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Neurobiology of Aging 27 (2006) 490–500 Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats Jiri Popelar a,∗ , Daniel Groh a,b , Jana Pelánová a , Barbara Canlon c , Josef Syka a a Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic b ENT Department, Charles University, 2nd Medical Faculty, 150 06 Prague 5, Czech Republic c Department of Physiology, Karolinska Institute, Stockholm 171 77, Sweden Received 12 November 2004; received in revised form 11 February 2005; accepted 10 March 2005 Abstract Auditory function in Fischer 344 (F344) and Long Evans (LE) rats was monitored during their lifespan by evaluating hair cell loss, middle-ear compliance and the recording of otoacoustic emissions and auditory brainstem responses. The results revealed a faster deterioration of hearing function in F344 rats compared with LE rats, resulting in larger hearing threshold shifts, a decrease in the latency and amplitude of click-evoked auditory brainstem responses, diminution of the distortion product otoacoustic emissions and a decrease in middle-ear compliance. However, hair cell loss, observed only at the most basal and apical parts of the organ of Corti, was comparable in older individuals of both rat strains. The results suggest involvement of cochlear (stria vascularis) and extracochlear (middle-ear) pathological changes during ageing. Thus, F344 rats represent a complex mix of conductive hearing loss (with low-frequency threshold shift, declining parameters of the middle-ear admittance and asymmetric otoacoustic emissions) and sensorineural hearing loss (with a decrease in the amplitudes of auditory brainstem response and a high-frequency threshold shift). © 2005 Elsevier Inc. All rights reserved. Keywords: Fischer 344 rats; Hearing threshold; Otoacoustic emissions; Auditory brainstem responses; Tympanometry; Cytocochleogram 1. Introduction The investigation of age-related changes in hearing and their possible prevention, studied in a normal animal populations, requires time consuming, long-lasting observations. Our previous studies, performed with Long Evans (LE) rats, revealed only limited hearing losses, small deteriorations in frequency difference limens and in the parameters of the middle latency response in very old LE rats (36 months old) compared with young rats [50,51,63]. Thus, special mutant strains of rats and mice, characterized by relatively faster aging, have been suggested for preferential use for age-related studies. C57BL/6 mice [24,37,39,60] and Sprague–Dawley and Fischer 344 (F344) rats [9,27,49,56] are most frequently used for ∗ Corresponding author. Tel.: +420 24106 2689; fax: +420 24106 2787. E-mail address: [email protected] (J. Popelar). 0197-4580/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2005.03.001 age-related studies in auditory research (for review see [19,62,66]). 1.1. Fischer 344 and Long Evans rats Since their introduction in 1920, F344 rats have been a widely used inbred rat strain for age-related studies as well as for studies in toxicology and cancer research. The advantage of inbreeding is small inter-subject variability due to the genetic homogeneity of the population; the disadvantage of inbred animals is the accumulation of recessive traits. LE rats are outbred, colored rats (hooded), introduced in 1915 by Drs. Long and Evans as a cross of several Wistar white females with a wild grey male. LE rats are used for neurological, toxicological and behavioral studies (including auditory learning tasks). Aged LE rats exhibit a small hearing loss [3,12], reduced temporal processing capabilities [12,38,50] and a tendency to increased values of frequency difference limens and gap detection thresholds [50]. J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 1.2. Hearing threshold The auditory sensitivity of different rat strains has been previously determined by behavioral techniques [18,26,63], auditory brainstem responses (ABRs) [9,44–47] and middle latency responses recorded from the auditory cortex [64]. However, due to the poor sensitivity of F344 rats at low frequencies, the hearing threshold in this rat strain is usually evaluated in the frequency range starting from 3–4 to 32 kHz. 491 relatively small number of missing hair cells, ganglion cell loss and strial degeneration [10,27–29,65]. A significantly larger hair cell loss was observed in the C57BL/6 mouse strain [23,60]. The aim of the present study was to monitor age-related changes in hearing function in LE and F344 rats by determining hearing thresholds, recording of otoacoustic emissions and middle-ear admittance, and correlating these physiological values with the morphological status of the cochlea by quantifying hair cell loss. 1.3. Auditory brainstem evoked responses It has been shown previously that the level of the inhibitory neurotransmitters glycine and ␥-aminobutyric acid (GABA) may decrease with age in central auditory structures [6,21,40], altering the relative balance of excitation and inhibition in aged animals. Such neurochemical changes can be reflected in the ABRs recorded from aged animals as a prolongation of wave latencies and shifts in the ABR amplitudeintensity function [2,56]. 2. Materials and methods The acoustical measurements were made in a sound proof and anechoic room. The walls and ceiling inside the room were covered by cones from phono-absorbent material; the attenuation was 55 dB at 250 Hz and 60–70 dB for frequencies above 500 Hz. 2.1. Animals 1.4. Otoacoustic emissions The cochlea not only receives sound, but also produces acoustic energy in the form of low-level sounds that are reemited from the cochlea through the middle-ear system to the outer-ear canal, where they can be measured by a sensitive microphone as otoacoustic emissions [30]. Among individual types of otoacoustic emissions, distortion product otoacoustic emissions (DPOAEs) have been reported to be easily recorded in the rat [31]. Otoacoustic emissions are susceptible to both temporary and permanent damage to the organ of Corti induced by noise trauma, ototoxic agents or aging, thereby making them a sensitive indicator of hair cell pathology [13,17,35,48,57]. Otoacoustic emissions are also very sensitive to any type of conductive losses caused by changes in the middle-ear components’ motility. Otoacoustic emissions have been recorded in different strains of rat, including adult F344 rats [46]. However, to date, no evaluation of agerelated changes of otoacoustic emissions in F344 rats have been reported in the literature. 