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Histopathologic observations of the aging gerbil cochlea
ItBI ELSEVIER
Hearing Research 104 (1997) 101 111
Histopathologic observations of the aging gerbil cochlea Joe C. Adams a, Bradley A. Schulte b,, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA b Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA Received 29 March 1996; revised 22 October 1996; accepted 29 October 1996
Abstract
Age-related histopathologic changes were examined in cochleas from 17 gerbils born and kept in a quiet environment until near the end of their life expectancy. Hearing loss varied greatly as did the loss of outer hair cells (OHC). Inner hair cells (IHC) were seldom missing even in cochleas with severe hearing losses. Flask- and spherical-shaped OHCs were frequently seen in the apical turn. Stereocilia were usually present and orderly on OHCs, but the tallest row of stereocilia on IHCs was often disarrayed and sometimes missing. Alterations in supporting cells were sometimes present in regions of extensive OHC loss. Although pillar cells were seldom missing, the nuclei of outer pillar cells were commonly displaced from their normal basal position. The density of radial fibers appeared similar to that in young gerbils except in the apical turn of one old ear where a marked loss of radial fibers occurred without an attendant loss of IHCs. All of the quiet-aged cochleas showed a characteristic clustering of epithelial cells lining the scala media surface of Reissner's membrane. This structural rearrangement was not accompanied by a significant decrease in the total number of cells forming Reissner's membrane and did not appear to be associated with hearing loss. The findings confirm and extend earlier work showing that several different types of cells are susceptible to histopathologic changes in old ears. The extent of histopathologic changes varied widely as did the degree of hearing loss in animals with a restricted genetic background and maintained under carefully controlled environmental conditions. It was not possible, based on these initial findings, to relate specific structural to specific functional changes in the aging cochlea. Further light and electron microscopic analysis of other regions from these aged cochleas may provide more conclusive data.
Keywords: Pathology; Presbycusis; Organ of Corti; Hair cell
1. Introduction
Various degrees and forms of cell loss and degeneration have been reported to occur with age in animal and human inner ears (see Cohen and Park, 1989; Gulya, 1990; Schuknecht and Gacek, 1993). Differences in emphasis and technique have resulted in reports of a wide range of age-related histopathologic changes, but few studies have attempted a comprehensive description of conditions that characterize the aged cochlea. In most previous studies, inner ears were processed to optimize visualization of a particular region or cell-type, and observations were usually made on only a few cochleas, often with unknown histories of exposure to noise and ototoxic drugs. In addition, most previous studies re* Corresponding author. Fax: +1 (803) 792 0368.
ported only histologic findings and did not include measures of the functional state of the ears before they were prepared for microscopic examination. The present report stems from a multifaceted study of presbycusis using the Mongolian gerbil as an animal model. The animals were maintained in a quiet environment and their cochleas were collected near the end of their life expectancy. Physiological measures of cochlear function and counts of hair cells (HC) from these animals have been reported elsewhere (Hellstrom and Schmiedt, 1990, 1991 ; Mills et al., 1990; Schmiedt et al., 1990; Mills, 1991; Schmiedt and Schulte, 1992; Tarnowski et al., 1991). We describe here pathologic changes that were observable at the light microscopic level, with emphasis upon the organ of Corti and Reissner's membrane. A somewhat unconventional surface preparation was employed to retain as much tissue as
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Fig. I. A: Surface view of the organ of Corti. *, Space previously occupied by an IHC. The darkly stained stereocilia of the O H C s appear orderly. An O H C is missing from row 1 and four O H C s are missing from rows 2 and 3. B a r = 12 p.m. B: Similar view from another cochlea showing a flask-shaped O H C in row 1 (arrow). *, Missing O H C in row 3. M a n y other O H C s are missing in adjacent corresponding areas. Calibration same as (A). C: The arrow points to a row 1 0 H C that has assumed a spherical shape and still retains its stereocilia. The adjacent O H C s in row 1 are missing, as are m a n y from the other rows. Calibration same as (A). D: A lone O H C (arrow) has assumed a spherical shape and apparently is still attached to a Deiters' cell. The cells along the right margin are Deiters' cells and those along the left margin are outer pillar cells. B a r = 10 [.tm.
possible for inspection by both light and electron microscopy. Ganglion cells and their fibers, the organ of Corti, and structures within the lateral wall all can be examined by re-embedding and processing different portions of the specimens to optimize visualization of given structures. This approach offers the advantage of permitting high resolution assessment of virtually all cell types within the cochlea, although the present report is largely limited to observations of surface preparations. When all phases of the analysis are complete, the results should offer an unprecedented broad perspective of cellular changes that occur with age and permit establishment of correlations between age-related hearing losses and corresponding cellular pathologies. Preliminary findings have been reported in abstract form (Adams et al., 1989).
2. Materials and methods
Cochleas were obtained from 17 gerbils, aged 33-36 months, following a terminal physiological experiment. The animals were born and raised in an especially quiet vivarium wherein the noise level rarely exceeded 41 dBA. Procedures for the care and use of animals were approved by the Animal Use Committee of the Medical University of South Carolina under NIH Grant P01 DC00422. The deeply anesthetized animals (1.5 g/kg urethane, i.p.) were perfused transcardially with 0.12 M phosphate buffer, pH 7.3, near body temperature, containing 0.1% sodium nitrite followed by approximately 100 ml of fixative. The fixative contained 4% freshly depolymerized paraformaldehyde and 2% glutaraldehyde in the same buffer that was used for the rinse. Each bulla was opened quickly; the round window was pierced and the stapes removed. Fixative was infused gently through the oval window until it escaped through the round window. The cochleas were then removed from the skull, immersed in fixative, and left refrigerated overnight. The fixative was flushed from the scalae using phosphate buffer and the inner ears were immersed in a solution of 0.12 M EDTA (pH 7.0) containing 0.2% glutaraldehyde. The cochleas were agitated gently at room temperature in the EDTA-fixative solution, which
was changed every 48 h until decalcification was complete ( ~ 2 weeks). The decalcified cochleas were bisected along the plane of the modiolus using a razor blade. To reduce compressive distortion of the organ of Corti during the bisection, the scalae were perfused with warm 10% gelatin and then chilled on an ice bath until the gelatin congealed. Following bisection, the gelatin was removed from the half cochleas by agitation in warm buffer. The tissue was immersed in 0.8% orcein in acetic acid/alcohol for 3 rain (Clark, 1960), rinsed, and placed in 1% OsO4 in phosphate buffer for 30 min, washed in water, then dehydrated and embedded in Epon resin. Prior to complete polymerization of the resin, the cochleas were dissected into individual halfturns and re-embedded in resin in a slide mold to facilitate subsequent viewing of the tissue as a surface preparation. Most of the microscopy was done with a planachromat 40 x oil immersion objective (Zeiss Epiplan, N.A. 0.85) with an exceptionally long working distance ( ~ 1.5 mm). The above procedures allowed visualization of the cochleas as surface preparations for examination of the organ of Corti as well as Reissner's membrane. Neither the lateral wall, including the stria vascularis, nor cells within the modiolus were readily observable in this material. However, these regions were available for study by light and electron microscopy in radial sections which were cut from the individual half-turns. Findings from observations of these radial sections will be reported elsewhere. Preliminary examination of the surface preparations demonstrated considerable differences in the appearance of Reissner's membrane between young and old ears. Unfortunately, the surface preparation did not provide a high resolution view of Reissner's membrane in all specimens. To better study the cells within Reissner's membrane, cochleas from several young gerbils ( ~ 6 weeks old) and several older animals (23-24 months old) were fixed, decalcified, and dissected in the same manner as described above, except that instead of being osmicated and embedded in plastic, Reissner's membrane was removed with fine forceps, flat-mounted on a glass slide, stained with Azure A, and coverslipped. This procedure provided a high resolution, orthogonal view of the membrane and facilitated evaluation of the cell types, numbers, and their distribution.
