Hypothermia protects the cochlea from noise damage

Hypothermia protects the cochlea from noise damage

225 HRK 00549 Hypothermia protects Kenneth the cochlea from noise damage R. Henry ‘,* and Richard A. Chole ’ Thresholds of the cochlear action...

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HRK 00549




the cochlea from noise damage

R. Henry ‘,* and Richard

A. Chole ’

Thresholds of the cochlear action potential were obtained from rodents at euthermrc (38°C) and hypothermic (30” .ind 25°C) rectal temperaturea. In the gerbil. low and middle frequency (1-X kHz) thresholds increased an average of 2.3 dB per “C’ decrusc of hody temperature; at 16 kHz. 3.5 dB/OC: and at 32 kHz, an increase of 4.4 dB/“C. In the mouw. these vaIuc’\ wwc: 2 -16 ktj7. I.4 dB increase per “c‘ decrease; 32 kHz. 2.7 dB/“C; 64 kHz, 3.X dB/“C. When suhjectz. maximally susceptible to permanent threshold shift (PTS) at low and middle frequencies (anesthetlrcd, immature mice) were exposed to 115 dB noise. hvpothcrml;l reduced PTS at these most susceptible frequencies (2-16 kHr). When awake adult mice sue exposed tc) this noise. hvpnthermi;l protected them from PTS at their most vulnerable frequency (32 kHz). permanent


shift. hypothermia.

mouse. gerbil, whole nrrve action

Introduction Cooling the entire body or the cochlea produces temperature-dependent loss of a reversible, cochlear sensitivity [29.19,20]. In the few species tested with a wide range of tones, this decrement is greatest in response to high frequency stimuli [22,4,16.6]. Neural (auditory nerve or brain) responses are affected more than sensory (cochlear microphonic) functions [29,8,18]. This hypothermic effect may offer insights into the mechanisms of cochlear processing and noise-induced permanent threshold shift (PTS). Dresher [14,15] reported that lowered cochlear or body temperature reduces the ability of 90 dB noise to produce a temporary threshold shift (TTS) of the cochlear microphonic which was generated in response to a low frequency tone. This pioneer experiment did not evaluate the influence of hypothermia on PTS. Nor did it measure neural auditory responses, which are now believed to be a more accurate estimate of hearing than the CM [lo]. The use of a single tone frequency also precluded a determination of which frequencies are most protected. * Fro &horn reprint requests should he addressed. 0378.5955/84/$03.00

” 1984 Elsevier Science Puhhshera





PTS is frequency-specific, with losses typically being greatest at or above the center frequency of the exposure noise [12.23.26]. In the mouse. susceptibility to PTS shifts with age. Young animals have the greatest total loss. with a wide range 01 frequencies being affected. Older mice are onI> affected at higher frequencies [26]. In the mouse. barbiturate anesthesia will also increase the magnitude of the PTS [24]. Therefore. if hypothermia protects from PTS, its influence might be frequency-dependent. The present experiment uses both age and anesthesia to alter the type of PTS in order to more adequately determine the possible protective effect of hypothermia. Materials and Methods Seven Mongolian gerbils (42-79 days old) and 68 laboratory mice (22-425 days old) were used for these experiments. The mice ( Mu nzuscxdus) were of two genotypes (AUS/s and CBA), with 36 of them being sexually immature (22.~33 days). Hypothermia was produced by immersing the body, up to the neck, in 10°C water. When rectal temperatures dropped approximately S”C. they were periodically removed for 5-10 min. and reimmersed in order to reduce the rate of tempera-


ture decrease. Pilot studies, using microthermocoupies implanted within the cochlea. revealed that this resulted in stable inner ear temperatures which were accurately reflected by rectal measurements. In the first experiment (Figs. 1 and 2) the animals were anesthetized (60 mg/kg pentobarbital, i.p.) during the immersion. The 36 young mice of the second experiment (Fig. 3) were anesthetized during cooling and their subsequent exposure to noise (5 min. 8-16 kHz octave band, 115 dB SPL). For the 17 adult mice of the second experiment which were exposed to noise when hypothermic immersion occurred when awake (Fig. 4). Cochlear action potentials (AP) were obtained by a modified form of the electrocochleographic technique [26]. This utilized surface electrodes (scalp vertex and soft palate) temporarily applied to an anesthetized (pentobarbital) animal, while a single ear was stimulated by tone bursts at frequencies ranging from 2 to 64 kHz in the mouse and 1-32 kHz in the gerbil. The 1 ms duration (200 ~LSrise and fall time) tone bursts were delivered at a repetition rate of 20/s using a quasifree field technique to stimulate the ear. The driver was located 2.5 cm from the ipsilateral ear, directed toward the opening of the ear canal. The contralateral ear was occluded. A 0.63 cm calibrated Briiel & Kjaer microphone was positioned adjacent to the tragus for calibration of the driver output. The sine wave output was not phase-locked. so that cochlear microphonics rapidly cancelled out over the 512-1024 responses per averaged evoked potential. After a clear response was obtained at 65 dB SPL or higher, an output attenuator was adjusted in 10 dB steps until the response neared threshold, after which 5 dB increases and decreases were made. Threshold was extrapolated as being between the highest SPL at which it could not reliably be elicited. and the lowest SPL at which it was constantly seen. This value was generally less than 1 pV, although it varied somewhat as a function of the background noise level. The noise-exposed mice were tested 4 days after exposure, a time at which PTS has stabilized in the mouse [26]. Rectal temperatures were constantly monitored, and thresholds were only obtained after the desired temperature had been stable (50.2”C) for 10 min or longer. The effects of noise and hypothermia were

