absent. All animals had some threshold shift, especially at frequencies above 4 kHz. These shifts ranged from 1 to 68 dB. At high frequencies, threshold shifts often occurred without hair-cell losses at corresponding cochlear locations. At low frequencies, threshold shifts seldom reflected the losses of hair cells commonly found in the cochlear apex. Thus, the correlation of specific hair-cell losses and CAP threshold shifts at corresponding frequencies was poor. On the other hand, the total number of missing hair cells, irrespective of location, was a good, general indicator of the hearing capacity in a given ear. It appears that the factor or factors that makes cochleas susceptible to hair-cell loss with increasing age also affects other cochlear mechanisms that are necessary for normal functioning of the ear.

Cochlea;

Aging;

Hair cell; Compound

action

potential;

Presbycusis;

Introduction

Decreases in human hearing sensitivity associated with aging (presbycusis) are well-documented (Schuknecht, 1964, 1974), but data are often difficult to interpret due to unknown and uncontrolled environmental variables. Consequently, in an effort to minimize the effects of disease, drugs and noise exposure, animal models have been used to study age-related changes in hearing (Henry and Chole, 1980; Bhattacharyya and Dayal, 1985; Willott et al., 1987; Keithley et al., 1989). In old animals, outer hair cells (OHCs) and inner hair cells (IHCs) are characteristically lost

Correspondence to: Betty I. Tarnowski, Department ogy, The Medical University of South Carolina, Avenue, Charleston, SC 29425, U.S.A.

of Pathol171 Ashley

Hearing

loss; Mongolian

gerbil

at one or both extreme ends of the cochlea. The loss of OHCs occurs particularly in the apex in the guinea pig (Federspil, 1972; Ulehlova, 1973; Coleman, 19761, in the albino rat (Keithley and Feldman, 1982) and in the chinchilla (Bhattacharyya and Dayal, 1985). IHC and some OHC losses are observed in the base in chinchillas and rabbits (Bhattacharyya and Dayal, 1985, 1989). A few studies have attempted to correlate hair-cell loss and hearing sensitivity. In the rat, hearing losses at 16 and 20 kHz, as assessed by auditory brainstem responses, were accompanied by hair-cell losses in the basal cochlea (Borg and Viberg, 1987). In other studies of aging rats, Crowley et al. (1972a,b) found that, although masked and unmasked VIII nerve action potentials and cochlear microphonic (CM) sensitivity levels were consistent with hair-cell losses, the decrease in CM could not be attributed exclusively to hair-cell loss. In the mouse, hearing

124

declined during aging, but the hearing losses correlated poorly with hair-cell counts (Henry and Chole, 1980; Henry, 1983) and were probably due to factors other than sensory cell losses (Chole and Henry, 1983). We have selected Mongolian gerbils to study effects of aging on hearing. The advantages and disadvantages of this animal as a model for presbycusis have been discussed by Mills et al. (1990). Unlike many rodents, the gerbil has low-frequency auditory sensitivity that is similar to humans. A substantial data base on the auditory electrophysiology of young gerbils provides a basis for interpretation of data from aging animals (Schmiedt, 1982, 1986, 1989). Additionally, the animals have a relatively short life expectancy (36 months) and are easy to breed and maintain in a noise-controlled environment (Cheal, 1986; Mills et al., 1990). The purpose of this paper is to quantify sensory hair-cell losses in quiet-aged gerbils and to relate these findings with electrophysiological measurements of sensitivity. Compared with previous studies, we have considerably more animals in our sample and the terminal testing of our animals was carried out at an age close to their expected lifetime. At this age, about 50% of the ears are lost to impaction of the external ear or to animal death. This is one facet of a comprehensive study of the aging gerbil auditory system. In addition to results reported here, more extensive physiological measurements and more detailed anatomical studies, including histopathological observations by electron microscopy, will be reported elsewhere. We chose to report first on cytocochleograms and CAP thresholds because of their obvious relevance and consequent popularity among researchers. The results make it clear that more extensive measurements than cytocochleograms and hearing thresholds are necessary for the adequate assessment of hearing loss associated with aging.

raised in a quiet vivarium where level was less than 41 dBA.