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Fig. 2. A: Short stereocilia on IHCs appear in a column down the center of the field, and are darkly stained. The tallest row of stereocilia is not visible in this image owing to the absence of orcein staining. Instances of missing and displaced short stereocilia as shown here were exceptional. B a r = 10 I.tm. B: The arrow indicates the position previously occupied by the head of an outer pillar cell. Note the spaces at and above the arrow that were previously occupied by first and second row OHCs. C: This field demonstrates cellular disarray within an ear that showed large threshold shifts and m a n y missing HCs. The large filled arrow indicates three outer pillar cell nuclei that are not in their normal position, The small filled arrow at the b o t t o m shows an outer pillar cell nucleus in its normal position. The open arrow points to what appear to be three Deiters' cells that are displaced to the center of the tunnel of Corti. The arrowhead points to mesenchymal cells on the b o t t o m surface of the basilar m e m b r a n e which are in focus due to tilting of the specimen. D: Outer pillar cells from the same specimen shown in (C). The nuclei o f the pillar cells (arrows) are high up the pillar process near the cuticular plate. The nuclei at the right are those of Deiters' cells.
Staining of the tissue with orcein greatly facilitated examination of stereocilia. The surface preparations were thicker than most such preparations because the spiral ligament was retained. The thicker specimens limited the usefulness of phase-contrast and Nomarski optics so the added contrast afforded by the orcein showed cellular detail that would otherwise not have been visible. In addition to stereocilia, other structures made apparent by orcein were cell nuclei and cell boundaries. All rows of O H C stereocilia were stained with orcein, as were the shorter rows on IHCs. However, the tallest row of stereocilia on IHCs failed to stain, possibly indicating a chemical difference between this row and all others.
derly (Fig. 1A). Misaligned and missing short IHC stereocilia such as those shown in Fig. 2A were exceptional. A more common finding was disarrayed and missing stereocilia in the tallest row on IHCs. Because these did not stain and showed little contrast, they were difficult to photograph and are not illustrated. There was not a good correlation of locations of disrupted stereocilia and frequencies at which hearing losses were found. Regions where stereocilia were disrupted sometimes corresponded to places where threshold shifts of evoked potentials indicated the presence of some abnormality. In other cases there were disrupted stereocilia at cochlea places where thresholds were normal. There was no evidence of artifactual disruption of stereocilia that, for example, may have been present at or near sites where the cochleas were bisected.
3.1. Hair cells
3.2. Pillar cells
Data concerning the number of missing and obviously abnormal hair cells have been reported elsewhere (Tarnowski et al., 1991). Few IHCs were missing. Even in cases that showed the most advanced stages of degeneration, the IHCs appeared normal except for stereocilia disarray (see below). Fig. 1A shows a rare instance in which there is a missing IHC. Also shown is the common finding of considerably more missing OHCs than IHCs. In contrast to IHCs, OHCs occasionally assumed abnormal flask (Fig. 1B) or rounded (Fig. 1C,D) shapes. These changes in shape were accompanied by detachment of the cells at either their apical or basal pole. In some cases, spherical-shaped OHCs which remained attached at the cuticular plate still retained their stereocilia (Fig. 1C). Another, less frequently observed alteration of OHCs, was their detachment from the cuticular plate and assumption of a spherical shape while apparently remaining attached to Deiters' cells (Fig. 1D). Under the latter condition, intact stereocilia were never observed. Spherical and flask-shaped OHCs were observed exclusively in the apical half of the cochlea, with most being present in the extreme apex (see Tarnowski et al., 1991). Differences in the state of orderliness of the stereocilia were seen among cochleas, as well as between regions within a given cochlea. In general, the darkly stained O H C and short IHC stereocilia were quite or-
Pillar cells constituted the other major cell type within the organ of Corti that showed obvious pathological changes. Although seldom missing (Fig. 2B shows an exception) the pillar cell nuclei were often misaligned, but with no apparent apical migration of the nuclei (Fig. 2C). The two cochleas with the greatest hearing loss, as assessed by evoked potential audiograms, showed the greatest losses of hair cells (see Tarnowski et al., 1991) and also the greatest number of abnormal outer pillar cells. Fig. 2D illustrates an extreme example where the nuclei of the outer pillar cells have migrated to the cells' apices (see also Fig. 4B). Even in these cases, pillar cells were seldom missing. In one instance a few transformed Deiters cells also were displaced into the tunnel of Corti (Fig. 2C).
3. Results
3.3. Reissner's membrane
Examination of surface preparations, radial sections and flat mounts showed that the cells comprising Reissner's membrane in young animals were spaced relatively uniformly (Fig. 3A) as compared to older animals where clusters of epithelial ceils separated by areas of lower cell density were routinely observed (Fig. 3B,C). Counts of cell nuclei were made in surface preparations from young and old animals to assess changes in cellularity. The density of all cells within Reissner's
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Fig. 3. A: Flat-mounted Reissner's membrane from a 6-week-old gerbil. The arrowhead points to a cell whose cytoplasm has been made visible by the azure stain. This cell also contains a few pigment granules. Only nuclei of other cells are stained. Nuclei of epithelial cells lining the scala media appear fairly uniform in size and most are euchromatic. Bar--70 [am. B: Reissner's membrane in a 36-month-old gerbil, embedded in epoxy. The orcein-stained nuclei are clearly visible. The arrows indicate clusters of epithelial cells with small nuclei. Bar= 60 [am. C: Flatmounted Reissner's membrane from a 24-month-old gerbil. Clustering of smaller, more heterochromatic nuclei is present in the upper left portion of the field. The small, faint dots are lipofuscin granules. Calibration same as (A). D: Surface view of OHCs (top) and Claudius cells (below). The arrowhead points to a shrunken Claudius cell with dark cytoplasm. Bar = 25 [am.
m e m b r a n e was measured in the basal, middle, and apical turns of eight cochleas taken from animals aged 3 months or less. These counts were compared to measures made in seven cochleas taken from animals that were approximately 3 years old and selected because the preparations allowed a favorable view of Reissner's m e m b r a n e in all three turns. Counts in the old animals were made from areas that included both clusters of cells and adjacent, cell sparse areas (see Fig. 3B) in an attempt to obtain a measure of the average cell density. The results showed that cell density was significantly greater in the apical as compared with the basal turn (t test, P < 0.05), but no significant differences in cell density were found between old and young animals when respective turns were compared. Age-related changes in Reissner's m e m b r a n e also were evaluated in Azure A-stained flat mounts from young and from 2-year-old gerbils. At least three different cell populations were identified, based on the size and staining properties of their nuclei and cytoplasm (Fig. 3). These consisted of clusters of epithelial cells with smaller more dense heterochromatic nuclei interspersed with less densely packed cells with larger, more euchromatic nuclei. A third cell type characteristically showed cytoplasmic azurophilia and contained varying amounts of pigment granules (Fig. 3A). The pigmented cells were a small minority of the cells present. The tissue depicted in Fig. 3C was from an animal only 2 years old but the clustering of epithelial cells, which was pronounced in 3-year-old gerbils (Fig. 3B) already was evident. Lipofuscin granules were abundant within cells of Reissner's m e m b r a n e in the stained flat-mount preparations of 2-year-old animals (Fig. 3C) but not those of young animals (Fig. 3A). 3.4. Miscellaneous Other forms of histopathology were occasionally observed. For example, Fig. 3D illustrates what appears to be a degenerating Claudius cell. This cell was within a field where other cells appeared normal. Another infrequent observation was a marked decrease in radial fiber density in the apical half-turn of one 36-month-old gerbil (Fig. 4A). In contrast, Fig. 4C illustrates the high density of radial fibers in the same region from the ear of another 36-month-old gerbil that showed only a small hearing loss and a minimal loss of hair cells at
any site along the cochlear partition. The C A P threshold in response to 500 Hz tone bursts was greater than 50 dB higher for the ear shown in Fig. 4A as compared with that shown in Fig. 4C. In the whole mount, I H C s appeared to be present at the locations corresponding to the loss of radial fibers shown in Fig. 4A, but overlying tissue obscured the surface view somewhat. Radial sections were therefore cut through this region and these showed the presence of I H C s (Fig. 4B). N o radial fibers were seen in the osseous spiral lamina of the radial sections. In the region of missing radial fibers, O H C s were missing and the positions of the outer pillar cell nuclei were also abnormal (Fig. 4B). This report includes the cochlea of one old gerbil that was excluded in the previous paper on H C counts (Tarnowski et al., 1991). The animal was 36 months old at the termination of the experiment and the cochlea was dramatically different from that of all other animals. Physiological measurements showed no endocochlear potential and no evoked response to high levels of sound stimulation. When the cochlea was bisected, it was found to be filled with fibrous connective tissue. The tissue was osmicated and embedded in plastic without further dissection. Semithin sections (Fig. 4D) showed that the organ of Corti, the entire lateral wall, the auditory nerve and the ganglion cells had been replaced by highly vascularized connective tissue and focal accumulations of inflammatory cells. Blood vessels in the bone were atrophied and replaced by flocculent material. In addition, there was new bone growth in the basal and middle turns (Fig. 4D). Neither the external nor middle ear of this animal showed unusual features.