1.5. Tympanometry Tympanometry is a simple non-invasive test to measure the admittance characteristics and to detect various pathologies of the outer- and middle-ear. Tympanometry belongs to a basic battery of audiological tests used in clinical practice. However, to our knowledge, there is only one report measuring the acoustic impedance properties of the middle-ear in Sabra rats and their changes after body cooling [14]. 1.6. Cochlear pathology Previous studies in old Sprague–Dawley, F344 and a cross of Brown-Norway and F344 (BNF) rats have documented a Recordings of auditory brainstem responses and otoacoustic emissions were performed in 23 male rats of the inbred albino strain Fischer 344 and in 25 Long Evans rats (21 males, 4 females). F344 rats were tested repeatedly several times during their lifespan from the age of 1 or 3 month to 21 months. Testing of hearing function in LE rats was performed in several groups of animals ranging from 1 to 30 months of age. The F344 rats were obtained from Charles River Deutschland (Charles River Wiga GmbH, Sulzfeld, Germany); the LE rats were bred in a local facility. 2.2. ABR recording The rats were anesthetized with an intramuscular injection of 38 mg/kg body weight of ketamine (Calypsol, Gedeon Richter Ltd.) and 5 mg/kg body weight of xylazine (Sedazine, Fort Dodge) and placed on a heating pad that automatically maintained body temperature at 38 ◦ C. During testing the pinnae and the outer-ear cannals were allowed to be free, head movements were restricted by a sliding ring placed over the nose and by fixing the teeth in a headholder. The ABRs were recorded, under anesthesia, by three stainless-steel needle electrodes, placed subdermally over the vertex (positive) and the right and left mastoids (negative and ground electrodes) of the animal. The signal from the electrodes was amplified by a WPI DAM 60 differential amplifier (filters 300 Hz–3 kHz, gain 80 dB) and processed with a TDT data acquisition system (Tucker-Davis Technologies, Gainesville, FL; 16-bit A/D converter, sampling rate 50 kHz) using BioSig software. Acoustic stimuli for the ABR recordings were generated by a PC-based TDT system and presented in free-field conditions via two loudspeakers (Tesla ARN 5614 woofer and Motorola KSN-1005 tweeter) placed 70 cm in front of the 492 J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 animal’s head. To measure the ABR input–output function, the animal was stimulated with clicks (alternating monopolar electrical pulses 100 s) with a repetition rate of 11 Hz and decreasing intensity in 5 dB steps. For assessing hearing thresholds (ABR audiograms), clicks and tone bursts (burst duration 5 ms, 2 ms rise/fall times) in one-octave steps, ranging from 1 to 3 kHz were used. The hearing threshold at each frequency was determined visually by reducing the stimulus intensity from the suprathreshold level in 5 dB steps to obtain a just-detectable response. As a rule, responses to 500 acoustical stimuli were averaged. Older rats in which otoacoustic emission testing (see later) showed bilateral ear response differences, the ABR audiograms were measured separately in individual each ear. Sound stimuli were presented under free-field conditions, but the ABRs were evoked by the stimulation of the tested ear because the opposite ear was masked by broad-band noise (intensity 40–50 dB SPL) presented to the outer-ear canal by a piezoelectric stimulator. 2.3. DPOAE recording Animals were tested with an ILO 96 otoacoustic emission analyser (Otodynamics Ltd.) with H-probe for neonate ears using a plastic adapter to fit the probe into the external ear canal. Cubic 2f1 –f2 DPOAEs were recorded using two primary tones, f1 and f2 (ratio f2 /f1 = 1.22), presented with f1 and f2 primary tone levels of L1 /L2 = 60/50 dB SPL. DP-grams (the function of DPOAE level on increasing stimulus frequency) were recorded with a resolution of four points per octave over the frequency range 1–6.3 kHz. DPOAEs were measured in each ear of an animal. 2.4. Tympanometry A handheld impedance audiometer (MT 10, Interacoustics, Denmark) was used to measure the middle-ear compliance, outer-ear canal volume, the gradient of the tympanometric curve and middle-ear pressure. Probe-tone frequency was 226 Hz, amplitude 85 dB SPL, tympanometry range +200 to −300 daPa, pump speed 250–350 daPa/s. 2.5. Morphological analysis of the cochlea At the end of the experiment the rats were sacrificed by an overdose of narcotic (pentobarbital, Pentobarbital Spofa, 200 mg/kg i.p.), and the cochleae were removed from the temporal bone and processed for histological examination. The oval window was perforated and the cochlea gently perfused through the round window with fixative (10% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4), then placed in the fixative for 1–2 h before rinsing with PBS. The cochlea was then exposed to 0.3% Triton X-100 for 10 min, rinsed in PBS, and incubated in fluorescently labeled Phalloidin (tetramethylrhodamine isothiocyanate conjugate (TRITC)) (1:100) (Molecular Probes, USA) for 45 min and rinsed several times in PBS. The organ of Corti was dissected into 1/2–3/4 coils and placed on a microscope slide in Citi-flour, covered with a cover slip and sealed with non-fluorescent nail polish. TRITC-conjugated phalloidin intensely labels filamentous actin in the stereocilia, the cuticular plate, and the adherens junctions that connect adjacent sensory and supporting cells, and thus phalangeal scar formations are easily identifiable. Inner and outer hair cells were examined using a Zeiss Axiovert microscope equipped with an oil immersion 40× objective. The number of existing and missing hair cells in each 0.25 mm section was recorded and plotted on a cochleogram (the cochleogram shows the percentage of hair cell loss for each millimeter region along the length of the basilar membrane). 