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6J mouse
Hearing Research 141 (2000) 12^18 www.elsevier.com/locate/heares
Pattern of degeneration of the spiral ganglion cell and its processes in the C57BL/6J mouse Judith A. White b
a;
*, Barbara J. Burgess b , Robert D. Hall b , Joseph B. Nadol
b
a Department of Otolaryngology ^ Head and Neck Surgery, Lahey Clinic Medical Center, 41 Mall Rd., Burlington, MA 01805, USA Department of Otolaryngology, Massachusetts Eye and Ear In¢rmary, and Department of Otology and Laryngology, Harvard Medical School, Boston, MA, USA
Received 7 January 1999; received in revised form 22 October 1999; accepted 27 October 1999
Abstract Although degeneration of spiral ganglion cells has been described as a histopathologic correlate of hearing loss both in animals and humans, the pattern and sequence of this degeneration remain controversial. Degeneration of hair cells and of spiral ganglion cells and their dendritic processes was evaluated in the C57BL/6J mouse, in which there is a genetically determined progressive sensorineural loss starting in the high frequencies that is similar to the pattern commonly seen in the human. Auditory function was evaluated by brainstem evoked responses, and degeneration of hair cells, ganglion cells and their dendrites was evaluated histologically at 3, 8, 12 and 18 months of age. Progressive loss of auditory sensitivity was correlated with the loss of outer and inner hair cells and spiral ganglion cells and their dendritic processes. In addition, dendritic counts were consistently lower at a distal location in the osseous spiral lamina (i.e. near the organ of Corti) than at a proximal location (i.e. near the spiral ganglion), and the difference between the number of distal dendrites and the number of proximal dendrites tended to be greater with advancing age. These observations suggest an age-related progressive retrograde degeneration of spiral ganglion cells. Thus, in degenerating cochleas, some remaining spiral ganglion cells may have no distal dendritic processes near the organ of Corti. This may have implications for successful stimulation of the cochlear neuron in cochlear implantation. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Neural degeneration; Spiral ganglion; C57BL/6J mouse; Deafness
1. Introduction Although a progressive loss of spiral ganglion cells has been described in a variety of degenerative disorders of the inner ear in animals (Spoendlin, 1971, 1975 ; Keithley and Feldman, 1979) and in the human (Bredberg, 1968; Kerr and Schuknecht, 1968; Lindsay and Hinojosa, 1978; Suzuka and Schuknecht, 1988; Nadol et al., 1989), the sequence of degeneration of the components of the ¢rst order cochlear neuron remains controversial. Thus, as a result of various lesions to the organ of Corti in the cat, Spoendlin (1971, 1975, 1984) found a rapid degeneration of the spiral ganglion cells and their processes. A similar pattern of degener-
* Corresponding author. Tel.: +1-781-744-8451; Fax: +1-781-744-5248; E-mail: [email protected]
ation was seen in humans (Spoendlin and Schrott, 1989, 1990), with no di¡erence observed between the number of remaining dendrites in the osseous spiral lamina and the number of spiral ganglion cells or axons. This suggested that retrograde degeneration initiated at the periphery progresses at a rapid rate. In contrast, in human studies, the number of remaining spiral ganglion cells far exceeded the number of residual dendritic ¢bers (Suzuka and Schuknecht, 1988 ; Felix et al., 1990; Nadol et al., 1990; Felder et al., 1997), suggesting that spiral ganglion cells may survive losses of their dendritic processes for periods of time measured in years. In addition, Ylikoski et al. (1978, 1981) suggested that the axon of the human spiral ganglion cell may actually persist even after destruction of both the peripheral dendritic process and the cell body, suggesting an extremely slow rate of retrograde degeneration of this cell.
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A pattern of progressive sensorineural loss starting in the high frequencies, which is similar to the degenerative pattern in a number of disorders that a¡ect the human inner ear, develops in the C57BL/6J mouse. This inbred mouse has been used as an animal model for degeneration in the human auditory system (Willott et al., 1995). The genetics of the age-related hearing loss in this mouse has been elucidated in a series of papers by Erway and co-workers (Erway et al., 1993; Willott et al., 1995; Erway et al., 1996; Johnson et al., 1997). The AHL (age-related hearing loss) gene maps to chromosome 10. Hearing loss can begin as early as 30 days for high frequency stimuli (Li and Borg, 1991; Shnerson and Pujol, 1982). It progresses to include low frequency stimuli by six to seven months, severe loss across all frequencies by one year and near total loss of hearing by 18 months of age (e.g., Henry and Chole, 1980 ; Mikaelian, 1979). Degeneration of outer hair cells in the basal turn begins during the ¢rst several months of life, followed by progressive loss of inner hair cells and spiral ganglion cells (Mikaelian, 1979). At an ultrastructural level, evidence of degeneration of spiral ganglion cells includes demyelination of ¢bers and clumping and fusion of spiral ganglion somas (Cohen et al., 1990). This study was performed to test the hypothesis that dendritic processes of spiral ganglion cells degenerate more rapidly near the organ of Corti than near the spiral ganglion cell. Such a pattern would suggest a progressive retrograde degeneration. 2. Materials and methods Thirteen female C57BL/6J mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA) at age 3 months and maintained in a colony until auditory testing and killing at ages 3, 8, 12 and 18 months. At these ages, the animals were deeply anesthetized with sodium pentobarbital using an initial dose of 60 mg/kg injected intraperitoneally and additional doses of approximately half that strength as needed. They were restrained in a head clamp, and auditory thresholds were measured using the brainstem evoked response technique. Stimuli were generated by Modular Instruments auditory signal generators and ampli¢ed by a Crown D75 ampli¢er. They were presented through Radio Shack tweeters, Cat. No. 40-1377, mounted in earphones with tubes that ¢t snugly in the ear canal. The earphones were specially designed for small rodents by Michael E. Ravicz of the Eaton Peabody Laboratory, MEEI. The acoustic stimuli were tone bursts, shaped by a Blackman window, with frequencies of 2, 4, 8, 15, 33 and 50 kHz, rise and fall times of 0.5 ms and a plateau of 2 ms. For each frequency the sound