4. Discussion
This report is part of a comprehensive study of hearing losses and histopathological changes in cochleas from animals near the end of their life expectancy. More animals were included than in previous aging studies and they were kept in an especially quiet environment and had no exposure to any known ototoxins throughout their lives. The most remarkable findings were: (1) the great variability in the type and severity of histopathological changes seen a m o n g animals of the same age, raised under carefully controlled environmental conditions; and (2) the lack of a close correspondence between the loci of cellular disruptions and the
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Fig. 4. A: Radial fibers in the osseous spiral lamina in the apical turn of a 36-month-old gerbil that showed marked threshold elevations at all frequencies. The number of fibers is reduced considerably as compared with an age-matched control shown in (C). Bar= 12 gm. B: Semithin radial section through the same tissue that was shown in (A). The open arrow points to the head of an IHC. The arrowhead points to a nucleus of an outer pillar cell that is apparently migrating up the pillar. *, Osseous spiral lamina which is devoid of myelinated fibers in this section. Bar = 44 gm. C: Radial fibers in the apical turn of a 36-month-old gerbil that showed little threshold elevation at any frequency. The density of the osmicated fibers is clearly greater than those shown in (A). Calibration same as (A). D: Cross-section through the middle turn of the 36-month-old gerbil that showed no physiological signs of hearing. The arrowhead points to what was originally a blood vessel in bone. Such vessels were filled with a dense precipitate. The blood vessels of the fibrous connective tissue and cellular infiltrate that has replaced the normal cochlear tissue appear as clear areas and contain no precipitate. Blood cells were removed from these vessels by the cardiac perfusion. The asterisk lies over new bone growing in the region that was formerly scala vestibuli. The blood vessel indicated by the arrowhead is 116 gm long,
frequencies at which h e a r i n g losses were present. T h e range o f h i s t o p a t h o l o g i c changes is in a g r e e m e n t with studies showing large differences in h e a r i n g losses a m o n g similarly aged a n i m a l s from o u r o u t b r e d gerbil c o l o n y (Hellstrom a n d Schmiedt, 1990; Mills et al., 1990) a n d p o i n t to genetic variability as a d e t e r m i n a n t in presbycusis. The lack o f c o r r e s p o n d e n c e of histop a t h o l o g y a n d h e a r i n g loss is in agreement with previous o b s e r v a t i o n s that d i s r u p t i o n s of the o r g a n of Corti a c c o u n t for only some o f the h e a r i n g losses that occur due to aging. T h e p a t t e r n of cell loss a n d d e g e n e r a t i o n within the
o r g a n of Corti indicated that O H C s are most vulnerable to age-related degenerative changes. A previous analysis of cytocochleograms from the cochleas examined here revealed a good correlation between overall h e a r i n g loss a n d total H C loss, b u t there was a p o o r c o r r e l a t i o n between H C losses a n d C A P threshold shifts at specific locations a l o n g the frequency-place m a p ( T a r n o w s k i et al., 1991). Thus, a l t h o u g h the extent of H C loss provides a gross assessment of the general state of the cochlea, other forms of cellular p a t h o l o g y must be sought to explain the large variability in hearing loss at specific frequencies. T h e present o b s e r v a t i o n s ex-
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tended previous findings to include assessment of stereocilia disarray and of the condition of non-sensory cells. These additional observations revealed that organ of Corti pathology, as seen with the light microscope, cannot account for the hearing losses that were found in the previously reported physiological tests. Pathological changes in supporting cells were present only in regions where there were relatively large numbers of missing OHCs. It is therefore apparent that degenerative changes in supporting cells cannot account for hearing losses that were not associated with O H C loss. Nevertheless, the nature of the degeneration of the supporting cells differed somewhat from previous reports and merits discussion. The most prevalent abnormality of supporting cells was disruption of outer pillar cells. This was usually manifest as a misalignment or displacement of nuclei and was found in cochleas that showed the largest O H C losses. In contrast to reports in other mammalian species (Covell and Rogers, 1957; Bredberg, 1968; Schuknecht, 1993; Nadol, 1980), in the present study supporting cells were seldom missing. The lack of missing supporting cells may be because the gerbils were raised in a quiet environment, whereas in the animal study cited above there were apparently no special precautions taken against acoustic trauma and in the studies of human material, adequate documentation of acoustic and medical histories was not available. Bohne et al. (1990) reported a decrease in the number of pillar cells with advancing age in chinchillas but migration of pillar cell nuclei was uncommon. Our material consisted of ears from 17 gerbils near the end of their life expectancy, whereas the oldest chinchillas studied by Bohne et al. (1990) were from animals in the last 40% of the average lifespan of their colony, with very few cases at the extreme of their expected lifespan. Perhaps changes in pillar cells, such as we observed in the gerbil would be more evident in a population of chinchillas that included more cases of advanced age. Changes in the appearance of the heads of pillar cells were common in aging chinchillas (Bohne et al., 1990), but no such alterations were seen in the present material. The use of different tissue processing methods in the two studies may account for this apparent discrepancy. A clear but not necessarily causal relationship has been established between loss of sensitivity of eighth nerve fibers and disruption of stereocilia induced by acoustic trauma (see Liberman, 1987). To our knowledge, the present study is the first analysis of stereocilia disarray in aged animals raised in a quiet environment. In general, O H C stereocilia appeared orderly, with little evidence of disarray except on obviously abnormal OHCs. Unfortunately, our preparation was not optimal for assessing IHC stereocilia because the tallest row failed to stain with orcein. Nevertheless, IHC stereocilia disarray occasionally was observed in cochlear locations
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where CAP thresholds were normal. Conversely, IHC stereocilia usually appeared normal even at locations with markedly elevated CAP thresholds. Although no firm conclusions can be drawn concerning the relationship of stereocilia disarray and threshold shift, the present findings, along with previous results (Tarnowski et al., 1991), strongly suggest that organ of Corti dysfunction cannot account for a sizable portion of hearing losses in quiet-aged gerbils. The most consistent age-related structural change in the gerbil cochlea was the development of densely packed clusters of epithelial cells in Reissner's membrane. This phenomenon was not a definitive sign of extreme age, however, because it was apparent even in 2-year-old cochleas. Mesenchymal cells on the scala vestibuli side of Reissner's membrane were difficult to positively identify in surface preparations and flat mounts, so the identity of the clustered cells was confirmed by ultrastructural examination of radial sections taken from the same specimens (unpublished observations). The clustering of epithelial cells in the gerbil may correspond to the formation of 'whorls' of epithelial cells, first described by Retzius (1884), and shown by Johnsson (1971) to be present in humans as young as 16 years. Watanuki et al. (1981) and Quijano et al. (1990) have also noted increased irregularity in the arrangement of the epithelial surface of Reissner's membrane with increasing age in human ears. Studies in both rats (Feldman, 1990) and humans (Yoon et al., 1991) have reported an age-related decrease in the cellularity of Reissner's membrane. Counts of all cell types in Reissner's membrane in humans showed a decrease in cell density at a very young age, followed by a long period of stable cell numbers, and a further decrease beyond the age of 75 years (Quijano et al., 1990). Our results in gerbil differ in that we did not find a decrease in total cell number with age. Perhaps the 'young' gerbils in our control group were beyond the developmental stage of the 'young' cases of Quijano et al. (1990), or methodological differences may be responsible for the apparent disparity in the results. It also is possible that this disparity reflects differences between species in the population dynamics of mesenchymal cells lining the scala vestibular side of Reissner's membrane. In the gerbil the mesenchymal cells form a densely packed layer during postnatal development but rapidly decline in number and become sparse by 1 month of age (unpublished observations). Although the arrangement and perhaps also the composition of cells in Reissner's membrane changes with age, the changes evidenced by clustering of epithelial cells were present in 2- and 3-year-old gerbils with normal or near-normal hearing, as well as in adolescent humans (Johnsson, 1971). Moreover, the consistent structural changes seen in Reissner's membrane in all 3-year-old gerbils contrasts with the wide variability in
