Breed differences in cochlear integrity in adult, commercially raised chickens

Hearing Research 166 (2002) 82^95 www.elsevier.com/locate/heares

Breed di¡erences in cochlear integrity in adult, commercially raised chickens Dianne Durham a

a;b;

, Debra L. Park a , Douglas A. Girod

a;b

Department of Otolaryngology and the Smith Mental Retardation Research Center, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7380, USA b Kansas City Veterans A¡airs Medical Center, Kansas City, MO, USA Received 25 May 1999; accepted 3 January 2002

Abstract Two types of chickens are commercially available. Broiler birds are bred to develop quickly for meat production, while egg layers are bred to attain a smaller adult size. Because we have observed breed differences in the response of central auditory neurons to cochlear ablation in adult birds [Edmonds et al. (1999) Hear. Res. 127, 62^76], we examined cochleae from the two breeds for differences in integrity. We evaluated cochlear hair cell structure using scanning electron microscopy and cochlear hair cell function using distortion product otoacoustic emissions (DPOAEs) and the auditory brainstem response. We observed striking breed differences in cochlear integrity in adult but not hatchling birds. In adult broiler birds, all cochleae showed damage, encompassing at least the basal 29% of the cochlea. In 15 of 18 broiler ears, damage was observed throughout the basal 60% of the cochlea. In contrast, cochleae from egg layer adults were largely normal. Two thirds of egg layer ears showed no anatomical abnormalities, while in the remainder cochlear damage was seen within the basal 48% of the cochlea. DPOAEs recorded from egg layer birds showed loss of high frequency emissions in every ear for which the cochlea displayed anatomical damage. Average sound pressure levels in both commercial facilities were 90 dB, suggesting these two breeds may exhibit differential susceptibility to noise damage. = 2002 Elsevier Science B.V. All rights reserved. Key words: Presbycusis; Hair cell regeneration; Aging; Auditory ; Deafness

1. Introduction Hearing loss is a signi¢cant health problem in the USA, particularly among older Americans. Most hearing loss can be traced to damage to or actual degeneration of sensory hair cells in the cochlea and is often more pronounced for high frequencies. Loss of sensory cells in the cochlea can be caused by aging itself, longterm exposure to damaging agents such as noise, or can have a genetic basis (Willott, 1991; Mills et al., 1996; Steel and Brown, 1996; Lonsbury-Martin et al., 1998). In addition to preventing the transduction of sound, cochlear dysfunction also results in a frequency-dependent decrease in a¡erent input impinging on neurons in the central nervous system (CNS). Many studies have * Corresponding author. Tel.: +1 (913) 588-6731; Fax: +1 (913) 588-6708. E-mail address: [email protected] (D. Durham).

shown that CNS neurons rely on continuous a¡erent input both for development of connections as well as maintenance of normal adult neuronal function and metabolism (Rubel et al., 1990; Linden, 1994; Mattson, 1996). Particularly in sensory systems, the manner in which alterations in a¡erent input a¡ect CNS neurons changes with age. Neurons in young animals undergo more profound changes than those in adult animals (Born and Rubel, 1985; Hashisaki and Rubel, 1989; Tierney et al., 1997; Edmonds et al., 1999). Knowledge of the nature of these CNS changes, particularly the extent to which they are reversible, is becoming increasingly important as our ability to improve function of the auditory periphery with cochlear implants (Niparko, 1998) or regeneration of hair cells (Stone et al., 1998; Smolders, 1999; Cotanche, 1999) increases. Several animal models exist for evaluating age-related changes in cochlear structure and function, including cochlear degeneration observed in di¡erent strains of

0378-5955 / 02 / $ ^ see front matter = 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 2 ) 0 0 3 0 1 - 5