the mean

sound

Materials and Methods

Physiology The surgical preparation and physiological procedures have been described previously (Schmiedt, 1989; Schmiedt et al., 1990). All procedures were done in a double-walled sound and vibration isolated booth. Compound action potential (CAP) thresholds at 12 frequencies were determined for each ear and threshold shifts were computed relative to the averages from 21 young (4-7 months) ears which served as the standard. All ears were examined for middle-ear disorders and were calibrated for ear-canal SPL on an individual basis. Immediately following the physiological experiments, animals were perfused intracardially with 0.1% sodium nitrite in 120 mM phosphate buffer (pH 7.2, 37°C) followed by a fixative consisting of 4% paraformaldehyde and 2% glutaraldehyde in 120 mM phosphate buffer (pH 7.2) at room temperature. The round window was opened and the same fixative was injected into the Scala vestibuli via the oval window. The cochleas were immersed in fixative overnight at 4°C. The fixative was then rinsed out with phosphate buffer and the cochleas were gently agitated at room temperature in 120 mM EDTA containing 0.2% glutaraldehyde. Decalcification took approximately 2 weeks. In preparation for dissection, warm 10% gelatin was injected into the Scala vestibuli. The infiltrated cochlea was cooled on an ice bath to congeal the gelatin so that it provided a firm matrix to support the organ of Corti. The cochlea was bisected through the modiolus with a razor blade. The halves were rinsed in warm buffer to remove the stained with 0.8% orcein in acetic gelatin, acid/alcohol, post-fixed in 1% osmium in the same phosphate buffer, dehydrated in an ascending series of ethanols, and embedded in Epon resin. Following polymerization, each bisected cochlea was dissected into half turns and re-embedded in Epon resin as a surface preparation.

Animals Ears from 16 gerbils of both genders, aged 33 to 36 months, were used for histological and physiological evaluation. Animals were born and

Frequency-distance map A frequency-cochlear distance gerbil cochlea has been inferred microphonic measurements made

map for the from cochlear in Scala media

125

(Sokolich et al., 1976; Schmiedt and Zwislocki, 1977). Since an exact frequency-distance map for the gerbil cochlea has not yet been established, we have applied Greenwood’s (1961, 1990) formula and fit it to the microphonic map. The mapping function is:

ting hair-cell data in each of the 50 bins against the frequency location calculated from the frequency-distance map. CAP thresholds at the 0.5, 0.7, 1, 1.4, 2, 2.8, 4, 5.6, 8, 11.3, 16, and 20 kHz locations were plotted on the same axes to facilitate comparisons of hair-cell counts and thresholds.

Frequency = 0.40. [ lO^( 0.021. Distance)] Results

where frequency is in kHz, and distance is the percent distance normalized to the total length of the cochlea and is referred to the apex. The resulting curve provides a close fit to the data points obtained with the previous microphonic measurements, and correlates well with more recent electrophysiological data (Echteler et al., 1989).

To compare young and old cochleas with regard to percent hair-cell loss as a function of

166 140

Cytocochleograms

126

Surface preparations were used for counting the number of hair cells both missing and present. The specimens were observed with a long working distance 40 x oil-immersion objective (Zeiss Epiplan, N.A. 0.85). Distances were measured using a calibrated ocular reticule. The numbers of IHCs and OHCs present within reticule units were recorded for each cochlear segment. Hair cells were scored as absent based on the spaces created by the missing cells. Data for each segment were arranged in an array which represents hair-cell counts and actual length for the total length of the cochlea. The average total length traced from the 16 quiet-aged ears was 11.46 mm ( & 0.25 mm SD). The cochlea was divided into 50 bins of equal length (0.229 + 0.005 mm SD), and an averaging algorithm was applied to calculate cell densities. The algorithm estimates hair-cell counts in each bin by averaging all raw counts within the bin, weighted by the distance over which they were made. When cells could not be counted due to processing difficulties, counts of adjacent cells within the same bin were used to estimate the counts for the whole bin. Note that small ‘wipeouts’ of all three rows of OHCs occurring over a smaller distance than that corresponding to a bin are somewhat obscured by this averaging technique. However, such wipeouts were almost never seen in these quietaged gerbils. Cytocochleograms were constructed by plot-

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Fig. 1. Hair-cell densities as a function of frequency along the gerbil cochlea. The number of hair cells is the raw count per mm of basilar membrane. The cochlear length was divided into 50 bins of equal length and cell densities were calculated using an averaging algorithm. The cochlear map was used to convert cochlear distance to frequency. Top: mean densities from 7 young gerbils. Bottom: mean densities from 21 quietaged gerbils. Hair-cell density does not change with age in the gerbil.