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pressure level was measured for a reference voltage speaker input using a Larson Davis model 2530 1/4 in. microphone, a model 910B microphone preampli¢er and a specially designed coupler between microphone and ear tubes. Speaker outputs were periodically checked to ensure repeatability. The auditory brainstem response was recorded di¡erentially between a screw electrode threaded into the skull at the `vertex', the biteboard of the headholder and a ground electrode at the anterior edge of the scalp incision. The signals were ampli¢ed by an Ithaco model 1201 preampli¢er with a gain of 25,000. High pass and low pass ¢lters were set at 30 Hz and 3 kHz respectively. Stimuli were presented at a rate of 50/s. Averages of 1000 responses were recorded for a descending series of stimulus levels until near threshold levels were reached ; then averages of 8000 responses were recorded to make the threshold determinations. Body temperature was maintained near 37³C by a warming pad. Thresholds were measured for both ears in each animal. There were three animals in each group at ages of 3, 8 and 18 months and four animals 12 months of age. On completion of the evoked response testing, right atrial cannulation and intravascular perfusion with heparin and saline solution were performed, followed by fresh ¢ltered Karnovsky's ¢xative. The cochleae were dissected, placed in fresh ¢xative overnight, decalci¢ed in 0.1 M EDTA with 1% glutaraldehyde, osmicated, dehydrated in alcohol, and embedded in Poly/Bed. The basal turn was dissected, and a radially oriented segment was obtained between 0.75 and 2.0 mm from the round window, including the spiral ganglion, dendritic ¢bers and organ of Corti. Hair cell counts were made in surface preparations. Dendritic ¢bers and spiral ganglion cells were counted in tangential serial sections, 2 Wm thick, mounted on glass slides, and stained with toluidine blue. Cross sections of dendritic ¢bers were counted at two locations: near the organ of Corti and near the spiral ganglion. In each specimen, several neural foramina were selected for counting the dendrites based on our ability to follow these foramina through serial tangential sections of the osseous spiral lamina from the proximal site to the distal site. The density of spiral ganglion cells was determined by counting cells with a visible nucleolus in approximately every tenth section through the spiral ganglion in the selected segment of the basal turn in each of the specimens of age 3, 8, 12 and 18 months. The area of the spiral ganglion was determined in each section by computerized planimetry, and a density was calculated. The average densities of spiral ganglion cells for each specimen were averaged again to calculate an average density for each age group as presented in Table 1. One ear from each animal was included in the statistical analysis.
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Table 1 Number of hair cells and density of spiral ganglion cells at four ages in the C57BL/6J mouse Location
3 Months n=3
OHC 1 OHC 2 OHC 3 IHC SGC
100 99 99 100 1 293
(1)a (1) (2) (0) (54)b
8 Months
12 Months
18 Months
n=3
n=4
n=3
0 0 13 (13) 93 (1) 1 048 (72)
1 0 1 92 830
(2) (1) (2) (3) (128)
0 0 0 29 (6) 477 (133)
OHC = outer hair cells; IHC = inner hair cells; SGC = spiral ganglion cells. a Mean percent surviving hair cells and S.D. b Mean density of spiral ganglion cells, number/mm2 , and S.D.
The use of animals in this study was approved by the Animal Care Committee (Institutional Animal Care and Use Committee) of the Massachusetts Eye and Ear In¢rmary, using the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and Title 9 of the Code of Federal Regulations, Chapter 1, Subchapter A-Animal Welfare, part 3, as issued and enforced by the U.S. Department of Agriculture. 3. Results 3.1. Auditory thresholds The 13 C57BL/6J mice showed clear evidence of high frequency sensorineural loss at 8 months of age (Fig. 1). By 12 months, there was signi¢cant loss across all frequencies, especially at high frequencies. At 18 months, there was essentially no measurable auditory response. This progression of hearing loss is consistent with that previously reported for this strain (Willott, 1991). 3.2. Hair cells
with age in spiral ganglion cell density (r = 30.96; n = 13; P 6 0.001), which is consistent with the high correlation with surviving inner hair cells already noted. A direct comparison of the number of surviving dendrites across the several age groups was not possible because of the way in which the dendrites were selected for counting. They were counted in the neural foramina only when those foramina could be followed with con¢dence in serial sections from the proximal site to the distal site. In each subject, the largest area meeting that criterion was used to obtain the largest possible sample of dendrites. Absolute counts of the dendrites were therefore not appropriately compared across animals. What was clear, however, in all but one of the 13 animals there was a higher count at the proximal site than at the distal site (Fig. 3). The di¡erences were very small in a few instances, especially in the 3-month-old group, but remarkably consistent. Because there was no strong indication of a trend across ages, the data were combined for a single Student's t-test of the di¡erence between proximal and distal counts in the related samples (t = 3.51; df = 12; P 6 0.01). To evaluate the relative loss of ganglion cell dendrites at the proximal and
Surface preparations of the organ of Corti in the basal turn revealed that degeneration occurred ¢rst in the outer hair cells (Table 1). By 8 months of age, only 4% of the outer hair cells remained. A substantial loss of inner hair cells was not seen until 18 months of age, and by that time, only 29% of the inner hair cells were present. The extensive degeneration of the organ of Corti at 18 months of age is illustrated in Fig. 2. The number of surviving inner hair cells was highly correlated with the density of surviving spiral ganglion cells (r = 0.85; n = 10; P 6 0.02) and with the number of ganglion cell dendrites (r = 0.90 ; n = 10; P 6 0.001). 3.3. Density of spiral ganglion cells and counts of dendritic processes As shown in Table 1, there was a marked decline
Fig. 1. Median auditory brainstem response thresholds for four groups of mice of di¡erent ages. Both ears of each animal were included in the analysis. Symbols with arrows indicate minimum estimates due to output limitations of the system and, in a few cases, inadvertent failure to test at the highest intensities.
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Fig. 2. Degeneration of hair cells (A,B), distal dendrites (C,D), proximal dendrites (E,F) and spiral ganglion cells (G,H) with age. At 3 months of age, three rows of outer hair cells and a single row of inner hair cells are easily seen in the surface preparation, whereas at 18 months of age (B), all outer hair cells in this segment have degenerated and only a few inner hair cells remain (arrow). The density of remaining spiral ganglion cells is clearly reduced at 18 months compared with 3 months. The density of dendrite cross sections, both distally and proximally, is far greater at 3 months than at 18 months. Magni¢cation for A and B, 600U; C^H, 400U.