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mice (Willott, 1996), in chinchillas (Clark, 1992), and in deaf white cats (Ryugo et al., 1998). An extensive literature also exists describing the development, adult structure and function of the chick auditory system, both in the periphery and the CNS (Rubel and Parks, 1988; Rubel et al., 1990; Cotanche et al., 1994; Parks, 1997; Stone et al., 1998). Study of the avian cochlea (basilar papilla) is of particular interest because its sensory hair cells have the ability to regenerate after damage from ototoxic drugs or noise (Chen et al., 1996; Corwin and Oberholtzer, 1997; Stone et al., 1998; Cotanche, 1999). Current evidence suggests that after damage, the support cells of the avian cochlea move into the cell cycle, complete mitosis, and di¡erentiate into new hair cells (Cotanche et al., 1994; Tsue et al., 1994; Stone and Rubel, 2000). In addition to anatomical recovery, multiple studies have shown that physiological function progressively improves as morphological recovery occurs (Tucci and Rubel, 1990; Girod et al., 1991; Marean et al., 1993; Muller et al., 1996; Dooling et al., 1997; Ding-Pfennigdor¡ et al., 1998; Smolders, 1999; Woolley et al., 2001). Despite extensive knowledge of the structure and function of the chick cochlea in hatchling birds, little is known about age-related changes in avian cochlear morphology or function. The response of hatchling avian brainstem auditory neurons to alterations in cochlear integrity has been well documented, both in response to cochlear ablation (Rubel et al., 1990) as well as more recent studies evaluating CNS changes during hair cell loss and regeneration (Lippe, 1991; Park et al., 1998, 1999; Saunders et al., 1998; Durham et al., 2000). Recent studies in our laboratory have shown age- and breed-dependent di¡erences in neuronal cell death and glial proliferation following cochlear ablation in adult animals (Edmonds et al., 1999; Lurie and Durham, 2000). Two types of chickens are commercially available. Broiler birds (e.g. avian, Arbor Acres, Cobb, Hubbard strains) are bred to develop quickly for meat production, while egg layers (e.g. Hy-Line, HpN or White Leghorn strains) are bred to attain a smaller adult size. In egg layer adults, 30% of second-order neurons in nucleus magnocellularis (NM) die following cochlear ablation; the same proportion of NM neurons die when the cochlea is removed in a hatchling bird. In broiler birds, however, neuronal cell death only occurs in hatchlings. In adult broilers, no cell death is seen following cochlear ablation (Edmonds et al., 1999). It is not known to what extent age- or breed-related di¡erences in cochlear integrity might in£uence the response of CNS auditory neurons to cochlear ablation in the adult animal. The purpose of this study was to evaluate whether di¡erences in cochlear integrity exist in the two breeds of adult birds shown previously to di¡er in the CNS response to cochlear ablation. We

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examined cochleae in commercially raised adult birds of two breeds using scanning electron microscopy (SEM) and found striking di¡erences in cochlear integrity. These di¡erences in cochlear integrity may underlie di¡erences in the CNS response to cochlear ablation.

2. Materials and methods 2.1. Experimental subjects Adult, commercially raised chickens were the subjects of this experiment. Retired female breeders were obtained at 66 weeks of age, housed in an AAALACapproved facility, and given ad libitum access to food and water for up to 2 weeks prior to sacri¢ce. Adult broiler hens (Avian strain, n = 5 or Cobb strain, n = 10) were obtained from ConAgra, Inc. (Batesville, AR, USA), and adult egg layer hens (Hy-Line strain, n = 16) were obtained from Linn Grove Hatchery (Linn Grove, IA, USA). Cochleae from hatchling birds of both breeds, processed as part of other studies (Park et al., 1998; Sands and Durham, 1999), were examined for comparison to cochleae from adult birds. Brains were also harvested from all the adult birds used in this study as part of additional ongoing experiments (Sands and Durham, 1999; Smittkamp et al., 2001). All experimental protocols were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee (IACUC). To provide an estimate of the daily sound levels to which these retired breeders were exposed, we measured both average sound pressure levels and frequency-speci¢c sound pressure levels at both commercial facilities from which we obtained adult birds. Measurements were made using a Model 1800/OB300 digital sound level meter (Quest Electronics, Oconomowoc, WI, USA) on both the linear and the dB C scale. Sound measurements were taken 10 min after the investigator entered the laying house (coop) and the chickens had become accustomed to her presence. 2.2. Assessment of cochlear function Prior to sacri¢ce, the majority of adult egg layer birds (14 of 16 birds) were evaluated with two physiological tests, the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs), to determine whether either non-invasive measure of cochlear function would be useful in predicting the anatomical status of the cochlea. Birds were anesthetized with an intraperitoneal injection of sodium pentobarbital (25 mg/kg) and an intramuscular injection of ketamine (100 mg/kg) and placed in a double-walled, sound-attenuating booth. For ABR measurements, pin

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Fig. 1. SEM photomicrographs showing the range of cochlear morphology in adult egg layer birds. Top micrograph shows a normal cochlea (found in 71% of ears), while the bottom panel shows a typical damaged cochlea (found in 29% of ears). Drawings indicate the area of damage (shaded) as well as the region from which photomicrographs were taken (area between the solid lines).