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the amount of O H C loss and in the degree of hearing loss among these same ears (Tarnowski et al., 1991). It therefore seems unlikely that the cellular changes in Reissner's membrane are closely related to hearing losses. A similar conclusion was reached by Quijano et al. (1990). It seems clear that a variety of histopathological changes are associated with age-related hearing loss even under carefully controlled environmental conditions. Schuknecht (1993) has classified presbycusis into four different categories and we have observed examples of three of these types in aging gerbils, although no one category appeared in a pure form. Sensory presbycusis was evidenced by loss of hair cells (Tarnowski et al., 1991). In contrast with findings in humans where H C loss is characteristically in the basal third of the cochlea (Schuknecht, 1993), the predominant loss in gerbils was in the apex with a less pronounced loss in the base. Consistent with our gerbil data, aged chinchillas (Bhattacharyya and Dayal, 1985; Bohne et al., 1990), guinea pigs (Dayal and Barek, 1975; Coleman, 1976), rats (Keithley and Feldman, 1982) and squirrel monkeys (Dayal and Bhattacharyya, 1986) all also show the earliest and largest losses of hair cells in the apex. The exception of basal HC losses in humans may be due to species differences. However, environmental variables, such as acoustic history or entry of toxins via the round window due to middle ear infections, must be considered as possible explanations of the basal degeneration seen in humans. We observed one example of primary neural presbycusis which involved loss of nerve fibers without a corresponding loss of associated hair cells. Bohne et al. (1990) reported that 5% of their chinchillas showed radial fiber loss without accompanying H C loss. The latter finding indicates that neural degeneration can occur at or before midlife and raises the question of whether this disorder should actually be considered a sign of presbycusis. We have no way of determining the age at which the neural loss occurred in one region of one gerbil ear. However, our results are in keeping with the estimate of 5% incidence and the apical location of the loss reported in chinchillas. Loss of spiral ganglion cells with age has been reported for several species including the gerbil (Covell and Rogers, 1957; Bredberg, 1968; Johnsson and Hawkins, 1972; Schuknecht and Gacek, 1993; Nadol, 1979, 1980; Keithley and Feldman, 1979; Keithley et al., 1989, 1992). Our preliminary findings based on examination of radial sections confirm this observation. However, with the exception of the extreme case shown in Fig. 4A, ganglion cell losses were not reflected by appreciable changes in radial fiber density. Metabolic presbycusis (strial degeneration) has been observed to varying degrees in the gerbils under study (Schmiedt and Schulte, 1992) and is associated with loss
of immunostaining for Na,K-ATPase and a decline in the endocochlear potential (Schulte and Schmiedt, 1992). Unfortunately the surface preparations, as viewed in the present work, were not suitable for evaluation of structural changes in the lateral wall. Light and electron microscopic morphologic evaluation of radial sections cut from the present material will provide further documentation of pathologic changes in the aging lateral wall. Degeneration of the stria vascularis occurs commonly in aged ears of many species, including humans (Johnsson and Hawkins, 1972; Schuknecht, 1993; Nadol, 1979, 1980; Keithley et al., 1989, 1992; Schuknecht and Gacek, 1993) and undoubtedly plays an important role in the pathophysiology of presbycusis. A fourth category of presbycusis has been termed 'cochlear conductive' loss (Schuknecht, 1993). No histopathological changes have been identified that characterize this hypothetical form of presbycusis, but are presumed to be present based on a characteristic audiometric finding. No animals in our study showed such an audiogram and we observed no evidence of histopathological changes that suggested the presence of possible changes in cochlear sound conductive abilities. Gerbils in our colony did, however, often develop a conductive hearing loss due to impaction of the external auditory canals (Mills et al., 1990; see also Henry et al., 1983). Such losses are not described here because only animals with clear external canals were selected for study. The replacement of all normal cochlear structures by fibrous connective tissue, new bone, and inflammatory cells in the case illustrated in Fig. 4D is not considered to be a normal aging phenomenon. The case presented here appears to be the end stage of suppurative labyrinthitis that follows bacterial infections (Schuknecht, 1993). It serves as a reminder of the importance of obtaining histological verification of hearing pathology, regardless of the presumed cause. The variety of changes in the cochlea of aging gerbils observed in this and previous studies confirms the contention that presbycusis is a multifaceted and complex phenomenon. The type and severity of histopathologic changes varied widely, even in animals with a restricted genetic background maintained under carefully controlled conditions throughout their lifespan. The finding that cochleas which showed the greatest hearing losses also showed morphological changes in a variety of cell types suggests that animals prone to hearing loss were not selectively losing sensory cells, but rather were subject to a more general deterioration of the ear and probably other tissues as well.
Acknowledgments The authors wish to thank Mrs. Leslie Harrelson for
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e d i t o r i a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d b y res e a r c h G r a n t P01 D C 0 0 4 2 2 f r o m t h e N a t i o n a l I n s t i t u t e on Deafness and Other Communication Disorders, National Institutes of Health.
References Adams, J.C., Tarnowski, B.I., Schmiedt, R.A., Hellstrom, L. and Schulte, B.A. (1989) Age related changes in the cochlea. Anat. Rec. 223, 6A. Bhattacharyya, T.K. and Dayal, V.S. (1985) Age-related cochlear hair cell loss in the chinchilla. Ann. Otol. Rhinol. Laryngol. 94, 75 80. Bohne, B.A., Gruner, M.M. and Harding, G.W. (1990) Morphological correlates of aging in the chinchilla cochlea. Hear. Res. 48, 7992. Bredberg, G. (1968) Cellular pattern and nerve supply of the human organ of Corti. Acta Otolaryngol. Supp. 236, 1-135. Clark, G. (1960) Staining Procedures Used by the Biological Stain Commission. Williams and Wilkins, Baltimore, MD, p. 63. Cohen, G.M. and Park, J.C. (1989) The developing and senescent inner ear: selected topics and models. CRC Crit. Rev. Neurobiol. 4, 179-199. Coleman, J.W. (1976) Hair cell loss as a function of age in the normal cochlea of the guinea pig. Acta Otolaryngol. 82, 3340. Covell. W.P. and Rogers, J.B. (1957) Pathologic changes in the inner ears of senile guinea pigs. Laryngoscope 67, 118-129. Dayal, V.S. and Barek, W.G. (1975) Cochlear changes from noise, kanamycin and aging. Laryngoscope 85 (Suppl.), 1 18. Dayal, V.S. and Bhattacharyya, T.K. (1986) Comparative study of age-related cochlear hair cell loss. Ann. Otol. Rhinol. Laryngol. 95, 510-513. Feldman, M.L. (1990) Reissner's membrane in aged rats. Abstr. Assoc. Res. Otolaryngol., 421422. Gulya, A.J. (1990) Aging: structural and physiological changes of the auditory and vestibular mechanisms. ASHA Rep. 13, 126-133. Hellstrom, L.I. and Schmiedt, R.A. (1990) Compound action potential input/output functions in young and quiet-aged gerbils. Hear. Res. 50, 163-174. Hellstrom, L.I. and Schmiedt, R.A. (1991) Rate-level functions of auditory-nerve fibers have similar slopes in young and old gerbils. Hear. Res. 53, 217 222. Henry, K.R., Chole, R.A. and McGinn, M.D. (1983) Age-related increase of spontaneous aural cholesteatoma in the Mongolian gerbil. Arch. Otolaryngol. 109, 19 21. Johnsson, L. (1971) Reissner's membrane in the human cochlea. Ann. Otol. Rhinol. Laryngol. 80, 425438. Johnsson, L. and Hawkins, J.E. Jr. (1972) Sensory and neural degeneration with aging, as seen in microdissections of the human inner ear. Ann. Otol. Rhinol. Laryngol. 81, 179 193. Keithley, E.M. and Feldman, M.L. (1979) Spiral ganglion cell counts