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distal locations as a function of age, we used a ratio of the distal/proximal counts. There is some indication in Fig. 4 that the ratio declines with age, i.e. the distal loss is proportionally greater than the proximal loss. The e¡ect is marginal from a statistical standpoint and would have to be veri¢ed in a larger sample before signi¢cance can be attributed to it. 4. Discussion The pattern of degeneration of spiral ganglion cells in the inner ear has been controversial and may be speci¢c to the cell type, species, and even cause of hearing loss. Thus, in the cat, Spoendlin (1971, 1975, 1984) reported rapid degeneration of all segments of the type I spiral ganglion cell following insult either to dendritic processes or to the organ of Corti. In contrast, type II ganglion cells may survive in the cat following transection of the cochlear nerve (Spoendlin, 1979) or following noise trauma (Spoendlin, 1975) or ototoxic lesions of the organ of Corti (Spoendlin, 1975; Bichler et al., 1983). In the rat, age-related degeneration of the spiral ganglion cell seemed to a¡ect both types I and II spiral ganglion cells equally (Keithley and Feldman, 1979). Similarly, in the human, there was no survival advantage for type II ganglion cells (Zimmermann et al., 1995). In a quantitative study of 9 human temporal bones with typical high frequency sensorineural hearing loss attributable to `presbycusis', Felder and SchrottFischer (1995) described an age-related loss of nerve ¢bers along the entire length of the cochlea that apparently was not strictly related to loss of either inner or outer hair cells. In human specimens, evidence for simultaneous or near simultaneous degeneration of spiral ganglion bodies and dendritic processes has been re-
Fig. 3. Dendritic counts taken in cross section in the osseous spiral lamina at a distal (near the organ of Corti) and at a proximal (near the spiral ganglion) location. In every subject but one (at age 3 months), the number of distal dendrites was less than the number at proximal sites.
Fig. 4. The mean ratio of the number of distal to the number of proximal dendrites tended to be lower at older, compared with younger, ages.
ported (Spoendlin and Scrott, 1989, 1990). However, other investigators have reported that the number of remaining spiral ganglion cells far exceeded the number of dendritic processes in the osseous spiral lamina in the human (Lindsay and Hinojosa, 1978; Kerr and Schuknecht, 1968; Suzuka and Schuknecht, 1988; Nadol et al., 1989 ; Felix et al., 1990; Nadol, 1997). In fact, selective loss of the dendritic arborization and synaptic contacts of spiral ganglion cells has been shown in presbycusis, Meniere's disease, and Usher's syndrome (Nadol, 1988). In animals (Kellerhals, 1967; Leake and Hradek, 1988) and in humans (Johnsson and Hawkins, 1972 ; Otte et al., 1978; Ylikoski et al., 1981 ; Suzuka and Schuknecht, 1988 ; Felix et al., 1990 ; Nadol, 1990; Felder et al., 1997), degeneration begins in the most peripheral processes of the spiral ganglion cell and progresses toward the cell body. This has been called the `dying back' process (Cavanagh, 1964). In the C57BL/6J mouse, hearing loss begins in the high frequencies at approximately 3^6 months of age and progresses to a total hearing loss at all frequencies by 15 months (Henry, 1983; Willott, 1991). The concomitant degeneration of the organ of Corti begins in the outer hair cells and later involves inner hair cells and spiral ganglion cells (Mikaelian, 1979). Therefore, the decrement in auditory function and its histopathologic correlates in the C57BL/6J mouse are similar to the pattern of degeneration seen in the human in a variety of disease processes. The present study suggests a retrograde pattern of degeneration of cochlear neurons in which degeneration begins in distal dendritic processes and progresses proximally toward the spiral ganglion cell, providing evidence of the dying back process in another species. In addition, the di¡erence between the number of distal and proximal dendrites may also increase with age, but that relationship is not yet certain. Success of cochlear implantation in the profoundly
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deaf probably depends, at least in part, on the number of residual spiral ganglion cells (Clopton et al., 1980). However, the exact site of excitation of surviving spiral ganglion cells by implanted electrodes is not known. Thus, it is not known whether successful stimulation requires the presence of peripheral dendritic processes or whether direct stimulation of the spiral ganglion cell body may occur in the absence of the dendrite. The demonstration of retrograde degeneration of spiral ganglion cells in the C57BL/6J mouse con¢rms ¢ndings in other animals and in humans and suggests that in the degenerating inner ear, all remaining spiral ganglion cells may not have dendritic ¢bers that extend peripherally to the level of the organ of Corti. In addition, the ¢nding that degeneration appears to occur in a retrograde fashion suggests that even among remaining degenerating dendrites, electrical properties may be more severely a¡ected distally than centrally. Acknowledgements Supported in part by NIDCD Grant #P01-DC00361.
References Bichler, E., Spoendlin, H., Raucheager, H., 1983. Degeneration of cochlear neurons after amikacin intoxication in the rat. Arch. Otorhinolaryngol. 237, 201^208. Bredberg, G., 1968. Cellular pattern and nerve supply of the human organ of Corti. Acta Otolaryngol. (Stockh.) 236 (Suppl.), 6^135. Cavanagh, J.B., 1964. The signi¢cance of the `dying back' process in experimental and human neurological disease. Int. Rev. Exp. Pathol. 3, 219^267. Clopton, B.M., Spelman, F.A., Miller, J.M., 1980. Estimates of essential neural elements for stimulation through a cochlear prosthesis. Ann. Otol. Rhinol. Laryngol. 89 (Suppl.), 5^7. Cohen, G.M., Park, J.C., Grasso, J.S., 1990. Comparison of demyelination and neural degeneration in spiral and Scarpa's ganglia of C57Bl/6 mice. J. Electron Microsc. Tech. 15, 165^172. Erway, L.C., Willott, J.F., Archer, J.R., Harrison, D.E., 1993. Genetics of age-related hearing loss in mice: I. Inbred and F1 hybrid strains. Hear. Res. 65, 125^132. Erway, L.C., Shiau, Y.W., Davis, R.R., Krieg, E.F., 1996. Genetics of age-related hearing loss in mice: III. Susceptibility of inbred and F1 hybrid strains to noise-induced hearing loss. Hear. Res. 93, 181^187. Felder, E., Schrott-Fischer, A., 1995. Quantitative evaluation of myelinated nerve ¢bers and hair cells in cochleae of humans with agerelated high-tone hearing loss. Hear. Res. 91, 19^32. Felder, E., Kanovier, G., Scholtz, A., Rask-Andersen, H., SchrottFischer, A., 1997. Quantitative evaluation of cochlear neurons and computer-aided three dimensional reconstruction of spiral ganglion cells in humans with a peripheral loss of nerve ¢bers. Hear. Res. 105, 183^190. Felix, H., Johnsson, L.G., Gleeson, M., Pollak, A., 1990. Quantitative analysis of cochlear sensory cells and neuronal elements in man. Acta Otolaryngol. (Stockh.) 470 (Suppl.), 71^79.