evaluated in terms of dB threshold change I’hi~ allowed a valid comparison between the gerbil and mouse, whose normal electrocochlear audiograms are quite different [26,27]. It also allowed the data of the two mouse strains to be combined because. over the age span of the second experiment (Figs. 3. 4) they respond to noise in a nearly identical fashion [25]. The statistical significance of the threshold changes was determined by Student’s t-tests subsequent to analysis of variance. The scanning electron microscope .(SEM) was used to assess the physical damage to the hair cells of the organ of Corti. After fixation with mixed aldehydes, dissected cochleas were processed by the ‘OTOTO’ method [35] by alternately immersing them in 1% osmium tetroxide and 0.5% thiocarbohydrazide. After critical point drying, the entire organ of Corti was photographed with an ISI-DSl30 SEM without sputter coating. Results Hypothermia produced a temperatureand frequency-dependent threshold elevation in both the gerbil and mouse. The age of the mice (22-420 days) did not appear to influence this effect, so Figs. 1 and 2 collapse this variable. The 30” gerbil

Figs. 1 and 2. Fig. 1 (top): Increase of the gerbil cochlear action potential (AP) threshold as a result of whole body hypothermia. The dotted line represents the threshold at euthermic (38°C) conditions. Fig. 2 (bottom): Increase of the mouse AP threshold during hypothermic condition (see legend to Fig. 1).

had an average increase of 19.2 dB, with this loss to 36.9 dB at 25°C (Fig. 1). The magnitude of this effect averaged 1.7 times greater at the highest (16 and 32 kHz) than at the tower f l-8 kHz) frequencies (all P < 0.001). These effects were very similar in the mouse (Fig. 2). At 30”. thresholds increased by an average of 11.6 dB: at 25°C. 2S.X dB. Higher frequencies (32 and 64 kHr) were elevated 2.6 times more than lower frequencies (ail P < 0.001). Hyp~~theri~ii~~ had a nearly identical effect on the four lowest frequencies for both species (i.e.. 1-X kHz thresholds declined by nearly the same amount at each temperature in the gerbit. as did 2-32 kHz thresholds in the mouse). But a different pattern was seen with the two higher frequencies, with the effect being 8 < I6 < 32 kHz in the gerbil. and 16 < 32 < 64 kHz in the mouse (P c 0.05 and 0.01. respectively). Hypothermia protected mice against permanent threshold shift (PTS). and this influence varied as increasing



4. Fig.










in kHz

3 (top):





harhital) Juvenile (22-33




mice which

maximal sensitivity


to low (pento-

days old) mice. (30°C)

mice which are setwtive



was achieved by anesthetizing

Fag. 4 (bc>ttom): Hypothermia adult


to noise trauma. This

and mid-frequency



to noise trauma



at the



region of the cochlea.

Fig. 5. Scanning electron micrographs of the mid-apical (A) and upper-basal (B) cochlear turns of an adult mou.\e expowd to n&r: under hypothermic (30°C) conditions. The apical turn appears normal. but scattered outer hair cell loss at the hasal turn. Hair ceil loss was seen in both the apical {C) and basal (D) turns of the euthermic (38°C) of the outer hair cells were missing

regions. becoming more frequent end. (Magnification X 1090.)

from C, and 90% are missing



noise exposed mouse. In this example. approximately Lightly

as the upper basal (32 kHr) region was approsched,


scattered inner hair cell loss was seen at apical

and bewming

les frequent

at tlx extreme


a function of frequency and the magnitude of PTS. The young (22-33 days old), anesthetized mice (Fig. 3) that were exposed at normal body temperature (38°C) had an average of 37.2 dB PTS (P < 0.001). This effect was greatest (49.8 dB. P < 0.005) at 8 and 16 kHz. The mice exposed at a rectal temperature of 30°C had an average PTS of 26 dB, with this effect being approximately the same at all frequencies. When comparisons were made with euthermic (38°C) exposed mice, hypothermia protected by an average of 16.9 dB at frequencies up to 16 kHz (P < 0.05), but had no significant influence at higher frequencies. PTS was less extreme in the adult (90- 120 days old) mice which were exposed to noise when awake (Fig. 4). The euthermic exposed mice had an average PTS of 16.5 dB, with the greatest elevation (46.9 dB) being at 32 kHz (P < 0.001). Hypothermia protected the adult mice from PTS (3.7 dB less PTS, average), and this effect was only significant at 32 kHz (P < 0.005). Scanning electron microscopy supports these electrophysiological findings (Fig. 5). In the euthermic exposed adult mice, there was little damage at the apex: approximately 5% loss of outer hair cells (OHCs) and negligible loss of inner hair cells (IHCs) of the organ of Corti. At the middle of Corti’s organ, the loss became progressively larger, with approximately 20% of the OHCs missing at a region which roughly corresponds to 16 kHz. At the basal region, approximately 95% of the OHCs and 10% of the IHCs were missing. These losses were all less for the hypothermic exposed mice. This was especially evident at the upper portion of the basal level of the organ of Corti. a region corresponding to 32 kHz. Discussion