2.6. Experimental protocol For the otoacoustic emission testing, each rat was anesthetized, put on a heating pad and its head was fixed in the headholder. The outer- and middle-ear were otoscopically checked for the presence of cerumen, fluids or infections. The ILO probe was inserted into the outer-ear canal and the DPOAEs were measured. Then the ILO probe was fixed in the opposite ear and the same procedure was repeated. Following DPOAE testing, the ABRs to click and tone stimulation in free-field conditions were recorded. At the end of the entire test session, tympanometry was performed in either ear. The anesthesia was usually effective for 1 h, which was sufficient to perform all recordings. 2.7. Statistical evaluation of data The pairwise differences in hearing thresholds between age groups at each frequency were tested by a two-tailed ttest with the significance level of individual tests adjusted according to Bonferoni’s correction. An unpaired t-test was used to test the differences between ABR audiograms and DPOAE levels at individual frequency bands between F344 and Long Evans rat strains. The relationship between tympanometric parameters, ABR thresholds and DPOAE values were tested by a correlation test. Statistical evaluation of data were performed using the GraphPad Prism 4 software. The care and use of animals reported on in this study were approved by the Ethics Committee of the Institute of Experimental Medicine and followed the guidelines of the Declaration of Helsinki. 3. Results The hearing function of each F344 rat was monitored several times during its lifespan from a young age (1 or 3 months old) to old age (21 months old). The F344 rats were sacrificed and processed for cochlear histological examination at the age of 22 months. The hearing function of LE rats was J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 493 Fig. 1. Average hearing thresholds (ABR audiograms) and threshold shifts in young and old LE (A and C) and F344 (B and D) rats. The average ABR audiogram of 1-month-old LE rats is displayd in grey color in panel B for comparison. Bars represent ± S.E.M. measured in individual groups of animals aged 1, 6, 12, 24 and 30 months. 3.1. ABR audiograms The average hearing thresholds (ABR audiograms) and threshold shifts in LE and F344 rats as a function of age are shown in Fig. 1. Young, 1-month-old LE rats (LE 1 m, panel A) had the best hearing between 8 and 16 kHz; at lower and higher frequencies the ABR threshold increased. The small S.E.M. values at individual frequencies reflect the small interindividual variation in LE rats. Average ABR audiograms of 24- and 30-month-old LE rats were shifted to higher intensities. Age-related threshold shifts (panel C) were very small at low frequencies (less than 10 dB at frequencies 1–4 kHz) and larger at higher frequencies (a maximum value of 19 dB at 16 kHz in 30-month-old LE rats). The average ABR audiogram of 1-month-old F344 rats (F 1 m, Fig. 1B) was approximately 20 dB higher at low frequencies (1–2 kHz, p < 0.001) and 5–10 dB higher at middle and high frequencies between 4 and 32 kHz (p < 0.05) compared with the average audiogram of young LE rats. Hearing thresholds for adult (12-month-old) F344 rats increased significantly by 5–7 dB at high frequencies (16 and 32 kHz, p < 0.001). A pronounced, fast deterioration in hearing thresholds, across the whole frequency range, ocurred in F344 rats older than 12 months (18 and 20 months old). The average TS at individual frequencies in individual age categories are shown in panel D of Fig. 1. The TS in 12-monthold F344 rats varied from ±3 dB at the lower frequencies to almost 10 dB between 16 and 32 kHz, thus resembling TS values in 2-year-old LE rats. However, the average TS in the oldest, 20-month-old F344 rats ranged from 20 to 23 dB at the low frequencies to 38 dB at frequencies between 16 and 32 kHz. Data presented in Fig. 1B and D were obtained using freefield acoustical stimulation, which means that they represent the threshold values of the more sensitive ear. However, in half of the F344 rats older than 12 months, testing of individual ears (i.e. free-field acoustical stimulation, but one ear was masked by wide-band noise) demonstrated significantly bilateral differences in thresholds. Bilateral differences often reached 10–20 dB at several frequencies. Thus, the average TS evaluated in individual ears can be even higher than the presented average TS during binaural stimulation. 3.2. Click-evoked ABRs ABR parameters as a function of absolute click intensity (dB pSPL, left column) or click intensity above hearing threshold (dB sensation level, dB SL, right column) and age are displayed in Fig. 2. The shape of click-evoked ABRs was almost uniform in both F344 and LE rats (Fig. 2A, inserted schema). ABRs consisted of five vertex-positive peaks, labeled I–V. As a rule, the latency of peak I exponentially decreased with increasing stimulus intensity (the ABR latency was reduced by 2 ms as the correction for the acoustic travel time from the loudspeakers to the ear). The average peak I latencyintensity curves (Fig. 2A) were almost identical in F344 (open symbols) and LE (filled symbols) rats of all age categories. In the oldest animals, 20-month-old F344 rats and 30-month- 494 J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 Fig. 2. Age-related changes of the parameters of ABRs in LE and F344 rats as a function of absolute click intensity in dB pSPL (A, C and E) and intensity in dB above threshold (dB sensation level, dB SL; B, D and F). Bars represent ± S.E.M. Inserted schemas in panels A and E show the ABR shape and the principle of the ABR amplitude evaluation. old LE rats, the average peak I latencies tended to be longer than latencies in younger animals, but the differences were not significant. However, when displaying peak I latencies at intensities relative to hearing threshold in dB SL (Fig. 2B), the average peak I latency-intensity curve in 20-month-old F344 rats decreased more rapidly than those in other animals. At higher suprathreshold intensities, the peak I latencies of 20-month-old F344 rats