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Henry, K.R., Chole, R.A., 1980. Genotypic di¡erences in, behavioral, physiological and anatomical expressions of age-related hearing loss in the laboratory mouse. Audiology 19, 369^383. Henry, K., 1983. Aging and audition. In: Willott, J.F. (Ed.), In: The Auditory Psychobiology of the Mouse. Charles C. Thomas, Spring¢eld, IL, pp. 470^493. Johnson, K.R., Erway, L.C., Cook, S.A., Willott, J.F., Zheng, Q.Y., 1997. A major gene a¡ecting age-related hearing loss in C57B1/6J mice. Hear. Res. 114, 83^92. Johnsson, L.G., 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., Feldman, M.L., 1979. Spiral ganglion cell counts in an age-graded series of rat cochleas. J. Comp. Neurol. 188, 429^ 442. Kellerhals, B., 1967. Die Morphologie des ganglion spirale Cochleae. Acta Otolaryngol. (Stockh.) 226 (Suppl.), 1^78. Kerr, A., Schuknecht, H.F., 1968. The spiral ganglion in profound deafness. Acta Otolaryngol. (Stockh.) 65, 586^598. Leake, P.A., Hradek, G.T., 1988. Cochlear pathology of long term neomycin induced deafness in cats. Hear. Res. 33, 11^33. Li, H.-S., Borg, E., 1991. Age-related loss of auditory sensitivity in two mouse genotypes. Acta Otolaryngol. (Stockh) 111, 827^ 834. Lindsay, J.R., Hinojosa, R., 1978. Ear anomalies associated with renal dysplasia and immunode¢ciency disease. A histopathological study. Ann. Otol. Rhinol. Laryngol. 87, 10^17. Mikaelian, D.O., 1979. Development and degeneration of hearing in the C57/bl6 mouse: relation of electrophysiologic responses from the round window and cochlear nucleus to cochlear anatomy and behavioral responses. Laryngoscope 89, 1^15. Nadol, J.B., Jr., 1988. Innervation densities of inner and outer hair cells of the human organ of Corti. Evidence for auditory neural degeneration in a case of Usher's syndrome. ORL J. Otorhinolaryngol. Relat. Spec. 50, 363^370. Nadol, J.B., Jr., 1990. Degeneration of cochlear neurons as seen in the spiral ganglion of man. Hear. Res. 49, 141^154. Nadol, J.B., Jr., 1997. Patterns of neural degeneration in the human cochlea and auditory nerve: implications for cochlear implantation. Otolaryngol. Head Neck Surg. 117, 220^228. Nadol, J.B., Jr., Eavey, R.D., Liberfarb, R.M., Merchant, S.N., Williams, R., Climenhager, D., Albert, D.M., 1990. Histopathology of the ears, eye, and brain in Norrie's disease. Am. J. Otolaryngol. 11, 112^124. Nadol, J.B., Jr., Young, Y.S., Glynn, R.J., 1989. Survival of spiral ganglion cells in profound sensorineural hearing loss: implications for cochlear implantation. Ann. Otol. Rhinol. Laryngol. 98, 411^ 416. Otte, J., Schuknecht, H.F., Kerr, A.G., 1978. Ganglion cell population in normal and pathological human cochleae: implications for cochlear implantation. Laryngoscope 88, 1231^1246. Shnerson, A., Pujol, R., 1982. Age-related changes in the C57BL/6J mouse cochlea. I. Physiological Findings. Develop. Brain Res. 2, 65^75. Spoendlin, H., 1971. Degeneration behavior of the cochlear nerve. Ann. Otol. Rhinol. Laryngol. 112 (Suppl.), 76^82. Spoendlin, H., 1975. Retrograde degeneration of the cochlear nerve. Acta Otolaryngol. 79, 266^275. Spoendlin, H., 1979. Sensory neural organization of the cochlea. J. Laryngol. Otol. 93, 853^877. Spoendlin, H., 1984. Factors inducing retrograde degeneration of the cochlear nerve. Ann. Otol. Rhinol. Laryngol. 112 (Suppl.), 76^82. Spoendlin, H., Schrott, A., 1989. Analysis of the human auditory nerve. Hear. Res. 43, 25^38. Spoendlin, H., Schrott, A., 1990. Quantitative evaluation of the hu-
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man cochlear nerve. Acta Otolaryngol. (Stockh.) 470 (Suppl.), 61^ 69. Suzuka, Y., Schuknecht, H.F., 1988. Retrograde cochlear neuronal degeneration in human subjects. Acta Otolaryngol. (Stockh.) 450 (Suppl.), 1^20. Willott, J.F., 1991. Aging and the Auditory System: Anatomy, Physiology and Psychophysics. Singular Publishing, San Diego, CA. Ylikoski, J., Belal, A., Jr., House, W.F., 1981. Morphology of human
cochlear nerve after labyrinthectomy. Acta Otolaryngol. (Stockh.) 91, 161^171. Ylikoski, J., Collan, Y., Palva, T., 1978. Pathologic features of the cochlear nerve in profound deafness. Arch. Otolaryngol. 104, 202^ 207. Zimmermann, C.E., Burgess, B.J., Nadol, J.B., Jr., 1995. Patterns of degeneration in the human cochlear nerve. Hear. Res. 90, 192^201.