electrodes were placed subcutaneously behind each ear and over the vertex of the skull, and the animal’s head was positioned between two TDX-35 headphones. Using a Nicolet Path¢nder electrodiagnostic system (Madison, WI, USA), evoked potentials were measured in response to a 10 ms click stimulus (rise/fall time of 4 ms; frequency range 2142^6300 Hz). Responses were averaged over 250 repetitions and measured at 5 dB intervals. The ABR threshold was de¢ned as the lowest intensity click stimulus which elicited an identi¢able and repeatable ABR waveform. At the conclusion of ABR measurements, DPOAE measurements were obtained from each ear using an ILO 92 otoacoustic emission system (Otodynamics, Ltd., Hats¢eld, Herts, UK). An infant testing probe was placed into the external ear canal so that an airtight ¢t was achieved. Subsequently, a 2f1 3f2 DPOAE audiogram (DPgram) was obtained over a frequency range of 698^6023 Hz. Two sinusoidal stimuli of di¡erent frequencies, f1 and f2 (f1 6 f2 ), and equal amplitude

(70 dB SPL) were presented simultaneously through separate channels. DPOAEs were recorded at three steps per octave at a frequency ratio (f2 /f1 ) of 1.22 for 120 s. The 2f1 3f2 distortion product was plotted as a function of the f2 stimulus. For each cochlea, we used the highest frequency of emission recorded to compare physiological responses among animals. The highest frequency emission was de¢ned as the highest frequency response with an amplitude greater than two standard deviations above the noise £oor. This measure was chosen for this analysis because we observed that damage always occurred at the base of the cochlea and extended apically. 2.3. Assessment of cochlear integrity Either immediately after physiological tests were completed or up to 5 days later, animals were killed and their cochleae were prepared for SEM to assess cochlear integrity. Birds not already anesthetized at

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Fig. 2. SEM photomicrographs showing the range of cochlear morphology in adult broiler birds. Top micrograph shows a cochlea with damage con¢ned to the base (found in 37% of ears), while the bottom panel shows a cochlea in which damage extends into apical regions (found in 63% of ears). Drawings indicate area of damage (shaded) as well as the region from which photomicrographs were taken (area between the solid lines).

the time of sacri¢ce were given an overdose of Euthasol. For half of the egg layer hens, animals were decapitated and brains were quickly removed and frozen for other analyses. In each ear the columella was removed and the ear was perfused with 3.5% glutaraldehyde in phosphate-bu¡ered saline (PBS; 0.1 M phosphate bu¡er, pH 7.4) by slowly perfusing 3 ml of this solution through the round window (Girod et al., 1989). For the remaining egg layer hens and all of the broiler hens, bodies were ¢rst perfused transcardially with PBS containing 1 U/ml heparin followed by either 10% phosphate-bu¡ered formalin (0.1 M, pH 7.4) or mixed aldehydes (glutaraldehyde and paraformaldehyde in PBS) (Hyde and Durham, 1994). Immediately following the systemic perfusion (10 min after death), the ears were perfused as described above and the brains were removed from the heads to be used for other analyses. Following cochlear perfusion and brain removal, the

head was placed in the 3.5% glutaraldehyde ¢x at 4‡C for at least 10 days. The temporal bone containing the cochlea was then dissected free from the head and post¢xed with 1% osmium in distilled water. The ¢nal dissection included removing the tectorial membrane, and preparation for the scanning electron microscope included dehydration with a graded series of ethanol, critical point drying in carbon dioxide, and sputtercoating with palladium gold. SEM photographs of the cochlear surface were taken at a magni¢cation of 110U using a Hitachi S2700 microscope. Montages were made of the entire cochlear surface and used to document the damaged region of the cochlea as a percentage of the total cochlear length (percent length damage). Using a planimeter, the total cochlear length and the length of the damaged region were measured; percent length damage was calculated as described previously (Husmann et al., 1998). For

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regions and regenerating hair cells were made from SEM images taken at 400^1500U magni¢cation.

3. Results 3.1. Anatomical evaluation

Fig. 3. SEM photomicrographs depicting the range of hair cell abnormalities seen in egg layer adult birds. All photomicrographs are from the base of the cochlea, at a location 25% of the distance from the base to the apex. (A) Normal hair cells in an undamaged cochlea, (B) moderately damaged hair cells, (C) severely damaged region with no hair cells present. Arrows in B indicate hair cells being extruded from the sensory epithelium. Scale bar = 20 Wm.

these measurements, cochlear regions were considered damaged if hair cells were completely missing or if hair cells were present but showed abnormalities on their apical surfaces or in their arrangement along the basilar membrane. Additional qualitative analyses of damaged