were significantly shorter than these measured in younger F344 rats or in LE rats of any age category (p < 0.001). Waveform interpeak latencies (the total duration of the response measured between peak I and peak IV, Fig. 2C and D) were significantly longer in the youngest (1-month-old) LE and F344 rats than in older animals (p < 0.01). In 30-monthold LE and 20-month-old F344 rats the average interpeak latencies again increased, but this interpeak latency shift did not reach statistical significance (p < 0.05) at all intensity values. However, the time course of these changes was faster in F344 rats than in LE rats. The interpeak latencies measured in 12-month-old F344 rats were comparable with those obtained in 24-month-old LE rats, and the interpeak latencies obtained in 20-month-old F344 rats were comparable with those obtained in 30-month-old LE rats. Pronounced age-related changes in ABR amplitudes were observed in F344 rats (Fig. 2E and F). ABR amplitudes were measured as the difference between the value of the positive peak II and the negative through between the waves III and IV (see inserted schema in Fig. 2E). Whereas the average ABR amplitudes in all age categories of LE rats were almost identical, in F344 rats the maximal ABR amplitudes decreased continuously during their life (Fig. 2F). The average maximal ABR amplitudes obtained in 20-month-old F344 reached less than one-half of the values measured in 1-month-old F344 rats. 3.3. Distortion product otoacoustic emissions In all animals, the DPOAE response amplitudes increased with increasing stimulus frequency, and the shape of the DPgram (the function of DPOAE response amplitude on stimulus frequency) was uniform and typical for the individual rat strains. The average DP-grams of F344 and LE rats of different ages are demonstrated in Fig. 3. The average DP-grams of LE rats (dashed lines) exceeded the background noise level at frequencies above 1–1.5 kHz and reached a plateau (maximal amplitudes of 20–30 dB SPL) usually at a frequency of J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 495 Fig. 3. The average DP-grams of F344 and LE rats of different ages. Grey area represents DPOAE background noise. Bars represent ± S.E.M. 5.6 kHz. The DPOAE response amplitudes in LE rats did not change during aging, and the averaged DP-grams obtained in 3-month-old and 30-month-old LE rats were almost identical. In contrast to LE rats, the DPOAE amplitudes decreased with aging in F344 rats (solid lines). In 3-month-old F344 rats, no DPOAEs were measurable at frequencies below 2–2.5 kHz. At higher frequencies the DPOAE amplitudes increased and reached a plateau of 22–29 dB SPL at similar frequencies as did DPOAEs in LE rats. DPOAE amplitudes in 6-month-old F344 rats slightly decreased and resulted in a frequency shift of the average DP-gram to higher frequencies. The testing of F344 rats at the age of 12 months revealed large differences in DPOAE amplitudes in individual ears. In half of the 12month-old F344 rats (9 of 18), the DPOAEs were recordable in only one ear, whereas in the opposite ear the DPOAEs were totally absent. In the remaining nine F344 rats, the DPOAEs were either absent in both ears (three animals) or present symmetrically in both ears (six animals). At the age of 18 months small DPOAEs were measurable in only six ears and the average DP-gram did not exceed the background noise. To demonstrate the time course of DPOAE changes in individual ears, the maximal DPOAE amplitudes measured at a frequency of 5.6 kHz in 18 ears of 9 F344 rats are displayed as a function of age in Fig. 4. In 3-month-old, F344 rats the maximal DPOAE amplitudes reached a very uniform value in all ears, ranging between 22 and 29 dB SPL. In older animals the DPOAE amplitudes started to decrease almost linearly with different slopes in individual ears. The faster Fig. 5. The average cytocochleograms of 30-month-old LE rats (n = 4) and 22-month-old F344 rats (n = 12) showing the percent hair cell loss. the decrease at a young age, the sooner the disappearance of the DPOAEs in the older animal. In six ears of the evaluated sample, DPOAEs were not measurable at the age of 12 months, in six ears the DPOAEs disappeared at the age of 18 months, in five ears the DPOAEs disappeared at the age of 20 months and in one ear the relatively high DPOAE amplitudes were still measurable at 22 months of age. 3.4. Hair cell loss The middle-ear cavity of all but two ears were processed for histologic evaluation. Two ears in which the middle-ear was filled with secret resulting from otitis media infection were excluded from the physiological and histological evaluations. The hair cell loss in the organ of Corti was determined in 12 ears of 22-month-old F344 rats and in 4 ears of 30month-old LE rats. The data are displayed in Fig. 5 as average cytocochleograms. Individual curves in these diagrams represent the percentage of missing inner hair cells (IHC) and outer hair cells (OHC) in the organ of Corti in each strain. In the 22-month-old F344 rats (open symbols), the pronounced OHC loss was observed mainly at the apical and basal ends of the cochlea (50–60%), whereas only a few OHCs were missing from the middle part of the cochlea (less than 10%). The number of missing IHCs was very low and did not exceed 10%. In 30-month-old LE rats (filled symbols) the pattern of OHC and IHC loss was similar, but the number of missing IHCs and OHCs at the basal end of the organ of Corti was larger in LE rats than in F344 rats. These results document a similar pattern of the age-related degeneration in the organ of Corti between the old LE and F344 rats. 3.5. Tympanometry Fig. 4. The time course of DPOAE maximum amplitudes in individual ears of F344 rats. To check the parameters of middle-ear admittance, tympanometric recordings in both ears in several age groups of F344 and LE rats. The average parameters of the tympanometric curves, characterized by the compliance (maximal value of the tympanometric curve), gradient (the slope of the tym- 496 J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 Fig. 6. The average middle-ear compliance, gradient of the tympanometric curve and middle-ear pressure