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Pattern of degeneration of the spiral ganglion cell and its processes in the C57BL/6J mouse Judith A. White b
a;
*, Barbara J. Burgess b , Robert D. Hall b , Joseph B. Nadol
b
a Department of Otolaryngology ^ Head and Neck Surgery, Lahey Clinic Medical Center, 41 Mall Rd., Burlington, MA 01805, USA Department of Otolaryngology, Massachusetts Eye and Ear In¢rmary, and Department of Otology and Laryngology, Harvard Medical School, Boston, MA, USA
Received 7 January 1999; received in revised form 22 October 1999; accepted 27 October 1999
Abstract Although degeneration of spiral ganglion cells has been described as a histopathologic correlate of hearing loss both in animals and humans, the pattern and sequence of this degeneration remain controversial. Degeneration of hair cells and of spiral ganglion cells and their dendritic processes was evaluated in the C57BL/6J mouse, in which there is a genetically determined progressive sensorineural loss starting in the high frequencies that is similar to the pattern commonly seen in the human. Auditory function was evaluated by brainstem evoked responses, and degeneration of hair cells, ganglion cells and their dendrites was evaluated histologically at 3, 8, 12 and 18 months of age. Progressive loss of auditory sensitivity was correlated with the loss of outer and inner hair cells and spiral ganglion cells and their dendritic processes. In addition, dendritic counts were consistently lower at a distal location in the osseous spiral lamina (i.e. near the organ of Corti) than at a proximal location (i.e. near the spiral ganglion), and the difference between the number of distal dendrites and the number of proximal dendrites tended to be greater with advancing age. These observations suggest an age-related progressive retrograde degeneration of spiral ganglion cells. Thus, in degenerating cochleas, some remaining spiral ganglion cells may have no distal dendritic processes near the organ of Corti. This may have implications for successful stimulation of the cochlear neuron in cochlear implantation. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Neural degeneration; Spiral ganglion; C57BL/6J mouse; Deafness
1. Introduction Although a progressive loss of spiral ganglion cells has been described in a variety of degenerative disorders of the inner ear in animals (Spoendlin, 1971, 1975 ; Keithley and Feldman, 1979) and in the human (Bredberg, 1968; Kerr and Schuknecht, 1968; Lindsay and Hinojosa, 1978; Suzuka and Schuknecht, 1988; Nadol et al., 1989), the sequence of degeneration of the components of the ¢rst order cochlear neuron remains controversial. Thus, as a result of various lesions to the organ of Corti in the cat, Spoendlin (1971, 1975, 1984) found a rapid degeneration of the spiral ganglion cells and their processes. A similar pattern of degener-
* Corresponding author. Tel.: +1-781-744-8451; Fax: +1-781-744-5248; E-mail: [email protected]
ation was seen in humans (Spoendlin and Schrott, 1989, 1990), with no di¡erence observed between the number of remaining dendrites in the osseous spiral lamina and the number of spiral ganglion cells or axons. This suggested that retrograde degeneration initiated at the periphery progresses at a rapid rate. In contrast, in human studies, the number of remaining spiral ganglion cells far exceeded the number of residual dendritic ¢bers (Suzuka and Schuknecht, 1988 ; Felix et al., 1990; Nadol et al., 1990; Felder et al., 1997), suggesting that spiral ganglion cells may survive losses of their dendritic processes for periods of time measured in years. In addition, Ylikoski et al. (1978, 1981) suggested that the axon of the human spiral ganglion cell may actually persist even after destruction of both the peripheral dendritic process and the cell body, suggesting an extremely slow rate of retrograde degeneration of this cell.
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 9 ) 0 0 2 0 4 - X
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A pattern of progressive sensorineural loss starting in the high frequencies, which is similar to the degenerative pattern in a number of disorders that a¡ect the human inner ear, develops in the C57BL/6J mouse. This inbred mouse has been used as an animal model for degeneration in the human auditory system (Willott et al., 1995). The genetics of the age-related hearing loss in this mouse has been elucidated in a series of papers by Erway and co-workers (Erway et al., 1993; Willott et al., 1995; Erway et al., 1996; Johnson et al., 1997). The AHL (age-related hearing loss) gene maps to chromosome 10. Hearing loss can begin as early as 30 days for high frequency stimuli (Li and Borg, 1991; Shnerson and Pujol, 1982). It progresses to include low frequency stimuli by six to seven months, severe loss across all frequencies by one year and near total loss of hearing by 18 months of age (e.g., Henry and Chole, 1980 ; Mikaelian, 1979). Degeneration of outer hair cells in the basal turn begins during the ¢rst several months of life, followed by progressive loss of inner hair cells and spiral ganglion cells (Mikaelian, 1979). At an ultrastructural level, evidence of degeneration of spiral ganglion cells includes demyelination of ¢bers and clumping and fusion of spiral ganglion somas (Cohen et al., 1990). This study was performed to test the hypothesis that dendritic processes of spiral ganglion cells degenerate more rapidly near the organ of Corti than near the spiral ganglion cell. Such a pattern would suggest a progressive retrograde degeneration. 2. Materials and methods Thirteen female C57BL/6J mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA) at age 3 months and maintained in a colony until auditory testing and killing at ages 3, 8, 12 and 18 months. At these ages, the animals were deeply anesthetized with sodium pentobarbital using an initial dose of 60 mg/kg injected intraperitoneally and additional doses of approximately half that strength as needed. They were restrained in a head clamp, and auditory thresholds were measured using the brainstem evoked response technique. Stimuli were generated by Modular Instruments auditory signal generators and ampli¢ed by a Crown D75 ampli¢er. They were presented through Radio Shack tweeters, Cat. No. 40-1377, mounted in earphones with tubes that ¢t snugly in the ear canal. The earphones were specially designed for small rodents by Michael E. Ravicz of the Eaton Peabody Laboratory, MEEI. The acoustic stimuli were tone bursts, shaped by a Blackman window, with frequencies of 2, 4, 8, 15, 33 and 50 kHz, rise and fall times of 0.5 ms and a plateau of 2 ms. For each frequency the sound