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in an age-graded series of rat cochleas. J. Comp. Neurol. 188, 429442. Keithley, E.M. and Feldman, M.L. (1982) Hair cell counts in an agegraded series of rat cochleas. Hear. Res. 8, 249-262. Keithley, E.M., Ryan, A.F. and Woolf, N.K. (1989) Spiral ganglion cell density in young and old gerbils. Hear. Res. 38, 125 134. Keithley, E.M., Ryan, A.F. and Feldman, M.L. (1992) Cochlear degeneration in aged rats of four strains. Hear. Res. 59, 171-178. Liberman, M.C. (1987) Chronic ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates. Hear. Res. 26, 65 88. Mills, J.H. (1991) Effects of age and existing hearing loss. In: A. Dancer, D. Henderson, R.J. Salvi and R.P. Hamernick (Eds.), Noise Induced Hearing Loss, Mosby Year Book, St. Louis, MO, pp. 237-245. Mills, J.H., Schmiedt, R.A. and Kulish, L.F. (1990) Age-related changes in auditory potentials of Mongolian gerbil. Hear. Res. 46, 201 210. Nadol, J.B. Jr. (1979) Electron microscopic findings in presbyacusic degeneration of the basal turn of the human cochlea. Otolaryngol. Head Neck Surg. 87, 818-836. Nadol, J.B. Jr. (1980) The aging peripheral mechanism. In: D.S. Beasley and G.A. Davis (Eds.), Aging Communication Processing and Disorders. Grune and Stratton, Orlando, FL, pp. 63 85. Quijano, M.L., Kimura, R.S. and Fowler K.B. (1990) Reissner's membrane in aging, Meni~r~'s disease and profound sensorineural deafness. Oto-Rhino-Laryngol. 52, 273-280. Retzius, G. (1884) Das Geh6rorgan der Wirbelthiere. III. Das Geh6rorgan der Reptilien, der V6gel, and der S~iugethiere. Samson and Walin, Stockholm. Schmiedt, R.A. and Schulte, B.A. (1992) Physiologic and histopathologic changes in quiet- and noise-aged gerbil cochleas. In: A. Dancer, D. Henderson, R.J. Salvi and R.P. Hamernick (Eds.), Noise Induced Hearing Loss, Mosby Year Book, St. Louis, MO, pp. 246 256. Schmiedt, R.A., Mills, J.H. and Adams, J.C. (1990) Tuning and suppression in auditory nerve fibers of aged gerbils raised in quiet or noise. Hear. Res. 45, 221-236. Schuknecht, H.F. (1993) Pathology of the Ear. Lea and Febiger, Philadelphia, PA. Schuknecht, H.F. and Gacek, M.R. (1993) Cochlear pathology in presbycusis. Ann. Otol. Rhinol. Laryngol. 102, I 16. Schulte, B.A. and Schmiedt, R.A. (1992) Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Hear. Res. 61, 3546. Tarnowski, B.I., Schmiedt, R.A., Hellstrom, L.I., Lee, F. and Adams, J.C. (1991) Age-related changes in cochleas of Mongolian gerbils. Hear. Res. 54, 123-134. Watanuki, K., Sato, M., Kaku, Y., Shinkawa, S., Miyoshi, A. and Kawamoto, K. (1981) Reissner's membrane in human ears. Acta Otolaryngol. 91, 65 74. Yoon, T.H., Paparella, M.M., Schachern, P.A. and Le, C.T. (1991) Cellular changes in Reissner's membrane in endolymphatic hydrops. Ann. Otol. Rhinol. Laryngol. 100, 288-293.
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Histopathologic observations of the aging gerbil cochlea Joe C. Adams a, Bradley A. Schulte b,, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA b Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA Received 29 March 1996; revised 22 October 1996; accepted 29 October 1996
Abstract
Age-related histopathologic changes were examined in cochleas from 17 gerbils born and kept in a quiet environment until near the end of their life expectancy. Hearing loss varied greatly as did the loss of outer hair cells (OHC). Inner hair cells (IHC) were seldom missing even in cochleas with severe hearing losses. Flask- and spherical-shaped OHCs were frequently seen in the apical turn. Stereocilia were usually present and orderly on OHCs, but the tallest row of stereocilia on IHCs was often disarrayed and sometimes missing. Alterations in supporting cells were sometimes present in regions of extensive OHC loss. Although pillar cells were seldom missing, the nuclei of outer pillar cells were commonly displaced from their normal basal position. The density of radial fibers appeared similar to that in young gerbils except in the apical turn of one old ear where a marked loss of radial fibers occurred without an attendant loss of IHCs. All of the quiet-aged cochleas showed a characteristic clustering of epithelial cells lining the scala media surface of Reissner's membrane. This structural rearrangement was not accompanied by a significant decrease in the total number of cells forming Reissner's membrane and did not appear to be associated with hearing loss. The findings confirm and extend earlier work showing that several different types of cells are susceptible to histopathologic changes in old ears. The extent of histopathologic changes varied widely as did the degree of hearing loss in animals with a restricted genetic background and maintained under carefully controlled environmental conditions. It was not possible, based on these initial findings, to relate specific structural to specific functional changes in the aging cochlea. Further light and electron microscopic analysis of other regions from these aged cochleas may provide more conclusive data.
Keywords: Pathology; Presbycusis; Organ of Corti; Hair cell
1. Introduction
Various degrees and forms of cell loss and degeneration have been reported to occur with age in animal and human inner ears (see Cohen and Park, 1989; Gulya, 1990; Schuknecht and Gacek, 1993). Differences in emphasis and technique have resulted in reports of a wide range of age-related histopathologic changes, but few studies have attempted a comprehensive description of conditions that characterize the aged cochlea. In most previous studies, inner ears were processed to optimize visualization of a particular region or cell-type, and observations were usually made on only a few cochleas, often with unknown histories of exposure to noise and ototoxic drugs. In addition, most previous studies re* Corresponding author. Fax: +1 (803) 792 0368.
ported only histologic findings and did not include measures of the functional state of the ears before they were prepared for microscopic examination. The present report stems from a multifaceted study of presbycusis using the Mongolian gerbil as an animal model. The animals were maintained in a quiet environment and their cochleas were collected near the end of their life expectancy. Physiological measures of cochlear function and counts of hair cells (HC) from these animals have been reported elsewhere (Hellstrom and Schmiedt, 1990, 1991 ; Mills et al., 1990; Schmiedt et al., 1990; Mills, 1991; Schmiedt and Schulte, 1992; Tarnowski et al., 1991). We describe here pathologic changes that were observable at the light microscopic level, with emphasis upon the organ of Corti and Reissner's membrane. A somewhat unconventional surface preparation was employed to retain as much tissue as
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Fig. I. A: Surface view of the organ of Corti. *, Space previously occupied by an IHC. The darkly stained stereocilia of the O H C s appear orderly. An O H C is missing from row 1 and four O H C s are missing from rows 2 and 3. B a r = 12 p.m. B: Similar view from another cochlea showing a flask-shaped O H C in row 1 (arrow). *, Missing O H C in row 3. M a n y other O H C s are missing in adjacent corresponding areas. Calibration same as (A). C: The arrow points to a row 1 0 H C that has assumed a spherical shape and still retains its stereocilia. The adjacent O H C s in row 1 are missing, as are m a n y from the other rows. Calibration same as (A). D: A lone O H C (arrow) has assumed a spherical shape and apparently is still attached to a Deiters' cell. The cells along the right margin are Deiters' cells and those along the left margin are outer pillar cells. B a r = 10 [.tm.
possible for inspection by both light and electron microscopy. Ganglion cells and their fibers, the organ of Corti, and structures within the lateral wall all can be examined by re-embedding and processing different portions of the specimens to optimize visualization of given structures. This approach offers the advantage of permitting high resolution assessment of virtually all cell types within the cochlea, although the present report is largely limited to observations of surface preparations. When all phases of the analysis are complete, the results should offer an unprecedented broad perspective of cellular changes that occur with age and permit establishment of correlations between age-related hearing losses and corresponding cellular pathologies. Preliminary findings have been reported in abstract form (Adams et al., 1989).