measured in 6-month and 15-month-old LE and F344 rats. Bars represent ± S.E.M. LE rats. In general, many parameters of hearing function, such as ABR thresholds, some parameters of click-evoked ABRs and the number of missing hair cells, changed during aging almost twice as fast in F344 rats than in LE rats. In addition, DPOAEs were completely lacking in almost all F344 rats during their first 12 or 18 months of life, whereas DPOAEs in LE rats did not change until the age of 30 months. The decreases in DPOAE amplitudes proceeded at different rates in individual ears in half of the F344 rats. However, the different patterns of DPOAE changes observed in F344 and LE rats during aging did not correlate with the number of missing hair cells in the two strains of rats. The discrepancies between the functional and structural parameters of hearing function indicate that there are complex mechanisms underlying the age-related hearing loss in F344 rats. 4.1. Age-related changes in ABR audiograms Fig. 7. The function of the compliance values and maximal DPOAE amplitudes in individual ears of LE and F344 rats. panometric curve) and middle-ear pressure (resulting in the shift of the tympanometric curve to positive or negative pressure), are displayed in Fig. 6. Whereas the individual values of tympanometric recordings in young, 6-month-old rats of both strains, were almost identical, the values of middle-ear compliance, gradient of the tympanometric curve and middle-ear pressure, measured in 15-month-old F344 rats, were significantly reduced in comparison with LE rats of the same age (p < 0.0001, unpaired t-test). The close relationship between the parameters of middleear admittance and maximal DPOAE amplitude (usually measured at 5.6 kHz) in individual ears is demonstrated in Fig. 7. Whereas compliance values and maximal DPOAE amplitudes were relatively unchanged in 15-month-old LE rats in comparison with young animals, in F344 rats both compliance and maximal DPOAE amplitudes in individual ears decreased with age. The correlation between middle-ear admittance and maximal DPOAE amplitude is highly significant (p < 0.0001, Pearson correlation coefficients “r” for individual tympanometric parameters ranged between 0.63 and 0.77). Individual tympanometric parameters also correlate with hearing thresholds at all frequencies (p < 0.0001, r = 0.65–0.9). 4. Discussion The results document a faster deterioration of hearing function during the lifespan of F344 rats in comparison with ABR audiograms in LE rats demonstrated only small agerelated hearing threshold changes, ocurring mainly at high frequencies at the end of their life. A relatively small hearing loss with a more pronounced threshold shift at high frequencies was demonstrated in old LE rats by several authors [3,50]. In contrast to the findings in LE rats, the age-related threshold changes in F344 rats were more complex. Young F344 rats exhibited a significantly elevated hearing threshold with a larger threshold shift at low frequencies in comparison with LE rats [47]. During the 20 months of life, the hearing threshold in F344 rats significantly increased at all frequencies. The resulting TS in 12-month-old F344 rats was comparable with that obtained in LE rats at the age of 24 months and the TS in 20-month-old F344 rats were significantly larger than those measured in much older, 30-month-old LE rats. A similar pronounced, progressive age-related decline in hearing sensitivity in F344 rats was recently demonstrated by several authors [2,54–56,65], who suggested that the marked agerelated threshold changes during the relatively short lifespan of the F344 rat offers a suitable animal model for investigating the mechanisms underlying age-related hearing loss. However, the results of the present study demonstrate the complexity of pathological signs in F344 rat strain, and thus do not support this rat strain as a suitable model for the human presbycusis. 4.2. Click-evoked ABR The shape and features of the click-evoked ABRs measured in this study in young F344 and LE rats were very similar and correspond with those obtained in other strains of rat [2,7,44]. Small differencies were, however, found in the age-related changes of click-evoked ABR parameters between LE and F344 rats. The average peak I latency-intensity curve in 20-month-old F344 rats, displaying at intensities relative to hearing threshold (dB SL), decreased more rapidly than that measured in younger animals or LE rats of any age. J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 Reduced wave latencies are commonly reported in elderly subjects and are largely attributable to changes in peripheral auditory structures [8,16,58,68]. The course of age-related changes in interpeak latency (i.e. the central conduction time) was similar in both rat strains. The tendency towards the prolongation of the interpeak latencies in old LE and F344 rats comparison with younger animals can result from age-related neuronal degeneration in the cochlea and brainstem [5,28]. In F344 rats, interpeak latency prolongations were observed at 20 months of age, whereas in LE rats the interpeak latencies were prolonged no earlier than at 30 months. Whereas the ABR amplitudes did not change significantly during the lifespan of LE rats, in F344 rats the ABR amplitudes decreased with age. The ABR voltage changes in both rat strains were different despite their almost equal hair cell loss. Thus, there may be additional age-related changes in the auditory system such as, for example, neurochemical changes demonstrated in the brainstem of F344 rats [40,41,65]. 