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pressure level was measured for a reference voltage speaker input using a Larson Davis model 2530 1/4 in. microphone, a model 910B microphone preampli¢er and a specially designed coupler between microphone and ear tubes. Speaker outputs were periodically checked to ensure repeatability. The auditory brainstem response was recorded di¡erentially between a screw electrode threaded into the skull at the `vertex', the biteboard of the headholder and a ground electrode at the anterior edge of the scalp incision. The signals were ampli¢ed by an Ithaco model 1201 preampli¢er with a gain of 25,000. High pass and low pass ¢lters were set at 30 Hz and 3 kHz respectively. Stimuli were presented at a rate of 50/s. Averages of 1000 responses were recorded for a descending series of stimulus levels until near threshold levels were reached ; then averages of 8000 responses were recorded to make the threshold determinations. Body temperature was maintained near 37³C by a warming pad. Thresholds were measured for both ears in each animal. There were three animals in each group at ages of 3, 8 and 18 months and four animals 12 months of age. On completion of the evoked response testing, right atrial cannulation and intravascular perfusion with heparin and saline solution were performed, followed by fresh ¢ltered Karnovsky's ¢xative. The cochleae were dissected, placed in fresh ¢xative overnight, decalci¢ed in 0.1 M EDTA with 1% glutaraldehyde, osmicated, dehydrated in alcohol, and embedded in Poly/Bed. The basal turn was dissected, and a radially oriented segment was obtained between 0.75 and 2.0 mm from the round window, including the spiral ganglion, dendritic ¢bers and organ of Corti. Hair cell counts were made in surface preparations. Dendritic ¢bers and spiral ganglion cells were counted in tangential serial sections, 2 Wm thick, mounted on glass slides, and stained with toluidine blue. Cross sections of dendritic ¢bers were counted at two locations: near the organ of Corti and near the spiral ganglion. In each specimen, several neural foramina were selected for counting the dendrites based on our ability to follow these foramina through serial tangential sections of the osseous spiral lamina from the proximal site to the distal site. The density of spiral ganglion cells was determined by counting cells with a visible nucleolus in approximately every tenth section through the spiral ganglion in the selected segment of the basal turn in each of the specimens of age 3, 8, 12 and 18 months. The area of the spiral ganglion was determined in each section by computerized planimetry, and a density was calculated. The average densities of spiral ganglion cells for each specimen were averaged again to calculate an average density for each age group as presented in Table 1. One ear from each animal was included in the statistical analysis.
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Table 1 Number of hair cells and density of spiral ganglion cells at four ages in the C57BL/6J mouse Location
3 Months n=3
OHC 1 OHC 2 OHC 3 IHC SGC
100 99 99 100 1 293
(1)a (1) (2) (0) (54)b
8 Months
12 Months
18 Months
n=3
n=4
n=3
0 0 13 (13) 93 (1) 1 048 (72)
1 0 1 92 830
(2) (1) (2) (3) (128)
0 0 0 29 (6) 477 (133)
OHC = outer hair cells; IHC = inner hair cells; SGC = spiral ganglion cells. a Mean percent surviving hair cells and S.D. b Mean density of spiral ganglion cells, number/mm2 , and S.D.
The use of animals in this study was approved by the Animal Care Committee (Institutional Animal Care and Use Committee) of the Massachusetts Eye and Ear In¢rmary, using the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and Title 9 of the Code of Federal Regulations, Chapter 1, Subchapter A-Animal Welfare, part 3, as issued and enforced by the U.S. Department of Agriculture. 3. Results 3.1. Auditory thresholds The 13 C57BL/6J mice showed clear evidence of high frequency sensorineural loss at 8 months of age (Fig. 1). By 12 months, there was signi¢cant loss across all frequencies, especially at high frequencies. At 18 months, there was essentially no measurable auditory response. This progression of hearing loss is consistent with that previously reported for this strain (Willott, 1991). 3.2. Hair cells
with age in spiral ganglion cell density (r = 30.96; n = 13; P 6 0.001), which is consistent with the high correlation with surviving inner hair cells already noted. A direct comparison of the number of surviving dendrites across the several age groups was not possible because of the way in which the dendrites were selected for counting. They were counted in the neural foramina only when those foramina could be followed with con¢dence in serial sections from the proximal site to the distal site. In each subject, the largest area meeting that criterion was used to obtain the largest possible sample of dendrites. Absolute counts of the dendrites were therefore not appropriately compared across animals. What was clear, however, in all but one of the 13 animals there was a higher count at the proximal site than at the distal site (Fig. 3). The di¡erences were very small in a few instances, especially in the 3-month-old group, but remarkably consistent. Because there was no strong indication of a trend across ages, the data were combined for a single Student's t-test of the di¡erence between proximal and distal counts in the related samples (t = 3.51; df = 12; P 6 0.01). To evaluate the relative loss of ganglion cell dendrites at the proximal and
Surface preparations of the organ of Corti in the basal turn revealed that degeneration occurred ¢rst in the outer hair cells (Table 1). By 8 months of age, only 4% of the outer hair cells remained. A substantial loss of inner hair cells was not seen until 18 months of age, and by that time, only 29% of the inner hair cells were present. The extensive degeneration of the organ of Corti at 18 months of age is illustrated in Fig. 2. The number of surviving inner hair cells was highly correlated with the density of surviving spiral ganglion cells (r = 0.85; n = 10; P 6 0.02) and with the number of ganglion cell dendrites (r = 0.90 ; n = 10; P 6 0.001). 3.3. Density of spiral ganglion cells and counts of dendritic processes As shown in Table 1, there was a marked decline
Fig. 1. Median auditory brainstem response thresholds for four groups of mice of di¡erent ages. Both ears of each animal were included in the analysis. Symbols with arrows indicate minimum estimates due to output limitations of the system and, in a few cases, inadvertent failure to test at the highest intensities.
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Fig. 2. Degeneration of hair cells (A,B), distal dendrites (C,D), proximal dendrites (E,F) and spiral ganglion cells (G,H) with age. At 3 months of age, three rows of outer hair cells and a single row of inner hair cells are easily seen in the surface preparation, whereas at 18 months of age (B), all outer hair cells in this segment have degenerated and only a few inner hair cells remain (arrow). The density of remaining spiral ganglion cells is clearly reduced at 18 months compared with 3 months. The density of dendrite cross sections, both distally and proximally, is far greater at 3 months than at 18 months. Magni¢cation for A and B, 600U; C^H, 400U.