In normal untreated hatchling animals of either breed we did not observe any abnormalities in hair cell arrangement or morphology. However, in normal, otherwise untreated adult animals we observed di¡erences between breeds in the anatomical appearance of the cochleae. In 16 egg layer birds (Fig. 1), 15 of 21 (71%) ears examined anatomically showed no evidence of hair cell damage. In the remaining cochleae (six of 21, or 29% of ears), cochlear hair cell damage was evident, beginning at the basal, high frequency end of the cochlea and encompassing an average of 48% of the cochlear length (S.E.M. = 5.9%; range 31^68%). Damage was con¢ned to one ear in all of the birds in which anatomical cochlear abnormalities were seen (i.e. the two ears shown in Fig. 1 are from the same bird). In contrast, for broiler birds, all cochleae (n = 18 from 15 birds) showed damage beginning in the basal end of the cochlea (Fig. 2). The average percent length damage for broiler birds was 70% (S.E.M. = 4%, range 29^100%). In 15 of 18 broiler cochleae, damage encompassed 60% or more of the cochlea, while in the three remaining ears damage covered less than 45% of the cochlea. As with egg layer birds, the extent of damage was usually unequal in the two ears of a given animal. However, in broilers, all ears showed damage. Statistical analysis examining percent length damage as a function of breed showed a signi¢cant di¡erence between breeds in the extent of cochlear damage (M2 , P 6 0.0001). The criteria we use to determine the percent length damage in the cochlea do not di¡erentiate between mild and severe abnormalities of the sensory epithelium. Figs. 3 and 4 show examples of the range of hair cell damage seen in adult egg layer and broiler birds. All damage in egg layer birds was con¢ned to the basal half of the cochlea (Fig. 3). As described above, 71% of ears in egg layer birds showed no damage at all (Fig. 3A). In the majority of abnormal egg layer cochleae, hair cells were present in the damaged region but they often displayed abnormal morphological characteristics. We observed hair cells with either large apical surface areas, mis-oriented or short stereociliary bundles, or hair cells being extruded from the sensory epithelium (Fig. 3B). Within the damaged regions, abnormal hair cells were not arranged in the regular hexagonal array typical for normal animals. In two egg layer cochleae, damaged regions were devoid of hair cells altogether (Fig. 3C).

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Fig. 4. SEM photomicrographs depicting the range of hair cell abnormalities seen in adult broiler birds. Left three panels (A^C) are from the base of the cochlea (25% distance from base to apex) and the right three panels (D^F) are from the apical region (70% distance). All cochleae displayed abnormal hair cells in the base, while in the apex some cochleae contained normal hair cells (D). Arrows in B indicate damaged hair cells. Scale bar = 20 Wm.

Contained within the damaged epithelium were cells displaying a raised apical surface covered with microvilli. These cells resemble hyaline cells often seen in cochleae following severe noise damage (Oesterle et al., 1992; Cotanche et al., 1995; Bunting et al., 1996). In broiler birds, the basal end of the cochlea showed some damage in every ear (Fig. 4A^C). The least severely damaged basal regions contained hair cells with abnormally oriented stereocilia and large apical surfa-

ces. Hair cells in these cochleae lacked the usual hexagonal arrangement along the sensory epithelium (Fig. 4A). With more severe damage, large hair cells with absent stereocilia were observed scattered among hair cells with somewhat abnormal stereocilia (Fig. 4B). The most severely damaged basal regions displayed a total absence of any sensory cells along the epithelium (Fig. 4C). In these cochleae, like those in severely damaged egg layer ears, the epithelium was composed of cells

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Fig. 5. Higher magni¢cation SEM photomicrographs showing presence (A, C) or absence (B, D) of regenerating hair cells in a broiler bird (A, B) and an egg layer bird (C, D). Arrows indicate regenerating hair cells. Scale bar = 8.57 Wm.

Table 1 Summary of data from individual egg layer birds undergoing physiological analysis Animal number

98-3702 98-3705 98-3706 98-3707 98-3520 98-3521 98-2603 98-3701 98-3703 98-3704 98-3708 98-2600 98-2601 98-2602

Left ear

Right ear

DPOAE (f2 )

Damage

ABR

DPOAE (f2 )

Damage

ABR

5042 6348 6348 5042 5042 5042 4004 5042 2002 1587 5042 5042 2515 2002

0% 0% 0% 0% 0% 0% 0% 0% 40% 60% 0% 0% 31% 50%

45 U 40 35 35 U 40 45 45 45 30 30 40 70

5042 5042 6348 5042 6348 6348 5042 2002 5042 5042 2515 1587 5042 2515

U U 0% 0% U U G U U U 70% G G G

45 30 35 30 30 U 20 55 40 35 45 45 20 80

For each ear, values given are the highest f2 DPOAE frequency recorded, percent length damage determined from SEM, and the ABR threshold. U symbols indicate that data are not available for a given parameter. G in the ear damage column indicates prior topical unilateral administration of gentamicin as part of another experiment. Bold indicates ears with naturally occurring cochlear damage con¢rmed with SEM.