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-a- OHC Avorqp CAP Thrmhold 6htft Abnormal OHC * -5 IHC Fig. 2. Cochieograms and CAP threshold shifts from quiet-aged gerbils (N = 6) with minimal losses of ceils (S-8%) and small threshold shifts (O-25 dB). All graphs are plotted with respect to frequencies along the basilar membrane using the frequency-distance map for the gerbil (see text). Lower graphs represent IHC counts (squares) and combined OHC counts (pluses) expressed as percent present; CAPS (triangles) are threshold shift values relative to averages from 21 young control ears. The vertical axis is a dual scale representing percent hair cells present and threshold shift in dB of the CAP with respect to the control average. Note the general lack of correspondence between CAP threshold shifts and hair-ceil loss. Upper graphs represent the percent of abnormal OHCs. Most of the abnormal OHCs are in the apical portion of the cochlea. Abnormal cells were not counted in ear 82 L.

127

cochlear distance, it is assumed that the density of IHCs and OHCs along the organ of Corti remains constant with age. This assumption was tested and was found valid as Fig. 1 demonstrates: average hair-cell density was nearly identical for both groups of animals. Thus, comparisons on a percent basis between young and old gerbils are meaningful. The most striking finding was the extent to which individual cochleas differed with respect to hair-cell losses. Some ears showed remarkably little loss, whereas a few showed extreme losses. The cases fell into three obvious groups according to the degree of cell loss. The first group (N = 6) showed little cell loss (Fig. 2). There was a trend for OHCs to be missing in the apical turn and, to a lesser extent, in the extreme basal turn. For comparison, Fig. 3 shows a cochleogram of a young animal to demonstrate that animals in Fig. 2 actually do show some effects of aging. The second group (N = 8) was characterized as having moderate hair-cell loss (Fig. 4). The degree of cell loss in the apical turn was more extreme and obvious in this group and losses in the base were more consistent. The third group (N = 2) showed extreme losses of hair cells (Fig. 5). It was only in this group that obvious losses of IHCs occurred. There were only two cases with significant IHC loss so it is not possible to know if there may be places where IHCs are preferentially lost. In addition to showing loss of hair cells, Figs. 2-5 also show the positions of OHCs that were grossly abnormal (tops of figures). These cells assumed a spherical shape and were obviously greater in diameter than normal cells. Such cells were located exclusively in the apical turns and were randomly mixed with normal cells and OHC loss. None were found more basal than 43% of the distance from the apex (the 2.8 kHz place on the frequency-distance map). Also, no such abnormal cells were seen in young animals. Plotting CAP thresholds on the same figures with cochleograms facilitates comparison of haircell counts and hearing indices. A striking finding was the lack of correspondence of elevated thresholds and missing hair cells at frequencies of 3 kHz and below. Hair cells also were often missing in the extreme basal turn but the physical constraints of producing calibrated, high-level

acoustic signals made it impossible to measure thresholds for the corresponding frequencies (above 20 kHz). The two ears in which there were sizable hair-cell losses in the middle and upper basal turns were examples of extreme loss, and in these ears, there were uniform severe threshold shifts at all frequencies (Fig. 5). Fig. 6 shows OHC losses plotted against threshold shifts at different frequencies. Regression lines are least squares fits to these data. These plots illustrate the large range of values for threshold shifts and the absence of a corresponding range of values for missing hair cells. Consequently the correlation coefficients that describe these relations are generally poor. In those cases where the correlation coefficients reached statistically significant levels (1, 2 and 4 kHz), it is obvious that this was owing to extreme isolated points on the abscissa. Consequently, the reliability of the statistical significance is not compelling. On the other hand, the three categories that characterized hair-cell losses also characterized

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129

Discussion

threshold elevations in a general way. Animals with minimal hair-cell iosses (5-8%) showed corr~spondingly minimai threshold shifts (usually less than 20 dB at any frequency) (Fig. 2). Animals with moderate hair-cell losses (8-14%) showed greater threshold shifts (as much as 50 dB at some frequencies) (Fig. 4). Animals with severe hair-cell losses (41-54%) showed greater than 50 dI3 threshold shifts at all frequencies (Fig. 5). Thus, despite the lack of correspondence in the specific locations of these two measures, there was a general correspondence in the degree of effects measured, i.e. overall hair-cell loss and neural threshold shifts. Fig. 7 summarizes the central tendency for hair-cell counts and threshold shifts for these quiet-aged gerbils. There is on average almost no Ioss of IHCs, a scattered loss of OHCs with the greatest losses being at the extreme ends of the cochlea, and CAP threshold shifts greatest above 4 kHz.