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distal locations as a function of age, we used a ratio of the distal/proximal counts. There is some indication in Fig. 4 that the ratio declines with age, i.e. the distal loss is proportionally greater than the proximal loss. The e¡ect is marginal from a statistical standpoint and would have to be veri¢ed in a larger sample before signi¢cance can be attributed to it. 4. Discussion The pattern of degeneration of spiral ganglion cells in the inner ear has been controversial and may be speci¢c to the cell type, species, and even cause of hearing loss. Thus, in the cat, Spoendlin (1971, 1975, 1984) reported rapid degeneration of all segments of the type I spiral ganglion cell following insult either to dendritic processes or to the organ of Corti. In contrast, type II ganglion cells may survive in the cat following transection of the cochlear nerve (Spoendlin, 1979) or following noise trauma (Spoendlin, 1975) or ototoxic lesions of the organ of Corti (Spoendlin, 1975; Bichler et al., 1983). In the rat, age-related degeneration of the spiral ganglion cell seemed to a¡ect both types I and II spiral ganglion cells equally (Keithley and Feldman, 1979). Similarly, in the human, there was no survival advantage for type II ganglion cells (Zimmermann et al., 1995). In a quantitative study of 9 human temporal bones with typical high frequency sensorineural hearing loss attributable to `presbycusis', Felder and SchrottFischer (1995) described an age-related loss of nerve ¢bers along the entire length of the cochlea that apparently was not strictly related to loss of either inner or outer hair cells. In human specimens, evidence for simultaneous or near simultaneous degeneration of spiral ganglion bodies and dendritic processes has been re-
Fig. 3. Dendritic counts taken in cross section in the osseous spiral lamina at a distal (near the organ of Corti) and at a proximal (near the spiral ganglion) location. In every subject but one (at age 3 months), the number of distal dendrites was less than the number at proximal sites.
Fig. 4. The mean ratio of the number of distal to the number of proximal dendrites tended to be lower at older, compared with younger, ages.
ported (Spoendlin and Scrott, 1989, 1990). However, other investigators have reported that the number of remaining spiral ganglion cells far exceeded the number of dendritic processes in the osseous spiral lamina in the human (Lindsay and Hinojosa, 1978; Kerr and Schuknecht, 1968; Suzuka and Schuknecht, 1988; Nadol et al., 1989 ; Felix et al., 1990; Nadol, 1997). In fact, selective loss of the dendritic arborization and synaptic contacts of spiral ganglion cells has been shown in presbycusis, Meniere's disease, and Usher's syndrome (Nadol, 1988). In animals (Kellerhals, 1967; Leake and Hradek, 1988) and in humans (Johnsson and Hawkins, 1972 ; Otte et al., 1978; Ylikoski et al., 1981 ; Suzuka and Schuknecht, 1988 ; Felix et al., 1990 ; Nadol, 1990; Felder et al., 1997), degeneration begins in the most peripheral processes of the spiral ganglion cell and progresses toward the cell body. This has been called the `dying back' process (Cavanagh, 1964). In the C57BL/6J mouse, hearing loss begins in the high frequencies at approximately 3^6 months of age and progresses to a total hearing loss at all frequencies by 15 months (Henry, 1983; Willott, 1991). The concomitant degeneration of the organ of Corti begins in the outer hair cells and later involves inner hair cells and spiral ganglion cells (Mikaelian, 1979). Therefore, the decrement in auditory function and its histopathologic correlates in the C57BL/6J mouse are similar to the pattern of degeneration seen in the human in a variety of disease processes. The present study suggests a retrograde pattern of degeneration of cochlear neurons in which degeneration begins in distal dendritic processes and progresses proximally toward the spiral ganglion cell, providing evidence of the dying back process in another species. In addition, the di¡erence between the number of distal and proximal dendrites may also increase with age, but that relationship is not yet certain. Success of cochlear implantation in the profoundly
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deaf probably depends, at least in part, on the number of residual spiral ganglion cells (Clopton et al., 1980). However, the exact site of excitation of surviving spiral ganglion cells by implanted electrodes is not known. Thus, it is not known whether successful stimulation requires the presence of peripheral dendritic processes or whether direct stimulation of the spiral ganglion cell body may occur in the absence of the dendrite. The demonstration of retrograde degeneration of spiral ganglion cells in the C57BL/6J mouse con¢rms ¢ndings in other animals and in humans and suggests that in the degenerating inner ear, all remaining spiral ganglion cells may not have dendritic ¢bers that extend peripherally to the level of the organ of Corti. In addition, the ¢nding that degeneration appears to occur in a retrograde fashion suggests that even among remaining degenerating dendrites, electrical properties may be more severely a¡ected distally than centrally. Acknowledgements Supported in part by NIDCD Grant #P01-DC00361.
References Bichler, E., Spoendlin, H., Raucheager, H., 1983. Degeneration of cochlear neurons after amikacin intoxication in the rat. Arch. Otorhinolaryngol. 237, 201^208. Bredberg, G., 1968. Cellular pattern and nerve supply of the human organ of Corti. Acta Otolaryngol. (Stockh.) 236 (Suppl.), 6^135. Cavanagh, J.B., 1964. The signi¢cance of the `dying back' process in experimental and human neurological disease. Int. Rev. Exp. Pathol. 3, 219^267. Clopton, B.M., Spelman, F.A., Miller, J.M., 1980. Estimates of essential neural elements for stimulation through a cochlear prosthesis. Ann. Otol. Rhinol. Laryngol. 89 (Suppl.), 5^7. Cohen, G.M., Park, J.C., Grasso, J.S., 1990. Comparison of demyelination and neural degeneration in spiral and Scarpa's ganglia of C57Bl/6 mice. J. Electron Microsc. Tech. 15, 165^172. Erway, L.C., Willott, J.F., Archer, J.R., Harrison, D.E., 1993. Genetics of age-related hearing loss in mice: I. Inbred and F1 hybrid strains. Hear. Res. 65, 125^132. Erway, L.C., Shiau, Y.W., Davis, R.R., Krieg, E.F., 1996. Genetics of age-related hearing loss in mice: III. Susceptibility of inbred and F1 hybrid strains to noise-induced hearing loss. Hear. Res. 93, 181^187. Felder, E., Schrott-Fischer, A., 1995. Quantitative evaluation of myelinated nerve ¢bers and hair cells in cochleae of humans with agerelated high-tone hearing loss. Hear. Res. 91, 19^32. Felder, E., Kanovier, G., Scholtz, A., Rask-Andersen, H., SchrottFischer, A., 1997. Quantitative evaluation of cochlear neurons and computer-aided three dimensional reconstruction of spiral ganglion cells in humans with a peripheral loss of nerve ¢bers. Hear. Res. 105, 183^190. Felix, H., Johnsson, L.G., Gleeson, M., Pollak, A., 1990. Quantitative analysis of cochlear sensory cells and neuronal elements in man. Acta Otolaryngol. (Stockh.) 470 (Suppl.), 71^79.