2. Materials and methods
Cochleas were obtained from 17 gerbils, aged 33-36 months, following a terminal physiological experiment. The animals were born and raised in an especially quiet vivarium wherein the noise level rarely exceeded 41 dBA. Procedures for the care and use of animals were approved by the Animal Use Committee of the Medical University of South Carolina under NIH Grant P01 DC00422. The deeply anesthetized animals (1.5 g/kg urethane, i.p.) were perfused transcardially with 0.12 M phosphate buffer, pH 7.3, near body temperature, containing 0.1% sodium nitrite followed by approximately 100 ml of fixative. The fixative contained 4% freshly depolymerized paraformaldehyde and 2% glutaraldehyde in the same buffer that was used for the rinse. Each bulla was opened quickly; the round window was pierced and the stapes removed. Fixative was infused gently through the oval window until it escaped through the round window. The cochleas were then removed from the skull, immersed in fixative, and left refrigerated overnight. The fixative was flushed from the scalae using phosphate buffer and the inner ears were immersed in a solution of 0.12 M EDTA (pH 7.0) containing 0.2% glutaraldehyde. The cochleas were agitated gently at room temperature in the EDTA-fixative solution, which
was changed every 48 h until decalcification was complete ( ~ 2 weeks). The decalcified cochleas were bisected along the plane of the modiolus using a razor blade. To reduce compressive distortion of the organ of Corti during the bisection, the scalae were perfused with warm 10% gelatin and then chilled on an ice bath until the gelatin congealed. Following bisection, the gelatin was removed from the half cochleas by agitation in warm buffer. The tissue was immersed in 0.8% orcein in acetic acid/alcohol for 3 rain (Clark, 1960), rinsed, and placed in 1% OsO4 in phosphate buffer for 30 min, washed in water, then dehydrated and embedded in Epon resin. Prior to complete polymerization of the resin, the cochleas were dissected into individual halfturns and re-embedded in resin in a slide mold to facilitate subsequent viewing of the tissue as a surface preparation. Most of the microscopy was done with a planachromat 40 x oil immersion objective (Zeiss Epiplan, N.A. 0.85) with an exceptionally long working distance ( ~ 1.5 mm). The above procedures allowed visualization of the cochleas as surface preparations for examination of the organ of Corti as well as Reissner's membrane. Neither the lateral wall, including the stria vascularis, nor cells within the modiolus were readily observable in this material. However, these regions were available for study by light and electron microscopy in radial sections which were cut from the individual half-turns. Findings from observations of these radial sections will be reported elsewhere. Preliminary examination of the surface preparations demonstrated considerable differences in the appearance of Reissner's membrane between young and old ears. Unfortunately, the surface preparation did not provide a high resolution view of Reissner's membrane in all specimens. To better study the cells within Reissner's membrane, cochleas from several young gerbils ( ~ 6 weeks old) and several older animals (23-24 months old) were fixed, decalcified, and dissected in the same manner as described above, except that instead of being osmicated and embedded in plastic, Reissner's membrane was removed with fine forceps, flat-mounted on a glass slide, stained with Azure A, and coverslipped. This procedure provided a high resolution, orthogonal view of the membrane and facilitated evaluation of the cell types, numbers, and their distribution.
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Fig. 2. A: Short stereocilia on IHCs appear in a column down the center of the field, and are darkly stained. The tallest row of stereocilia is not visible in this image owing to the absence of orcein staining. Instances of missing and displaced short stereocilia as shown here were exceptional. B a r = 10 I.tm. B: The arrow indicates the position previously occupied by the head of an outer pillar cell. Note the spaces at and above the arrow that were previously occupied by first and second row OHCs. C: This field demonstrates cellular disarray within an ear that showed large threshold shifts and m a n y missing HCs. The large filled arrow indicates three outer pillar cell nuclei that are not in their normal position, The small filled arrow at the b o t t o m shows an outer pillar cell nucleus in its normal position. The open arrow points to what appear to be three Deiters' cells that are displaced to the center of the tunnel of Corti. The arrowhead points to mesenchymal cells on the b o t t o m surface of the basilar m e m b r a n e which are in focus due to tilting of the specimen. D: Outer pillar cells from the same specimen shown in (C). The nuclei o f the pillar cells (arrows) are high up the pillar process near the cuticular plate. The nuclei at the right are those of Deiters' cells.
Staining of the tissue with orcein greatly facilitated examination of stereocilia. The surface preparations were thicker than most such preparations because the spiral ligament was retained. The thicker specimens limited the usefulness of phase-contrast and Nomarski optics so the added contrast afforded by the orcein showed cellular detail that would otherwise not have been visible. In addition to stereocilia, other structures made apparent by orcein were cell nuclei and cell boundaries. All rows of O H C stereocilia were stained with orcein, as were the shorter rows on IHCs. However, the tallest row of stereocilia on IHCs failed to stain, possibly indicating a chemical difference between this row and all others.
derly (Fig. 1A). Misaligned and missing short IHC stereocilia such as those shown in Fig. 2A were exceptional. A more common finding was disarrayed and missing stereocilia in the tallest row on IHCs. Because these did not stain and showed little contrast, they were difficult to photograph and are not illustrated. There was not a good correlation of locations of disrupted stereocilia and frequencies at which hearing losses were found. Regions where stereocilia were disrupted sometimes corresponded to places where threshold shifts of evoked potentials indicated the presence of some abnormality. In other cases there were disrupted stereocilia at cochlea places where thresholds were normal. There was no evidence of artifactual disruption of stereocilia that, for example, may have been present at or near sites where the cochleas were bisected.
3.1. Hair cells
3.2. Pillar cells
Data concerning the number of missing and obviously abnormal hair cells have been reported elsewhere (Tarnowski et al., 1991). Few IHCs were missing. Even in cases that showed the most advanced stages of degeneration, the IHCs appeared normal except for stereocilia disarray (see below). Fig. 1A shows a rare instance in which there is a missing IHC. Also shown is the common finding of considerably more missing OHCs than IHCs. In contrast to IHCs, OHCs occasionally assumed abnormal flask (Fig. 1B) or rounded (Fig. 1C,D) shapes. These changes in shape were accompanied by detachment of the cells at either their apical or basal pole. In some cases, spherical-shaped OHCs which remained attached at the cuticular plate still retained their stereocilia (Fig. 1C). Another, less frequently observed alteration of OHCs, was their detachment from the cuticular plate and assumption of a spherical shape while apparently remaining attached to Deiters' cells (Fig. 1D). Under the latter condition, intact stereocilia were never observed. Spherical and flask-shaped OHCs were observed exclusively in the apical half of the cochlea, with most being present in the extreme apex (see Tarnowski et al., 1991). Differences in the state of orderliness of the stereocilia were seen among cochleas, as well as between regions within a given cochlea. In general, the darkly stained O H C and short IHC stereocilia were quite or-
Pillar cells constituted the other major cell type within the organ of Corti that showed obvious pathological changes. Although seldom missing (Fig. 2B shows an exception) the pillar cell nuclei were often misaligned, but with no apparent apical migration of the nuclei (Fig. 2C). The two cochleas with the greatest hearing loss, as assessed by evoked potential audiograms, showed the greatest losses of hair cells (see Tarnowski et al., 1991) and also the greatest number of abnormal outer pillar cells. Fig. 2D illustrates an extreme example where the nuclei of the outer pillar cells have migrated to the cells' apices (see also Fig. 4B). Even in these cases, pillar cells were seldom missing. In one instance a few transformed Deiters cells also were displaced into the tunnel of Corti (Fig. 2C).