4.3. Hair cell loss In 24-month-old Sprague–Dawley rats, Crowley et al. [10] found a small hair cell loss (5% of OHC lost, 1% of IHC) in the organ of Corti. Keithley and Feldman [27] demonstrated in 31–33-month-old Sprague–Dawley rats IHC losses ranging from 1.6 to 4.2% and OHC losses ranging from 2.1 to 23.3%. A significantly larger hair cell loss was observed in the C57BL/6 mouse strain. By 26 months of age, nearly all OHCs were missing throughout the cochlea, whereas IHC losses gradually tapered off from 100% near the base to around 20% near the apex [23,60]. Aged F344 rats (24 months old) displayed a small IHC loss (<10%) throughout the cochlea and a U-shaped loss of OHC, with the greatest losses (as high as 70%) confined to the apical and basal turns [65]. A similar U-shaped OHC loss has been observed in old F344 and LE rats in the present study. The number of hair cells lost in 22-month-old F344 rats corresponded to that seen in 30-month-old LE rats. 4.4. DPOAE The time course of the changes in DPOAE amplitudes with aging represents the most distinct difference between LE and F344 rats. In LE rats the DPOAE amplitudes did not change in any age group. This corresponds with the relatively low number of missing hair cells in the organ of Corti and the small TSs in the oldest, 30-month-old LE rats. In contrast to LE rats, in F344 rats the DPOAE amplitudes gradually decreased during the first months of their life, and between 12 and 18 months DPOAEs disappeared in almost all the ears of F344 rats. These finding contrasts with the fact that the TS values in 12-month-old F344 rats were almost identical with those obtained in 30-month-old LE rats with normal DPOAEs. Also, the number of missing OHCs (as a main generator of otoacoustic emissions) did not exceed that 497 seen in LE rats. In addition, in half of 12-month-old F344 rats, the DPOAEs were asymmetrical, i.e. DPOAEs were present in one ear while DPOAEs in the opposite ear were not measurable. The findings describing the age-related changes in DPOAE amplitudes in F344 rats have not previously been reported. 4.5. The possible mechanisms underlying age-related hearing loss The normal functioning of the cochlea depends on maintaining the physiological status of the ionic composition of the cochlear endolymph. The atrophy of the tissues of the cochlear lateral wall (stria vascularis and spiral ligament), those structures responsible for maintaining cochlear homeostasis, was shown previously in old animals [29,53,22]. Several authors demonstrated in old mice and gerbils a widespread degeneration of fibrocytes in the spiral ligament that preceded the loss of hair cells and/or neurons, suggesting that fibrocyte pathology may be a primary cause of hearing loss and ultimate sensory cell degeneration [20,33,43,53,59]. Similar histological analysis has yet to be performed in old F344 rats. The generation of endocochlear potential (EP) and signal transduction processes have been shown to be differentially affected by aging in different animals. Previous studies in the gerbil have shown that both the EP and the activity of Na,KATPase in the lateral wall decreases with age [15,52,53,61], whereas old C57BL/6J or CD-1 mice displayed a significantly reduced [K+]e (up to 30%) in cochlear turns and a pronounced threshold increase with no significant changes in normoxic EP [32,43,67]. Lautermann et al. [34] and Ogawa and Schacht [42] demonstrated in 24-month-old F344 rats that glutathione levels (as a part of the antioxidant system) and the inositol phosphate second messenger system remained unchanged in the cochlear lateral wall. Thus, some evidence exists that, despite the morphological changes, the functioning of the stria vascularis in some strains of mice and rats does not significantly change with aging. The DPOAEs measured in F344 rats in the present study were totally absent in half of the adult animals at the age of 12 months, in which the TS amounted to less than 20 dB. The same TS value in 30-month-old LE rats did not cause any DPOAE amplitude reduction. The reason for the disappearance of DPOAEs in F344 rats can be either altered OHC function (for example, by decreasing EP) or other, extracochlear mechanisms. 4.6. The role of the middle-ear in signal transmission The middle-ear may be an important, but so far unrecognized, factor contributing to hearing loss in the elderly. Several diseases are thought to influence the structure and function of the middle-ear, including otosclerosis (otospongiosis). Otosclerosis is a disease of unknown origin, progressive in nature (in many cases only monaural), that affects the 498 J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 middle-ear compartments and results in the appearence of otospongiotic foci on the labyrinthine capsule and fixation of the stapes. Microscopic observation of the middle-ear in incissor absent rat (an animal model of otosclerosis, [25]) revealed bony abnormalities of the ossicles and oval window and bony lesions in the incudostapedial joint. In the present study, macroscopic inspection of the bony cochleae revealed no evident signs of otospongiotic foci on the cochlea or any malformations of the incudostapedial joint in the old F344 rats. However, many of the metabolic mutations documented in F344 rats can result in another form of middle-ear dysfunction revealed by tympanometric testing. All parameters of the tympanometric test (the compliance, the gradient of the tympanometric curve and the middle-ear pressure) were found to be almost identical in young LE and F344 rats, but significantly changed in 15-month-old F344 rats in comparison with LE rats of the same age. Reduced values of compliance and the gradient of the tympanometric curve demonstrated increased impedance of the middle-ear compartments. A large negative pressure in the middle-ear capsule, often caused by altered function of the eustachian tube, limits the motility of the tympanic membrane and other middle-ear compartments [1,36]. Doan et al. [11] and Cannuscio et al. [4] demonstrated that deterioration in the structure of the tympanic membrane of aged mice or Brown-Norway rats resulted in deterioration in the tympanic membrane velocity transfer function. These authors speculated that this defect could be due to an altered collagen content of the tympanic membrane, which influences the ability of the tympanic membrane to replicate an acoustic signal. The collagen damage during aging was reported by Seidman et al. [55]. It may be that a collagen defect plays a crucial role in hearing loss in F344 rats, causing stria vascularis malfunction or altering middle-ear structures. All these changes can result in the substantial reduction of DPOAE amplitude and manifest as hearing loss. In summary, the aged LE