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Henry, K.R., Chole, R.A., 1980. Genotypic di¡erences in, behavioral, physiological and anatomical expressions of age-related hearing loss in the laboratory mouse. Audiology 19, 369^383. Henry, K., 1983. Aging and audition. In: Willott, J.F. (Ed.), In: The Auditory Psychobiology of the Mouse. Charles C. Thomas, Spring¢eld, IL, pp. 470^493. Johnson, K.R., Erway, L.C., Cook, S.A., Willott, J.F., Zheng, Q.Y., 1997. A major gene a¡ecting age-related hearing loss in C57B1/6J mice. Hear. Res. 114, 83^92. Johnsson, L.G., 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., Feldman, M.L., 1979. Spiral ganglion cell counts in an age-graded series of rat cochleas. J. Comp. Neurol. 188, 429^ 442. Kellerhals, B., 1967. Die Morphologie des ganglion spirale Cochleae. Acta Otolaryngol. (Stockh.) 226 (Suppl.), 1^78. Kerr, A., Schuknecht, H.F., 1968. The spiral ganglion in profound deafness. Acta Otolaryngol. (Stockh.) 65, 586^598. Leake, P.A., Hradek, G.T., 1988. Cochlear pathology of long term neomycin induced deafness in cats. Hear. Res. 33, 11^33. Li, H.-S., Borg, E., 1991. Age-related loss of auditory sensitivity in two mouse genotypes. Acta Otolaryngol. (Stockh) 111, 827^ 834. Lindsay, J.R., Hinojosa, R., 1978. Ear anomalies associated with renal dysplasia and immunode¢ciency disease. A histopathological study. Ann. Otol. Rhinol. Laryngol. 87, 10^17. Mikaelian, D.O., 1979. Development and degeneration of hearing in the C57/bl6 mouse: relation of electrophysiologic responses from the round window and cochlear nucleus to cochlear anatomy and behavioral responses. Laryngoscope 89, 1^15. Nadol, J.B., Jr., 1988. Innervation densities of inner and outer hair cells of the human organ of Corti. Evidence for auditory neural degeneration in a case of Usher's syndrome. ORL J. Otorhinolaryngol. Relat. Spec. 50, 363^370. Nadol, J.B., Jr., 1990. Degeneration of cochlear neurons as seen in the spiral ganglion of man. Hear. Res. 49, 141^154. Nadol, J.B., Jr., 1997. Patterns of neural degeneration in the human cochlea and auditory nerve: implications for cochlear implantation. Otolaryngol. Head Neck Surg. 117, 220^228. Nadol, J.B., Jr., Eavey, R.D., Liberfarb, R.M., Merchant, S.N., Williams, R., Climenhager, D., Albert, D.M., 1990. Histopathology of the ears, eye, and brain in Norrie's disease. Am. J. Otolaryngol. 11, 112^124. Nadol, J.B., Jr., Young, Y.S., Glynn, R.J., 1989. Survival of spiral ganglion cells in profound sensorineural hearing loss: implications for cochlear implantation. Ann. Otol. Rhinol. Laryngol. 98, 411^ 416. Otte, J., Schuknecht, H.F., Kerr, A.G., 1978. Ganglion cell population in normal and pathological human cochleae: implications for cochlear implantation. Laryngoscope 88, 1231^1246. Shnerson, A., Pujol, R., 1982. Age-related changes in the C57BL/6J mouse cochlea. I. Physiological Findings. Develop. Brain Res. 2, 65^75. Spoendlin, H., 1971. Degeneration behavior of the cochlear nerve. Ann. Otol. Rhinol. Laryngol. 112 (Suppl.), 76^82. Spoendlin, H., 1975. Retrograde degeneration of the cochlear nerve. Acta Otolaryngol. 79, 266^275. Spoendlin, H., 1979. Sensory neural organization of the cochlea. J. Laryngol. Otol. 93, 853^877. Spoendlin, H., 1984. Factors inducing retrograde degeneration of the cochlear nerve. Ann. Otol. Rhinol. Laryngol. 112 (Suppl.), 76^82. Spoendlin, H., Schrott, A., 1989. Analysis of the human auditory nerve. Hear. Res. 43, 25^38. Spoendlin, H., Schrott, A., 1990. Quantitative evaluation of the hu-
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man cochlear nerve. Acta Otolaryngol. (Stockh.) 470 (Suppl.), 61^ 69. Suzuka, Y., Schuknecht, H.F., 1988. Retrograde cochlear neuronal degeneration in human subjects. Acta Otolaryngol. (Stockh.) 450 (Suppl.), 1^20. Willott, J.F., 1991. Aging and the Auditory System: Anatomy, Physiology and Psychophysics. Singular Publishing, San Diego, CA. Ylikoski, J., Belal, A., Jr., House, W.F., 1981. Morphology of human
cochlear nerve after labyrinthectomy. Acta Otolaryngol. (Stockh.) 91, 161^171. Ylikoski, J., Collan, Y., Palva, T., 1978. Pathologic features of the cochlear nerve in profound deafness. Arch. Otolaryngol. 104, 202^ 207. Zimmermann, C.E., Burgess, B.J., Nadol, J.B., Jr., 1995. Patterns of degeneration in the human cochlear nerve. Hear. Res. 90, 192^201.
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