3. Results
3.3. Reissner's membrane
Examination of surface preparations, radial sections and flat mounts showed that the cells comprising Reissner's membrane in young animals were spaced relatively uniformly (Fig. 3A) as compared to older animals where clusters of epithelial ceils separated by areas of lower cell density were routinely observed (Fig. 3B,C). Counts of cell nuclei were made in surface preparations from young and old animals to assess changes in cellularity. The density of all cells within Reissner's
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Fig. 3. A: Flat-mounted Reissner's membrane from a 6-week-old gerbil. The arrowhead points to a cell whose cytoplasm has been made visible by the azure stain. This cell also contains a few pigment granules. Only nuclei of other cells are stained. Nuclei of epithelial cells lining the scala media appear fairly uniform in size and most are euchromatic. Bar--70 [am. B: Reissner's membrane in a 36-month-old gerbil, embedded in epoxy. The orcein-stained nuclei are clearly visible. The arrows indicate clusters of epithelial cells with small nuclei. Bar= 60 [am. C: Flatmounted Reissner's membrane from a 24-month-old gerbil. Clustering of smaller, more heterochromatic nuclei is present in the upper left portion of the field. The small, faint dots are lipofuscin granules. Calibration same as (A). D: Surface view of OHCs (top) and Claudius cells (below). The arrowhead points to a shrunken Claudius cell with dark cytoplasm. Bar = 25 [am.
m e m b r a n e was measured in the basal, middle, and apical turns of eight cochleas taken from animals aged 3 months or less. These counts were compared to measures made in seven cochleas taken from animals that were approximately 3 years old and selected because the preparations allowed a favorable view of Reissner's m e m b r a n e in all three turns. Counts in the old animals were made from areas that included both clusters of cells and adjacent, cell sparse areas (see Fig. 3B) in an attempt to obtain a measure of the average cell density. The results showed that cell density was significantly greater in the apical as compared with the basal turn (t test, P < 0.05), but no significant differences in cell density were found between old and young animals when respective turns were compared. Age-related changes in Reissner's m e m b r a n e also were evaluated in Azure A-stained flat mounts from young and from 2-year-old gerbils. At least three different cell populations were identified, based on the size and staining properties of their nuclei and cytoplasm (Fig. 3). These consisted of clusters of epithelial cells with smaller more dense heterochromatic nuclei interspersed with less densely packed cells with larger, more euchromatic nuclei. A third cell type characteristically showed cytoplasmic azurophilia and contained varying amounts of pigment granules (Fig. 3A). The pigmented cells were a small minority of the cells present. The tissue depicted in Fig. 3C was from an animal only 2 years old but the clustering of epithelial cells, which was pronounced in 3-year-old gerbils (Fig. 3B) already was evident. Lipofuscin granules were abundant within cells of Reissner's m e m b r a n e in the stained flat-mount preparations of 2-year-old animals (Fig. 3C) but not those of young animals (Fig. 3A). 3.4. Miscellaneous Other forms of histopathology were occasionally observed. For example, Fig. 3D illustrates what appears to be a degenerating Claudius cell. This cell was within a field where other cells appeared normal. Another infrequent observation was a marked decrease in radial fiber density in the apical half-turn of one 36-month-old gerbil (Fig. 4A). In contrast, Fig. 4C illustrates the high density of radial fibers in the same region from the ear of another 36-month-old gerbil that showed only a small hearing loss and a minimal loss of hair cells at
any site along the cochlear partition. The C A P threshold in response to 500 Hz tone bursts was greater than 50 dB higher for the ear shown in Fig. 4A as compared with that shown in Fig. 4C. In the whole mount, I H C s appeared to be present at the locations corresponding to the loss of radial fibers shown in Fig. 4A, but overlying tissue obscured the surface view somewhat. Radial sections were therefore cut through this region and these showed the presence of I H C s (Fig. 4B). N o radial fibers were seen in the osseous spiral lamina of the radial sections. In the region of missing radial fibers, O H C s were missing and the positions of the outer pillar cell nuclei were also abnormal (Fig. 4B). This report includes the cochlea of one old gerbil that was excluded in the previous paper on H C counts (Tarnowski et al., 1991). The animal was 36 months old at the termination of the experiment and the cochlea was dramatically different from that of all other animals. Physiological measurements showed no endocochlear potential and no evoked response to high levels of sound stimulation. When the cochlea was bisected, it was found to be filled with fibrous connective tissue. The tissue was osmicated and embedded in plastic without further dissection. Semithin sections (Fig. 4D) showed that the organ of Corti, the entire lateral wall, the auditory nerve and the ganglion cells had been replaced by highly vascularized connective tissue and focal accumulations of inflammatory cells. Blood vessels in the bone were atrophied and replaced by flocculent material. In addition, there was new bone growth in the basal and middle turns (Fig. 4D). Neither the external nor middle ear of this animal showed unusual features.
4. Discussion
This report is part of a comprehensive study of hearing losses and histopathological changes in cochleas from animals near the end of their life expectancy. More animals were included than in previous aging studies and they were kept in an especially quiet environment and had no exposure to any known ototoxins throughout their lives. The most remarkable findings were: (1) the great variability in the type and severity of histopathological changes seen a m o n g animals of the same age, raised under carefully controlled environmental conditions; and (2) the lack of a close correspondence between the loci of cellular disruptions and the
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Fig. 4. A: Radial fibers in the osseous spiral lamina in the apical turn of a 36-month-old gerbil that showed marked threshold elevations at all frequencies. The number of fibers is reduced considerably as compared with an age-matched control shown in (C). Bar= 12 gm. B: Semithin radial section through the same tissue that was shown in (A). The open arrow points to the head of an IHC. The arrowhead points to a nucleus of an outer pillar cell that is apparently migrating up the pillar. *, Osseous spiral lamina which is devoid of myelinated fibers in this section. Bar = 44 gm. C: Radial fibers in the apical turn of a 36-month-old gerbil that showed little threshold elevation at any frequency. The density of the osmicated fibers is clearly greater than those shown in (A). Calibration same as (A). D: Cross-section through the middle turn of the 36-month-old gerbil that showed no physiological signs of hearing. The arrowhead points to what was originally a blood vessel in bone. Such vessels were filled with a dense precipitate. The blood vessels of the fibrous connective tissue and cellular infiltrate that has replaced the normal cochlear tissue appear as clear areas and contain no precipitate. Blood cells were removed from these vessels by the cardiac perfusion. The asterisk lies over new bone growing in the region that was formerly scala vestibuli. The blood vessel indicated by the arrowhead is 116 gm long,
frequencies at which h e a r i n g losses were present. T h e range o f h i s t o p a t h o l o g i c changes is in a g r e e m e n t with studies showing large differences in h e a r i n g losses a m o n g similarly aged a n i m a l s from o u r o u t b r e d gerbil c o l o n y (Hellstrom a n d Schmiedt, 1990; Mills et al., 1990) a n d p o i n t to genetic variability as a d e t e r m i n a n t in presbycusis. The lack o f c o r r e s p o n d e n c e of histop a t h o l o g y a n d h e a r i n g loss is in agreement with previous o b s e r v a t i o n s that d i s r u p t i o n s of the o r g a n of Corti a c c o u n t for only some o f the h e a r i n g losses that occur due to aging. T h e p a t t e r n of cell loss a n d d e g e n e r a t i o n within the
o r g a n of Corti indicated that O H C s are most vulnerable to age-related degenerative changes. A previous analysis of cytocochleograms from the cochleas examined here revealed a good correlation between overall h e a r i n g loss a n d total H C loss, b u t there was a p o o r c o r r e l a t i o n between H C losses a n d C A P threshold shifts at specific locations a l o n g the frequency-place m a p ( T a r n o w s k i et al., 1991). Thus, a l t h o u g h the extent of H C loss provides a gross assessment of the general state of the cochlea, other forms of cellular p a t h o l o g y must be sought to explain the large variability in hearing loss at specific frequencies. T h e present o b s e r v a t i o n s ex-
