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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.
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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
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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-
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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-
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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
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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.