rats exhibit a moderately sloping ABR audiogram, a small hair cell loss confined to the apical and basal ends of the organ of Corti, no changes in DPOAE amplitudes or tympanometric parameters and almost no changes in click-evoked ABRs. Due to the rather small changes observed in the hearing function of LE rats during their life, this rat strain does not seem to be suitable animal model for the study of the mechanisms underlying age-related hearing loss in man. The age-related changes of hearing function in F344 rats are more pronounced, start to appear early after birth and accelerate at the end of their lifespan. However, discrepancies between individual parameters point on a complex hearing deterioration in F344 rats. Low-frequency hearing loss, declining sound transmission through the middle-ear, asymmetric DPOAE decrease represent a conductive type of hearing loss whereas decrease of the ABR amplitudes and high-frequency hearing loss reflect a sensorineural type of hearing loss. These results should be taken into account when using F344 rats as an animal model for investigating the cause, prevention and treatement of presbycusis. Acknowledgements Supported by grants AVOZ50390512, no. 309/04/1074 from the Grant Agency of the Czech Republic and no. NR/8113-4 from the Internal Grant Agency of the Czech Ministry of Health. The authors would like to acknowledge the valuable technical assistance of Agneta Viberg. References [1] Avan P, Buki B, Maat B, Dordain M, Wit HP. Middle ear influence on otoacoustic emissions. I. Noninvasive investigation of the human transmission apparatus and comparison with model results. Hear Res 2000;140:189–201. [2] Backoff PM, Caspary DM. Age-related changes in auditory brainstem responses in Fischer 344 rats: effects of rate and intensity. Hear Res 1994;73:163–72. [3] Campo P, Pouyatos B, Lataye R, Morel G. Is the aged rat ear more susceptible to noise or styrene damage than the young ear? Noise Health 2003;5:1–18. [4] Cannuscio JF, Saunders JC, Gratton MA. Middle ear transfer function: alterations in aged rats. Proceedings of the 27th Midwinter Meeting. Assoc Res Otolaryngol 2004:1181. [5] Casey MA, Feldman ML. Age-related loss of synaptic terminals in the rat medial nucleus of the trapezoid body. Neuroscience 1988;24(1):189–94. [6] Caspary DM, Raza A, Lawhorn Armour BA, Pippin J, Arneric SP. Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: Implications for neural presbycusis. J Neurosci 1990;10(7):2363–72. [7] Chen TJ, Chen SS. Brain stem auditory-evoked potentials in different strains of rodents. Acta Physiol Scand 1990;138:529–38. [8] Chu NS. Age-related latency changes in the brain-stem auditory evoked potentials. Electroencephalogr Clin Neurophysiol 1985;62(6):431–6. [9] Cooper Jr WA, Coleman JR, Newton EH. Auditory brainstem responses to tonal stimuli in young and aging rats. Hear Res 1990;43:171–9. [10] Crowley DE, Schramm VL, Swain RE, Swanson SN. Analysis of age-related changes in electric responses from the inner ear of rats. Ann Otol 1972;81:739–46. [11] Doan DE, Erulkar JS, Saunders JC. Functional changes in the aging mouse middle ear. Hear Res 1996;97(1–2):174–7. [12] Finlayson PG. Paired-tone stimuli reveal reductions and alterations in temporal processing in inferior colliculus neurons of aged animals. J Assoc Res Otolaryngol 2002;3:321–31. [13] Franklin DJ, Lonsbury-Martin BL, Stagner BB, Martin GK. Altered susceptibility of 2f1 –f2 acoustic-distortion products to the effects of repeated noise exposure in rabbits. Hear Res 1991;53(2):185–208. [14] Geal-Dor M, Khvoles R, Sohmer H. Cooling induces a decrease in middle ear compliance. J Basic Clin Physiol Pharmacol 1997;8(3):127–32. [15] Gratton MA, Schmiedt RA, Schulte BA. Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis. Hear Res 1996;102(1–2):181–90. [16] Harkins SW. Effects of age and interstimulus interval on the brainstem auditory evoked potential. Int J Neurosci 1981;15:107–18. [17] Hatzopoulos S, Petruccelli J, Laurell G, Avan P, Finesso M, Martini A. Ototoxic effects of cisplatin in a Sprague–Dawley rat animal model as revealed by ABR and transiently evoked otoacoustic emission measurements. Hear Res 2002;170:70–82. [18] Heffner HE, Heffner RS, Contos C, Ott T. Audiogram of the hooded Norway rat. Hear Res 1994;73:244–7. J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 [19] Henry KR. Aging and audition. In: Willott JF, editor. Auditory psychobiology of the mouse. Springfield, IL: Thomas; 1983. p. 470– 93. [20] Hequembourg S, Liberman MC. Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice. J Assoc Res Otolaryngol 2001;2:118–29. [21] Hunter C, Chung E, Van Woert MH. Age-dependent changes in brain glycine concentration and strychnine-induced seizures in the rat. Brain Res 1989;482(2):247–51. [22] Ichimiya I, Suzuki M, Mogi G. Age-related changes in the murine cochlear lateral wall. Hear Res 2000;139(1–2):116–22. [23] Idrizbegovic E, Bogdanovic N, Viberg A, Canlon B. Auditory peripheral influences on calcium binding protein immunoreactivity in the cochlear nucleus during aging in the C57BL/6J mouse. Hear Res 2003;179(1–2):33–42. [24] Idrizbegovic E, Bogdanovic N, Willott JF, Canlon B. Age-related increases in calcium-binding protein immunoreactivity in the cochlear nucleus of hearing impaired C57BL/6J mice. Neurobiol Aging 2004;25:1085–93. [25] Kaniff TE, Schneider G, Matz GJ. The incisor absent rat: an animal model for the study of otosclerosis. Otolaryngol Head Neck Surg 1990;103(3):406–12. [26] Kelly JB, Masterton B. Auditory sensitivity of the albino rat. J Comp Physiol Psychol 1977;91:930–6. [27] Keithley EM, Feldman ML. Hair cell counts in an age-graded series of rat cochleas. Hear Res 1982;28:249–62. [28] Keithley EM, Croskrey KL. Spiral ganglion cell endings in the cochlear nucleus of young and old rats. Hear Res 1990; 49(1–3):169–77. [29] Keithley EM, Ryan AF, Feldman ML. Cochlear degeneration in aged rats of four strains. Hear Res 1992;59:171–8. [30] Kemp DT. Stimulated acoustic emissions from within the human auditory system. J Acoust Soc Am 1978;64(5):1386–91. [31] Khvoles R, Freeman S, Sohmer H. Effect of temperature on the transient evoked and distortion product otoacoustic