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1987; Girod et al., 1989). These features include a small apical surface area and short stereociliary bundles centered on the apical surface, resembling embryonic hair cells (Cotanche and Sulik, 1984). We observed such hair cells in both egg layer (Fig. 5A) and broiler (Fig. 5C) birds, but only in less severely damaged regions of the cochlea. No qualitative di¡erences between breeds were observed in the number of regenerating hair cells. When damage was severe, no regenerating hair cells were observed in either breed (Fig. 5B,D). 3.2. Physiological analyses

Fig. 6. DPOAEs recorded from two adult egg layer birds. Top recording was made from an ear in which the cochlea was anatomically normal by SEM analysis. Bottom recording was made from an ear in which the cochlea showed damaged basal hair cells by SEM analysis. Highest f2 frequency recorded was 5042 Hz for the normal ear and 2002 Hz for the damaged ear. Shaded regions in each plot represent the noise £oor (dark shading) plus two standard deviations (lighter shading).

resembling hyaline cells. In three broiler cochleae, apical hair cells appeared normal (Fig. 4D). However, in 15 of 18 cochleae, damaged hair cells extended into the apical region. Damage was con¢ned to disorientation of stereocilia and enlargement of hair cell size (Fig. 4E) or in more severe cases loss of hair cells and more variability in hair cell size (Fig. 4F). Given the capacity for regeneration of hair cells in the hatchling chick (Cotanche et al., 1994; Stone et al., 1998), the adult quail (Ryals and Rubel, 1988), the adult starling (Marean et al., 1993), the adult pigeon (Muller et al., 1997; Muller and Smolders, 1998), and in the adult canary (Gleich et al., 1994, 1997), we examined damaged cochleae in these adult chickens for the presence of hair cells with morphological features characteristic of regenerating hair cells (Cotanche,

In egg layer birds, we evaluated the e⁄cacy of two physiological measures of auditory function, free-¢eld ABRs and DPOAEs, as a means to assess cochlear integrity non-invasively. DPOAE measurements proved to be an excellent method for predicting damage later con¢rmed with SEM anatomical analysis. Fig. 6 shows DPgrams from two ears, one in which the cochlea showed no damage as evaluated with SEM and another for which SEM analysis revealed damage along the basal 40% of the cochlea. In the anatomically normal ear, we obtained DPOAE responses throughout the expected frequency range for chickens (698^5042 Hz). In the damaged ear, however, the highest frequency at which we obtained a DPOAE response was 2002 Hz. As shown in Table 1, for every animal with an abnormal cochlea, the highest frequency DPOAE response we recorded was well below that for normal animals. For all left ears, in which both DPOAE responses and SEM evaluation were available, the mean highest DPOAE frequency was statistically signi¢cantly greater for ears

Fig. 7. Scatterplot showing the relationship between the highest frequency emission recorded (ln Hz) and the percent length damage (from the base) in the cochlea. Linear regression analysis showed a correlation of r2 = 0.81 for this relationship (P 6 0.01).

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Fig. 8. Measurements of sound levels as a function of frequency (linear dB scale) from laying houses at both commercial facilities. At both broiler and egg layer facilities elevations in sound level occur at low frequencies (likely due to machine noise) as well as within the normal chicken hearing range (due to the vocalizations of the birds).

with no anatomical damage as compared to ears with any amount of damage con¢rmed with SEM (unpaired t-test ; P 6 0.0001). Fig. 7 plots the relationship between the natural logarithm of the highest DPOAE frequency we recorded and the percent damage we observed in the cochlea for egg layer birds. A linear regression shows a signi¢cant correlation between ln frequency and the percent length damage (P 6 0.001; r2 = 0.81). This analysis suggests that DPOAEs are a reliable method for non-invasively assessing hair cell damage in these adult birds. In contrast, ABRs evoked with a click stimulus were not very sensitive predictors of damage to the cochlea, except when damage was present in both ears. As shown in Table 1, ABR thresholds were elevated only when both ears were damaged (animal 98-2602). In this animal, one ear showed naturally occurring damage, and the other ear was damaged by topical administration of gentamicin 5 days prior to the physiological measurements. This result is not surprising, given the interaural canal that connects the middle ears in birds (Rosowski and Saunders, 1980). 3.3. Environmental sound levels The anatomical features observed in damaged cochleae of broiler birds are very similar to those seen following exposure of hatchling birds to intense noise (Cotanche, 1987; Girod et al., 1989; Muller et al., 1996; Ryals et al., 1999). The breeding £ocks from which we obtained these adult birds are housed in a large metal structure (called a laying house), which measures roughly 640 feet in length and 40 feet in width. Each laying house contains approximately 18 000 chickens, whose vocalizations occur nearly continuously when the lights in the laying house are on (18 h/day). Based