Quiet-aged gerbils, all reared under identical conditions, show a remarkable range of both hair-cell losses and threshold shifts. The variability of these data was not completely unexpected as studies of this nature are inherently variable and require a large sample size. Sensory cell losses were predominantly of OHCs, and these losses occurred most frequently in the apex and base. In contrast, with the exception of the two ears with severe hearing losses, few IHCs were missing at any point along the cochlea. It has been suggested that architectural differences between the 1HCs and OHCs, with respect to their supporting celfs (Liberman, 19871, might account for differences in susceptibility to loss but this does not explain why the extreme ends of the cochlea manifest preferential OHC losses. The preferential loss of OHC in the apex and base agrees with the studies of other aging animals

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(Bredberg, 1968; Federspil, 1972; Crowley et al., 1972a; Johnsson and Hawkins, 1972; Ulehlova, 1973; Dayal and Barek, 197.5; Coleman, 1976; Henry and Chole, 1980; Keithley and Feldman, 1982; Chole and Henry, 1983; Bhattacharyya and Dayal, 1985; Borg and Viberg, 1987). The selective loss of OHCs in the extreme ends of the cochlea may be related to another change seen in aging ears: the degeneration of the stria vascularis. Strial degeneration has been shown to occur with aging in several species in the apex (Cove11 and Rogers, 1957; Schuknecht, 1974; Tachibana et al., 1984; Bhattacharyya and Dayal, 198.5). Keithley et al. (1989) have recently reported that oId gerbils (36-42 months) have considerable strial degeneration which is most marked in the apex but also present at the basal end of the cochlea. This agrees with the work of Schulte and Adams (1989a) which shows that strial atrophy in aging gerbils is accompanied by a decrease in the abundance of immunoreactive Na+-, K+-ATPase. This enzyme is known to direct transepithelial movement of fluid and ions in many sites (Burg, 1986; Koeppen and Giebisch, 19851, and undoubtedly plays a role in regulating the cation content of the cochlear Iymphs (Kerr et al., 1982; Mees, 1983; SchuIte and Adams, 1989b). The correspondence of the locations of

strial atrophy and hair-cell losses suggest the possibility that reduced ion pumping activity of this enzyme may somehow affect the viability of the sensory cells. On the other hand, degenerative OHC changes may represent a general ~lnerability of the extreme ends of the cochlea. Despite the high variabiiity of apical and basal degeneration, such changes are always present in quiet-aged gerbils. The selective degeneration of OHCs, the stria vascularis, and ganglion cells (Keithley et al., 1989) in the extreme ends of the cochlea may be related to a common aging process, but, there are no obvious reasons to suggest that the failure of any one of these cell types leads to the degeneration of the others. Another observation of interest was the oftenseen presence of abnormal or mo~hologically altered OHCs in the apex of quiet-aged gerbils. Swollen hair cells have frequently been observed following acoustic trauma (Mizukoshi et al., 1957; Beck and Michler, 1960; Beagley, 1965; Ward and Duvall, 1971; Spoendlin, 1971, 1976; Liberman and Kiang, 1978; Salvi et al., 1979; Liberman and Muiroy, 1982) so it does not seem likely that this pathologic change is unique to aging. Rather, cell swelling appears to represent one stage of OHC degeneration. The animals in this study showed a wide range of morphological and physiological changes with age. These differences reflect individual susceptibilities to the various aspects of the aging process. The range of values of the measures that were made should have provided a sufficient number of conditions to enable relations between the variables to be manifest, yet no strong relation was found. It was true that animals with few missing hair cells had only small threshold shifts and that those with many missing hair cells had profound threshold shifts. The intermediate cases frustrated efforts to establish more than a general relation between these two variables. The most obvious and consistent loss of hair cells was in the apical half of the cochlea, whereas the most consistent losses of sensitivity were at frequencies that are represented in the basal half of the cochlea. The lack of threshold shifts at lower frequencies demonstrates the inadequacy of CAP thresholds for detecting conspicuous OHC losses