emissions in rats. Audiol Neurootol 1998;3:349–60. [32] Lang H, Schulte BA, Schmiedt RA. Endocochlear potentials and compound action potential recovery: functions in the C57BL/6J mouse. Hear Res 2002;172(1–2):118–26. [33] Lang H, Schulte BA, Schmiedt RA. Effects of chronic furosemide treatment and age on cell division in the adult gerbil inner ear. J Assoc Res Otolaryngol 2003;4(2):164–75. [34] Lautermann J, Crann SA, McLaren J, Schacht J. Glutathionedependent antioxidant systems in the mammalian inner ear: effects of aging, ototoxic drugs and noise. Hear Res 1997;114:75– 82. [35] Lund SP, Jepsen GB, Simonsen L. Effect of long-term, low-level noise exposure on hearing thresholds. DPOAE and suppression of DPOAE in rats. Noise Health 2001;3(12):33–42. [36] Magnan P, Dancer A, Probst R, Smurzynski J, Avan P. Intracochlear acoustic pressure measurements: transfer functions of the middle ear and cochlear mechanics. Audiol Neurootol 1999;4(3–4):123–8. [37] McFadden SL, Ding D, Salvi R. Anatomical, metabolic and genetic aspects of age-related hearing loss in mice. Audiology 2001;40(6):313–21. [38] Mendelson JR, Ricketts C. Age-related temporal processing speed deterioration in auditory cortex. Hear Res 2001;158(1–2):84–94. [39] Mikaelian DO. Development and degeneration of hearing in the C57/b16 mouse: relation of electrophysiologic responses from the round window and cochlear nucleus to cochlear anatomy and behavioral responses. Laryngoscope 1979;89(1):1–15. [40] Milbrandt JC, Albin RL, Caspary DM. Age-related decrease in GABAB receptor binding in the Fischer 344 rat inferior colliculus. Neurobiol Aging 1994;15(6):699–703. [41] Milbrandt JC, Caspary DM. Age-related reduction of [3 H]strychnine binding sites in the cochlear nucleus of the Fischer 344 rat. Neuroscience 1995;67(3):713–9. 499 [42] Ogawa K, Schacht J. Aging does not alter phosphoinositide hydrolysis in the rat cochlear lateral wall. Auris Nasus Larynx 1999; 26:1–4. [43] Ohlemiller KK. Reduction in sharpness of frequency tuning but not endocochlear potential in aging and noise-exposed BALB/cJ mice. J Assoc Res Otolaryngol 2002;3(4):444–56. [44] Overbeck GW, Church MW. Effects of tone burst frequency and intensity on the auditory brainstem response (ABR) from albino and pigmented rats. Hear Res 1992;59:129–37. [45] Palombi PS, Caspary DM. Physiology of the young adult Fischer 344 rat inferior colliculus: responses to contralateral monaural stimuli. Hear Res 1996;100:41–58. [46] Polak M, Eshraghi AA, Nehme O, Ahsan S, Guzman J, Delgado RE, et al. Evaluation of hearing and auditory nerve function by combining ABR. DPOAE and eABR tests into a single recording session. J Neurosci Methods 2004;134:141–9. [47] Popelar J, Groh D, Mazelová J, Syka J. Cochlear function in young and adult Fischer F344 rats. Hear Res 2003;186:75–84. [48] Pouyatos B, Campo P, Lataye R. Use of DPOAEs for assessing hearing loss caused by styrene in the rat. Hear Res 2002;165: 156–64. [49] Raza A, Milbrandt JC, Arneric SP, Caspary DM. Age-related changes in brainstem auditory neurotransmitters: measures of GABA and acetylcholine function. Hear Res 1994;77(1–2):221–30. [50] Rybalko N, Popelar J, Syka J. Age-related changes in the hearing function in pigmented rats. Proceedings of the 21st Midwinter Meeting. Assoc Res Otolaryngol 1998:78. [51] Rybalko N, Syka J. Susceptibility to noise exposure during postnatal development in rats. Hear Res 2001;155:32–40. [52] Schmiedt RA. Effects of aging on potassium homeostasis and the endocochlear potential in the gerbil cochlea. Hear Res 1996;102(1–2):125–32. [53] Schulte BA, Schmiedt RA. Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Hear Res 1992;61(1–2):35–46. [54] Seidman MD, Khan MJ, Bai U, Shirwany N, Quirk WS. Biologic activity of mitochondrial metabolites on aging and age-related hearing loss. Am J Otol 2000;21:161–7. [55] Seidman MD, Ahmad N, Bai U. Molecular mechanisms of agerelated hearing loss. Ageing Res Rev 2002;1:331–43. [56] Simpson GV, Knight RT, Brailowsky S, Prospero-Garcia O, Scabini D. Altered peripheral and brainstem auditory function in aged rats. Brain Res 1985;348:28–35. [57] Sockalingam R, Freeman S, Cherny TL, Sohmer H. Effect of highdose cisplatin on auditory brainstem responses and otoacoustic emissions in laboratory animals. Am J Otol 2000;21:521–7. [58] Soucek S, Michaels L. Hearing loss in the elderly: audiometric, electrophysiological and histopathological aspects. London: SpringerVerlag; 1990. [59] Spicer SS, Schulte BA. Spiral ligament pathology in quiet-aged gerbils. Hear Res 2002;172(1–2):172–85. [60] Spongr VP, Flood DG, Frisina RD, Salvi RJ. Quantitative measures of hair cell loss in CBA and C57BL/6 mice throughout their life spans. J Acoust Soc Am 1997;101(6):3546–53. [61] Suryadevara AC, Schulte BA, Schmiedt RA, Slepecky NB. Auditory nerve fibers in young and quiet-aged gerbils: morphometric correlations with endocochlear potential. Hear Res 2001;161(1/2):45–53. [62] Syka J. Plastic changes in the central auditory system after hearing loss, restoration of function, and during learning. Physiol Rev 2002;82:601–36. [63] Syka J, Rybalko N, Brozek G, Jilek M. Auditory frequency and intensity discrimination in pigmented rats. Hear Res 1996;100:107–13. [64] Syka J, Rybalko N. Threshold shifts and enhancement of cortical evoked responses after noise exposure in rats. Hear Res 2000;139:59–68. [65] Turner JG, Caspary DM. Comparison of two rat models of aging. Peripheral pathology and GABA changes in the inferior colliculus. 500 J. Popelar et al. / Neurobiology of Aging 27 (2006) 490–500 In: Syka J, Merzenich MM, editors. Plasticity and signal representation in the auditory system. New York: Kluwer Plenum; 2005. p. 217–25. [66] Willot JF. Aging and the auditory system: anatomy, physiology, and psychophysics. San Diego: Singular Publishing Group Inc.; 1991. [67] Wu T, Marcus DC. Age-related changes in cochlear endolymphatic potassium and potential in CD-1 and CBA/CaJ mice. J Assoc Res Otolaryngol 2003;4(3):353–62. [68] Yamada O, Kodera K, Yagi T. Cochlear processes affecting wave V latency of the auditory evoked brain stem response. A study of patients with sensory hearing loss. Scand Audiol 1979;8:67–70.