on these observations we measured sound levels in laying houses at both commercial facilities. Average sound pressure levels were found to be 90 dB at both facilities. Fig. 8 shows sound levels as a function of frequency for both the egg layer and the broiler facilities. Sound levels are elevated at very low frequencies (below 100 Hz) and also between 700 and 7000 Hz, which is within the normal hearing range of these birds. As can be seen in Fig. 8, sound levels in the egg layer facility were very similar to those in the broiler facility. Thus it seems unlikely that a di¡erence in sound environment can explain the breed di¡erence in cochlear integrity. Rather, if long-term exposure to noise is a factor, these two breeds of birds likely di¡er in their susceptibility to noise.

4. Discussion 4.1. Breed-speci¢c cochlear abnormalities In this study we have described striking di¡erences in cochlear integrity between two breeds of commercially raised adult birds. In adult broiler birds, all cochleae showed some hair cell damage. The basal 29% of the cochlea (high frequency region) was always damaged, with damage extending up to 100% of the distance from the base. The severity of cochlear damage ranged from stereociliary abnormalities and departure from the normal hexagonal array of hair cells along the sensory epithelium to a total loss of hair cells and replacement of sensory cells with what appear to be hyaline cells. In egg layer birds, most cochleae were normal, while 29% showed hair cell abnormalities similar to those seen in broilers but occurring only in the base of the cochlea. Because of the auditory environment in which these birds live, in which average sound pressure levels are 90 dB, we suspect that noise contributes to this damage. However noise damage alone cannot be the only factor involved, as damage only occurs in one breed of chicken, even though sound levels at the two facilities are equally loud (Fig. 8). In addition, the pattern of damage is not characteristic of most noise damage in that it always encompasses the high frequency end of the cochlea and routinely involves both tall and short hair cells. Several features of the cochlear damage we observed deserve mention. First, unlike damage caused by systemic administration of ototoxic drugs or intense noise exposure in hatchling birds, damage was often markedly dissimilar in the two ears of a given animal. No egg layer birds showed damage in both ears, and in broiler birds damage was unequal in all three birds from which both ears were recovered. It will be interesting to determine whether any behavioral parameters

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(such as head position that protects one ear from noise) contribute to the unequal damage in the two ears. Second, the majority of our observations of adult broiler ears have been made in commercially raised birds at one adult age, 66 weeks, when these facilities routinely sacri¢ce breeding colonies. Preliminary data suggest that damage is not apparent in commercially raised birds until 30 weeks of age (Jaeckel et al., 2000). In broiler birds raised to 40 weeks of age in the KUMC animal facility, where average sound pressure levels are approximately 65 dB, much less severe damage is apparent (Smittkamp et al., 2001). These observations suggest that the cochlear damage seen in broilers is not likely to represent an early, developmental cochlear degeneration as seen in other models of age-related hearing loss (see below). Finally, because we observed what appear to be regenerating hair cells in the cochleae of both breeds of adult birds, the di¡erence in adult cochlear integrity cannot be merely due to an inability of adult broiler birds to regenerate new hair cells. In both breeds severely damaged regions contain no regenerating hair cells, suggesting that supporting cell damage may be so severe that no further regeneration is possible. Further study of the regeneration process itself will be needed to determine whether breed di¡erences exist in regeneration capacity.

unilateral stimulation. The ablation of the ipsilateral cochlea reduces the amplitude of the ABR but does not abolish the response. Thus the ABR is a reliable measure when (1) the contralateral cochlea is ablated, and (2) the anatomical status of both ears is equivalent. To date we have measured neither DPOAE nor ABR responses in adult broiler birds, but we anticipate that DPOAE measurements will allow accurate, non-invasive prediction of cochlear damage in these animals. Evaluations of functional recovery from severe noise trauma in adult pigeons (Muller et al., 1996, 1997; Ding-Pfennigdor¡ et al., 1998), chickens (Trautwein et al., 1996; Chen et al., 1996), budgerigers (Dooling et al., 1997), quail (Ryals et al., 1999), canaries (Ryals et al., 1999) and starlings (Marean et al., 1993, 1998) have consistently demonstrated incomplete recovery of physiological parameters, thought to be due to incomplete anatomical recovery of the sensory epithelium. Although these studies have utilized brief, intense noise exposure to create hair cell death and regeneration, the anatomical appearance of the residual cochlear damage as well as the functional de¢cits described in these studies are similar to those we have observed in adult broiler birds. More complete physiological evaluation of our adult birds will allow us to assess the extent of similarity in cochlear function.