132

at frequencies below about 3 kHz. A similar lack of correspondence between apical hair-cell loss and behavioral thresholds has been described in the chinchilla by Clark and Bohne (1986) with noise trauma and by Smith et al. (1987) with cryoprobe-induced injuries. On the other hand, CAP thresholds for high frequency stimuli can be a useful index of cochlear pathology Wang and Peake, 1960; Spoendlin and Baumgartner, 1977; Dallos et al., 1978; Robertson et al., 1980; Harrison, 1981). Unfortunately, we saw only two ears where there were significant losses of hair cells in the basal portion of the cochlea (excluding the extreme hook region where tone-evoked potentials were not feasible with our acoustic system) and these showed severe losses at all frequencies. Contrary to the case for the apex, CAP thresholds at high frequencies appeared to be a sensitive measure of basal cochlear pathology, but selective losses of hair cells in the upper basal cochlea appear not to be a normal manifestation of aging until there is a general loss of hair cells in all regions. The sensitivity losses recorded for higher frequencies must be due to factors other than hair-cell loss. Although no strong relation between loss of hair cells and increased thresholds was demonstrable, the consistency of these two effects suggests the presence of some more general process or processes that underlies both. The total number of missing hair cells was a good indicator of the degree to which thresholds at some frequency were elevated. This suggests that factors which predispose ears to vulnerability of hair-cell swelling and/or loss in the apical half of the cochlea also affect other mechanisms in more basal regions. It is clear that more refined measures will be necessary to enable measurement of functional losses associated with loss of apical hair cells and of the histopathological basis for the losses of high-frequency sensitivity. Summary Quiet-aged gerbils show a remarkable range of OHC loss whereas loss of IHCs is unusual except in cases of extreme degeneration. OHC losses are greatest at the extreme ends of the cochlea. These losses, particularly in the

apex, can occur without corresponding changes in CAP thresholds for frequencies below 3 kHz. CAP threshold elevations occur without concomitant hair-cell losses above the 4 kHz frequency location. The extent of hair-cell losses corresponded in a general way with increased thresholds, suggesting that a common phenomenon may be responsible for both changes. Cytocochleograms and CAP thresholds are shown to be inadequate measures for assessing pathologies of the cochlea that are associated with aging. Acknowledgements The authors wish to express their appreciation to Drs. J.H. Mills and B.A. Schulte for their contributions of ideas and comments concerning this research and to thank Larry Kulish and Sharon Heape for their technical assistance. This study was supported by NIH grant P50 DC00422. References Beagley. H. (1965) Acoustic trauma in the guinea pig: II. Electron microscopy including the morphology of cell junctions in the organ of Corti. Acta Otolaryngol. 60, 479-495. Beck, C. and Michler. H. (1960) Feinstrukturelle and histochemische veraenderagnen an den strukturen der cochlea beim meerschweinchen nach dosierter reintonbeschallung. Arch. Ohr. Nas., Kehlk. Heilk. 174, 496-567. Bhattacharyya. T.K. and Dayal. VS. (1985) Age-related cochlear hair cell loss in the chinchilla. Ann. Otol. Rhinol. Laryngol. 94, 75-80. Bhattacharyya. T.K. and Dayal. V.S. (1989) Age related hair cell loss in the organ of Corti of the rabbit. Abstr. Assoc. Res. Otolaryngol.. Clearwater Beach, FL, p. 356. Borg, E. and Viberg, A. (1987) Age-related hair cell loss in spontaneously hypertensive and normotensive rats. Hear. Res. 30, 111-118. Bredberg, G. (1968) Cellular pattern and nerve supply of the human organ of Corti. Acta Otolaryngol. (SuppI.) 236, 3-13s. Burg, M.B. (IY86) Renal handling of sodium, chloride. water, amino acids. and glucose. In: B.M. Brenner and F.C. Rector. Jr. (Eds.). The Kidney. 3rd ed., Saunders, Philadelphia, PA, p. 145. Cheal, M. (1986) The gerbil: a unique model for research on aging. Exp. Aging Res. 12, 3-21.

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