As body temperature decreases in the laboratory mouse and Mongolian gerbil, AP thresholds increase, with this effect being greater at higher frequencies. This has also been observed in the auditory system of the little brown bat [22], feral Mus, Clethrionomys [4], Tokay gecko [16] and guinea pig [6]. But some studies report a more uniform effect, regardless of frequency [21,38]. In the laboratory mouse and gerbil, this frequencyspecific effect appears to occur only on the basal

portion of the organ of Corti: hypothermia has ;I nearly identical effect on low and middle frequencies, but the effect is progressive on the two highest frequencies. The increased sensitivity of the high frequency portion of the cochlea may help explain several apparent contradictions in the experimental literature. When inferior collicular evoked potentials were measured from the mouse, its maximally sensitive frequency was approximately 15 kHz; yet when the same experimenters opened the bulla and recorded from the round window, 5 kHz was maximally sensitive [40]. Another study noted that acute round window recording (as opposed to using chronically implanted electrodes) is associated with elevated high frequency thresholds [7]. These differences could have resulted from localized cochlear cooling which occurs with an open bulla [4,42,37]. It would be interesting to determine whether local or whole body hypothermia is involved in the relative reduction of high frequency sensitivity responsiveness which occurs with some anesthetics [41], hydrocortisone [l] and during certain sleep stages in humans [34]. Hypothermia protects certain portions of the mouse cochlea from noise-induced damage. Dresher [14,15] first reported the interaction of hypothermia and noise on the guinea pig cochlea. The 200 Hz CM response declined with constant 90 dB noise stimulation, and the rate of decrease was slowed when the ear was cooled. A permanent effect was later briefly described by Henry [24], with hypothermia protecting the click-evoked AP from noise. The present report is the first, to our knowledge, which examines the frequencies which are most protected, and how this changes with the degree of susceptibility PTS. Recent experimental findings may help explain some of these effects. Konishi et al. [33] cooled guinea pigs, obtaining evidence of suppression of the active Kf transport and decreases of K’ permeability of the barrier between endolymph and perilymph. Corey and Hudspeth [9] measured receptor current from the saccular hair cells of the bullfrog, observing a longer time course for ionic flow through the transduction channels in the hypothermal condition. This could explain the elevated thresholds at all frequencies. High frequency transduction. which requires a more


rapid change of hair cell permeability, might be more affected than lower frequencies. But this does not explain why thresholds decline the same for low and middle frequencies, and progressively increase at higher frequencies. Perhaps local factors along the basilar membrane (differences of blood flow; changes of fluid viscosity or membrane stiffness; differences of afferents from or efferent connections to the hair cells) are responsible for this discontinuity. De Brey and Eggermont [13] have described a temperature-dependent discontinuity along the basilar membrane of the guinea pig: with cooling, the CM pattern shifted towards the base of the cochlea. Eggermont and his colleagues [ 17.381 noted a decrease of synchrony and adaptation of the AP, and a reduction of the ability of noise to mask the AP. Hypothermia offered some protection from PTS which was only evident at frequencies where noise elevated the thresholds by about 30 dB or more. In the mice whose youth and barbiturate anesthetization made them extremely vulnerable to PTS, this was quite evident at 2-16 kHz. In the less susceptible adult mice, this was only statistically significant at 32 kHz, even though OHC losses were seen at an area corresponding to 16 kHz. The best anatomical correlation with the AP PTS was IHC loss, which was greatest in the region corresponding to 32 kHz. Hypothermia also reduced IHC loss in this region. These observations agree with recent studies which show a much better correspondence of behavioral and neural auditory thresholds with 1HCs than with OHCs [39]. The protective effect of hypothermia may be at least partly due to metabolic changes. By reducing the requirements of cells within the organ of Corti, the magnitude of the metabolic exhaustion resulting from acoustic overstimulation might be reduced. Increasing the oxygen supply [28,2,5], infusions of low molecular weight sugars [30], and removal of the thyroid gland [2,3] prior to acoustic stress all protect the organ of Corti. The metabolic deficit may, in turn. affect proposed active cochlear processes [ 11,31,32.36,43] which could change the movement of the basilar membrane, thereby subjecting the hair cells to less mechanical stress.

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