4.2. Physiological evaluation of cochlear damage in adult birds

4.3. Comparison to models of age-related cochlear damage

Our physiological evaluation of egg layer birds suggests that DPOAE measurements are a very accurate predictor of not only the presence of hair cell damage (Table 1) but also the extent of damage along the length of the cochlea (Fig. 7). The variance (r2 = 0.81) of the correlation between percent length damage and the highest frequency of DPOAE that we report here for adults is similar to that obtained in hatchling birds following gentamicin-induced hair cell loss (Streubel et al., 1998), suggesting that it is a good predictor of the extent of hair cell damage. Because DPOAE measurements can be made unilaterally, this physiological test will be very valuable in assessing cochlear integrity prior to evaluation of CNS responses to unilateral cochlear ablation. More sophisticated DPOAE measurements such as input/output functions may provide additional information regarding hair cell function in these animals. ABR thresholds, at least those measured with subcutaneous scalp electrodes and free-¢eld stimuli, were not a particularly reliable measure of damage in one ear. This result is perhaps not surprising due to the presence of an interaural canal that connects the two middle ears in chickens (Rosowski and Saunders, 1980). Canady and colleagues (Canady et al., 1994) showed that ABR responses in the chick are bilateral despite

Several animal models of age-related degeneration of cochlear integrity or hearing levels have been evaluated. The hair cell abnormalities seen in Belgian Waterslager canaries (Gleich et al., 1994; Weisleder and Park, 1994; Ryals and Dooling, 1996; Weisleder et al., 1996) are similar to those observed here in that the severity of damage varies considerably among individual animals. In addition, functional de¢cits in Waterslagers, measured as frequency-speci¢c behavioral thresholds, CAP thresholds, or cochlear microphonic thresholds (Gleich et al., 1994, 1995), occur predominantly for high frequencies. Hair cell regeneration occurs in both adult chickens and Waterslager canaries, in which the rate of regeneration can be increased following ototoxic drug damage (Gleich et al., 1997). However, important di¡erences exist between damage seen in this breed of canaries and that reported here in adult chickens. First, damage in canaries is usually seen throughout the cochlea, often more prominent in apical regions than in basal regions. In adult chickens, damage always occurs in the base of the cochlea and extends apically. Rarely is damage in canaries as extensive in terms of hair cell loss as the severe damage seen in these adult chickens. Cochleae in Waterslager canaries always contain hair cells, albeit abnormal ones. Few extruding hair cells

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are observed in Waterslagers, while these dying hair cells are prominent features in the adult chicken (Figs. 3 and 4). Finally, the de¢cit in Waterslager canaries has a de¢nite genetic, congenital basis, in that young o¡spring of Waterslager and German Roller canaries (which have normal hearing) show hearing de¢cits intermediate to those of the two parent strains. Thus, it would appear that both the type of damage and the genetic basis for it are di¡erent in canaries and chickens. We have not evaluated the vestibular organs of adult broiler birds to determine whether damage to the saccule occurs as it does in Waterslagers (Weisleder and Park, 1994; Jones et al., 1998). Genetically programmed cochlear degeneration occurs in the deaf white cat, beginning at about the ¢rst post-natal week (Suga and Hattler, 1970) and mimics the Scheibe deformity seen in humans. In addition to cochlear abnormalities these animals exhibit loss of ganglion cells and altered morphology of brainstem auditory neurons (Schwartz and Higa, 1982; Larsen and Kirchho¡, 1992; Saada et al., 1996). Recent work has shown that a subset of these animals exhibit partial and not total functional deafness, and that anatomical abnormalities vary with the amount of hearing loss (Ryugo et al., 1998). The cochlear degeneration in deaf white cats is unlike that of adult chickens in that feline damage begins soon after birth in animals raised in a normal environment. Variability in damage among animals is similar to that seen in adult chickens. Perhaps the best-studied models for human hearing loss are speci¢c inbred mouse strains that exhibit di¡erent types of genetic hearing loss (Willott, 1996). In DBA/2J (DBA) mice the onset of cochlear degeneration occurs rapidly, within 3 weeks of birth (Willott, 1981; Willott et al., 1984). C57Bl/6J (C57) mice show normal cochlear development until 1^2 months of age, then demonstrate high frequency hearing loss and degeneration of outer then inner hair cells at the cochlear base. Cochlear degeneration proceeds apically, and aged C57 mice show behavioral and anatomical evidence for profound hearing loss (Willott, 1996). Balb/c mice show damage intermediate to that seen in DBA or C57 mice (Willott et al., 1998). C3H strains show very little damage in the adult cochlea (Erway and Willott, 1996). Genetic analyses indicate that hearing loss in these strains results from mutations at three separate loci (Erway et al., 1993). Hearing loss in these mouse strains resembles that in adult chickens in that it occurs ¢rst in high frequency regions and can progress to include total loss of hair cells. In both breeds of adult birds damage would appear to occur later in life than for either C57Bl/6J or DBA/2J mice. Interesting di¡erences among mouse strains are apparent in the e¡ects of these cochlear abnormalities on neuronal morphology in the anteroventral cochlear nucleus (AVCN). In both C57