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tended previous findings to include assessment of stereocilia disarray and of the condition of non-sensory cells. These additional observations revealed that organ of Corti pathology, as seen with the light microscope, cannot account for the hearing losses that were found in the previously reported physiological tests. Pathological changes in supporting cells were present only in regions where there were relatively large numbers of missing OHCs. It is therefore apparent that degenerative changes in supporting cells cannot account for hearing losses that were not associated with O H C loss. Nevertheless, the nature of the degeneration of the supporting cells differed somewhat from previous reports and merits discussion. The most prevalent abnormality of supporting cells was disruption of outer pillar cells. This was usually manifest as a misalignment or displacement of nuclei and was found in cochleas that showed the largest O H C losses. In contrast to reports in other mammalian species (Covell and Rogers, 1957; Bredberg, 1968; Schuknecht, 1993; Nadol, 1980), in the present study supporting cells were seldom missing. The lack of missing supporting cells may be because the gerbils were raised in a quiet environment, whereas in the animal study cited above there were apparently no special precautions taken against acoustic trauma and in the studies of human material, adequate documentation of acoustic and medical histories was not available. Bohne et al. (1990) reported a decrease in the number of pillar cells with advancing age in chinchillas but migration of pillar cell nuclei was uncommon. Our material consisted of ears from 17 gerbils near the end of their life expectancy, whereas the oldest chinchillas studied by Bohne et al. (1990) were from animals in the last 40% of the average lifespan of their colony, with very few cases at the extreme of their expected lifespan. Perhaps changes in pillar cells, such as we observed in the gerbil would be more evident in a population of chinchillas that included more cases of advanced age. Changes in the appearance of the heads of pillar cells were common in aging chinchillas (Bohne et al., 1990), but no such alterations were seen in the present material. The use of different tissue processing methods in the two studies may account for this apparent discrepancy. A clear but not necessarily causal relationship has been established between loss of sensitivity of eighth nerve fibers and disruption of stereocilia induced by acoustic trauma (see Liberman, 1987). To our knowledge, the present study is the first analysis of stereocilia disarray in aged animals raised in a quiet environment. In general, O H C stereocilia appeared orderly, with little evidence of disarray except on obviously abnormal OHCs. Unfortunately, our preparation was not optimal for assessing IHC stereocilia because the tallest row failed to stain with orcein. Nevertheless, IHC stereocilia disarray occasionally was observed in cochlear locations
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where CAP thresholds were normal. Conversely, IHC stereocilia usually appeared normal even at locations with markedly elevated CAP thresholds. Although no firm conclusions can be drawn concerning the relationship of stereocilia disarray and threshold shift, the present findings, along with previous results (Tarnowski et al., 1991), strongly suggest that organ of Corti dysfunction cannot account for a sizable portion of hearing losses in quiet-aged gerbils. The most consistent age-related structural change in the gerbil cochlea was the development of densely packed clusters of epithelial cells in Reissner's membrane. This phenomenon was not a definitive sign of extreme age, however, because it was apparent even in 2-year-old cochleas. Mesenchymal cells on the scala vestibuli side of Reissner's membrane were difficult to positively identify in surface preparations and flat mounts, so the identity of the clustered cells was confirmed by ultrastructural examination of radial sections taken from the same specimens (unpublished observations). The clustering of epithelial cells in the gerbil may correspond to the formation of 'whorls' of epithelial cells, first described by Retzius (1884), and shown by Johnsson (1971) to be present in humans as young as 16 years. Watanuki et al. (1981) and Quijano et al. (1990) have also noted increased irregularity in the arrangement of the epithelial surface of Reissner's membrane with increasing age in human ears. Studies in both rats (Feldman, 1990) and humans (Yoon et al., 1991) have reported an age-related decrease in the cellularity of Reissner's membrane. Counts of all cell types in Reissner's membrane in humans showed a decrease in cell density at a very young age, followed by a long period of stable cell numbers, and a further decrease beyond the age of 75 years (Quijano et al., 1990). Our results in gerbil differ in that we did not find a decrease in total cell number with age. Perhaps the 'young' gerbils in our control group were beyond the developmental stage of the 'young' cases of Quijano et al. (1990), or methodological differences may be responsible for the apparent disparity in the results. It also is possible that this disparity reflects differences between species in the population dynamics of mesenchymal cells lining the scala vestibular side of Reissner's membrane. In the gerbil the mesenchymal cells form a densely packed layer during postnatal development but rapidly decline in number and become sparse by 1 month of age (unpublished observations). Although the arrangement and perhaps also the composition of cells in Reissner's membrane changes with age, the changes evidenced by clustering of epithelial cells were present in 2- and 3-year-old gerbils with normal or near-normal hearing, as well as in adolescent humans (Johnsson, 1971). Moreover, the consistent structural changes seen in Reissner's membrane in all 3-year-old gerbils contrasts with the wide variability in
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the amount of O H C loss and in the degree of hearing loss among these same ears (Tarnowski et al., 1991). It therefore seems unlikely that the cellular changes in Reissner's membrane are closely related to hearing losses. A similar conclusion was reached by Quijano et al. (1990). It seems clear that a variety of histopathological changes are associated with age-related hearing loss even under carefully controlled environmental conditions. Schuknecht (1993) has classified presbycusis into four different categories and we have observed examples of three of these types in aging gerbils, although no one category appeared in a pure form. Sensory presbycusis was evidenced by loss of hair cells (Tarnowski et al., 1991). In contrast with findings in humans where H C loss is characteristically in the basal third of the cochlea (Schuknecht, 1993), the predominant loss in gerbils was in the apex with a less pronounced loss in the base. Consistent with our gerbil data, aged chinchillas (Bhattacharyya and Dayal, 1985; Bohne et al., 1990), guinea pigs (Dayal and Barek, 1975; Coleman, 1976), rats (Keithley and Feldman, 1982) and squirrel monkeys (Dayal and Bhattacharyya, 1986) all also show the earliest and largest losses of hair cells in the apex. The exception of basal HC losses in humans may be due to species differences. However, environmental variables, such as acoustic history or entry of toxins via the round window due to middle ear infections, must be considered as possible explanations of the basal degeneration seen in humans. We observed one example of primary neural presbycusis which involved loss of nerve fibers without a corresponding loss of associated hair cells. Bohne et al. (1990) reported that 5% of their chinchillas showed radial fiber loss without accompanying H C loss. The latter finding indicates that neural degeneration can occur at or before midlife and raises the question of whether this disorder should actually be considered a sign of presbycusis. We have no way of determining the age at which the neural loss occurred in one region of one gerbil ear. However, our results are in keeping with the estimate of 5% incidence and the apical location of the loss reported in chinchillas. Loss of spiral ganglion cells with age has been reported for several species including the gerbil (Covell and Rogers, 1957; Bredberg, 1968; Johnsson and Hawkins, 1972; Schuknecht and Gacek, 1993; Nadol, 1979, 1980; Keithley and Feldman, 1979; Keithley et al., 1989, 1992). Our preliminary findings based on examination of radial sections confirm this observation. However, with the exception of the extreme case shown in Fig. 4A, ganglion cell losses were not reflected by appreciable changes in radial fiber density. Metabolic presbycusis (strial degeneration) has been observed to varying degrees in the gerbils under study (Schmiedt and Schulte, 1992) and is associated with loss
of immunostaining for Na,K-ATPase and a decline in the endocochlear potential (Schulte and Schmiedt, 1992). Unfortunately the surface preparations, as viewed in the present work, were not suitable for evaluation of structural changes in the lateral wall. Light and electron microscopic morphologic evaluation of radial sections cut from the present material will provide further documentation of pathologic changes in the aging lateral wall. Degeneration of the stria vascularis occurs commonly in aged ears of many species, including humans (Johnsson and Hawkins, 1972; Schuknecht, 1993; Nadol, 1979, 1980; Keithley et al., 1989, 1992; Schuknecht and Gacek, 1993) and undoubtedly plays an important role in the pathophysiology of presbycusis. A fourth category of presbycusis has been termed 'cochlear conductive' loss (Schuknecht, 1993). No histopathological changes have been identified that characterize this hypothetical form of presbycusis, but are presumed to be present based on a characteristic audiometric finding. No animals in our study showed such an audiogram and we observed no evidence of histopathological changes that suggested the presence of possible changes in cochlear sound conductive abilities. Gerbils in our colony did, however, often develop a conductive hearing loss due to impaction of the external auditory canals (Mills et al., 1990; see also Henry et al., 1983). Such losses are not described here because only animals with clear external canals were selected for study. The replacement of all normal cochlear structures by fibrous connective tissue, new bone, and inflammatory cells in the case illustrated in Fig. 4D is not considered to be a normal aging phenomenon. The case presented here appears to be the end stage of suppurative labyrinthitis that follows bacterial infections (Schuknecht, 1993). It serves as a reminder of the importance of obtaining histological verification of hearing pathology, regardless of the presumed cause. The variety of changes in the cochlea of aging gerbils observed in this and previous studies confirms the contention that presbycusis is a multifaceted and complex phenomenon. The type and severity of histopathologic changes varied widely, even in animals with a restricted genetic background maintained under carefully controlled conditions throughout their lifespan. The finding that cochleas which showed the greatest hearing losses also showed morphological changes in a variety of cell types suggests that animals prone to hearing loss were not selectively losing sensory cells, but rather were subject to a more general deterioration of the ear and probably other tissues as well.
Acknowledgments The authors wish to thank Mrs. Leslie Harrelson for
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e d i t o r i a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d b y res e a r c h G r a n t P01 D C 0 0 4 2 2 f r o m t h e N a t i o n a l I n s t i t u t e on Deafness and Other Communication Disorders, National Institutes of Health.
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