and DBA strains, a signi¢cant loss of neurons is observed in AVCN, while Balb/c mice show a normal AVCN neuronal cell number (Willott et al., 1998). In adult chickens, neuronal cell number is similar to hatchlings in the cochlear nucleus of both broiler or egg layer adults (Born and Rubel, 1985; Edmonds et al., 1999). It is not known for either species how neuronal cell number is controlled by cochlear input, but these two strains of birds will allow us to investigate that mechanism. 4.4. Contribution of noise damage to cochlear abnormalities Based on the morphology of the most severely damaged cochleae, it seems most likely that the damage we observe in these adult birds is due to prolonged exposure to noise. In cochleae devoid of hair cells (Figs. 4 and 5), the surface of the epithelium is covered by what appear to be hyaline cells based on surface morphology. Following severe noise damage, hyaline cells have been shown to ¢ll in space on the sensory epithelium from which hair cells have been extruded and supporting cells are lost (Girod et al., 1995; Cotanche et al., 1995; Bunting et al., 1996). Preliminary anatomical evaluation of adult broiler cochleae suggests that regions devoid of hair cells do contain hyaline cells (Colgan et al., 2000). The anatomical features we observed in our most severely damaged cochleae resemble those seen in adult pigeons, quail, canaries and budgerigers after severe noise trauma (Muller et al., 1996; Ding-Pfennigdor¡ et al., 1998; Ryals et al., 1999). In these studies, hair cell regeneration was absent when damage to the sensory epithelium was severe enough, similar to our observations in adult broiler and egg layer chickens (Fig. 5). Our measurements of the sound levels in both commercial facilities showed average sound pressure levels of 90 dB during the daytime when birds are active. Frequency-speci¢c measurements (Fig. 8) show elevated sound levels both below and within the normal hearing range of these birds (100^6000 Hz). Such noise levels, if present for the majority of the lifespan of these birds, could surely produce the damage we observed with SEM. Although it is clear that hair cell regeneration can occur after multiple, brief exposures to intense noise (Adler and Saunders, 1995), it is not clear how long-term exposure to less intense noise will a¡ect the sensory epithelium. Sound levels in the commercial facilities do exceed those recently shown to ameliorate the age-related cochlear degeneration seen in mouse models (Willott and Turner, 1999; Willott et al., 2000). Examination of birds raised to adulthood in a quiet environment will allow us to determine whether noise is indeed a causative factor. Given that noise levels are equal in the two facilities, the di¡erence between these two

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breeds of birds would seem to represent a breed di¡erence in susceptibility to noise. Such breed-speci¢c di¡erences in susceptibility to noise have been demonstrated in C57Bl/6J mice (Erway and Willott, 1996).

5. Conclusions In summary, we have shown that profound di¡erences in cochlear integrity are present in two breeds of adult birds, perhaps the result of a breed-dependent susceptibility to noise damage. These di¡erences in cochlear integrity may contribute to di¡erences in the response of the central auditory pathway to unilateral cochlear ablation in egg layer vs broiler birds (Edmonds et al., 1999; Lurie and Durham, 2000; Smittkamp et al., 2001). In addition, evaluation of the breed di¡erences in susceptibility to age-related degeneration of cochlear hair cells in chickens, which have the capacity to regenerate damaged hair cells, may prove very valuable in understanding age-related hearing loss in humans.

Acknowledgements The authors thank Sandy Parsons and Virginia Morris for expert technical assistance, and Ralph Park and Elise Jaeckel for help in obtaining adult broiler birds. Judith Widen generously provided the ILO 92 otoacoustic emission system used for DPOAE measurements. Supported by NIDCD Grant DC01589 to D.D., the KUMC Department of Otolaryngology, and a Merit II grant to D.G. from the Kansas City Veterans A¡airs Research O⁄ce.

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