Age-related changes in auditory nerve-inner hair cell connections, hair cell numbers, auditory brain stem response and gap detection in UM-HET4 mice.

This study compared the timing of appearance of three components of age-related hearing loss that determine the pattern and severity of presbycusis: the functional and structural pathologies of sensory cells and neurons and changes in gap detection (GD), the latter as an indicator of auditory temporal processing. Using UM-HET4 mice, genetically heterogeneous mice derived from four inbred strains, we studied the integrity of inner and outer hair cells by position along the cochlear spiral, inner hair cell-auditory nerve connections, spiral ganglion neurons (SGN), and determined auditory thresholds, as well as pre-pulse and gap inhibition of the acoustic startle reflex (ASR). Comparisons were made between mice of 5-7, 22-24 and 27-29 months of age. There was individual variability among mice in the onset and extent of age-related auditory pathology. At 22-24 months of age a moderate to large loss of outer hair cells was restricted to the apical third of the cochlea and threshold shifts in the auditory brain stem response were minimal. There was also a large and significant loss of inner hair cell-auditory nerve connections and a significant reduction in GD. The expression of Ntf3 in the cochlea was significantly reduced. At 27-29 months of age there was no further change in the mean number of synaptic connections per inner hair cell or in GD, but a moderate to large loss of outer hair cells was found across all cochlear turns as well as significantly increased ABR threshold shifts at 4, 12, 24 and 48 kHz. A statistical analysis of correlations on an individual animal basis revealed that neither the hair cell loss nor the ABR threshold shifts correlated with loss of GD or with the loss of connections, consistent with independent pathological mechanisms.

Cochlear microphonic enhancement in two tone interactions.

Two tone interaction functions of the cochlear microphonic (CM) were obtained from pigmented guinea pigs. First (basal) cochlear turn recording locations show optimally enhanced levels of CM when the interfering tone (F2) was positioned about 4 kHz above probe tones (F1) of 12 kHz and 20 kHz. Maximum enhancement occurred for equal level tones. No enhancement was seen for a probe tone of 4 kHz. When basal turn cochlear sensitivity was compromised, CM enhancement caused by the interfering tone was altered and only CM reduction was then seen. The CM reduction was the typical characteristic described by many earlier studies. Guinea pigs with various changes in cochlear sensitivity were studied, providing evidence in support of earlier reports that CM interference (both reductions and enhancements) depends on far field vector summation of the outputs of hair cells from a restricted area of the basilar membrane. No CM enhancement was seen in micropipette recordings from within the organ of Corti.

The effects of efferent activation on the acoustically and electrically evoked otoacoustic emission.

The effects of efferent activation on the otoacoustic emission were measured in anesthetized guinea pigs. The otoacoustic emission (2F(1)-F(2)) was evoked by the conventional method of presenting either two continuous tones or a sinusoidal current to the round window (RW) of the cochlea. The efferent effects on the acoustically evoked emission are greatest at low stimulus levels and least for high levels. The efferent effects on the electrically evoked emission (EEOAE) are relatively constant across current levels. In each case, efferent activation resulted in an initial large reduction in the emission amplitude followed by a smaller and more constant reduction. Strychnine eliminated the efferent effects independent of the method of emission activation. Strychnine had no effect on the EEOAE, suggesting that the RW current did not evoke a local efferent effect. Slow versus fast efferent effects were observed in the recovery of the emission amplitude at the termination of efferent activation. Only a fast recovery in the emission amplitude was observed for stimuli below 10 kHz while the amplitude recovery had fast and slow components for stimuli presented above 10 kHz.

Chronic strychnine administration into the cochlea potentiates permanent threshold shift following noise exposure.

To investigate whether elimination of the medial efferent system influences permanent threshold shift following noise exposure, we developed an animal model in which strychnine was chronically delivered into the cochlea via an osmotic pump. Pigmented female guinea pigs were allocated into three groups: group I was treated with strychnine (50 microM, 0.5 microl/h, 14 days) in the left ear and exposed to noise (105 dB SPL broadband, 3 h) 3 weeks after the cessation of the strychnine perfusion; group II received strychnine in the left ear but no noise exposure; group III was treated with Ringer's solution in the left ear and exposed to noise. Animals in group II developed no hearing loss after the strychnine perfusion. The operated ears in group I demonstrated greatest hearing threshold shift 3 h after noise exposure. Hearing recovered during 2 weeks after noise exposure in both operated and non-operated ears in groups I and III. Two weeks after noise exposure, the operated ears in group I showed significantly greater threshold shift at 12, 16, and 20 kHz compared to the operated ears in group III and non-operated ears in groups I and III. These findings suggest that chronic strychnine administration into the cochlea inactivates the medial efferents without changing hearing threshold and that the medial efferents help to protect against permanent threshold shift following noise exposure.

The medial cochlear efferent system does not appear to contribute to the development of acquired resistance to acoustic trauma.

Noise-induced hearing loss (NIHL) was compared between sound conditioned and unconditioned guinea pigs, in which the left ear in both groups had been perfused with strychnine. Animals in the conditioned group were subjected to moderate sound (85 dB SPL broadband, 5 h/day, 10 days) and then exposed to intense sound (110 dB SPL broadband, 5 h). Unconditioned animals were exposed only to the intense sound. Following intense sound exposure, strychnine-treated ears showed greater NIHL than untreated ears in both unconditioned and conditioned animals, demonstrating the role of the medial efferents to reduce NIHL. Conditioned animals, however, showed smaller hearing loss and cochlear damage in both strychnine-treated and untreated ears compared to unconditioned animals; the protective effects given by conditioning were equivalent between the strychnine-treated and untreated ears. These results suggest that, although the medial efferent system acts to attenuate NIHL, it may not be necessary for the acquired resistance to NIHL provided by conditioning.

Laser Doppler velocimetry of basilar membrane vibration.

A method is described for the measurement of basilar membrane (BM) vibration velocimeter (LDV). The instrumentation was coupled to a compound microscope which served to visualize reflective glass microbeads placed on the BM. The laser beam of the LDV was focused in the microscope object plane and positioned over the reflective bead. We show examples of frequency tuning curves and displacement input/output intensity functions obtained with the technique.

Acquired resistance to acoustic trauma by sound conditioning is primarily mediated by changes restricted to the cochlea, not by systemic responses.

Hearing loss caused by intense sound exposure can be significantly reduced by pre-exposing subjects to moderate-level acoustic stimuli. This phenomenon occurs in a variety of mammals. We investigated whether sound conditioning provides acquired resistance to acoustic trauma through local mechanisms selectively in the conditioned ears or if systemic mechanisms are involved that would yield contralateral protection in unconditioned ears. Guinea pigs (group I) in which one external ear canal was occluded were exposed to conditioning sound (2-20 kHz, 85 dB SPL, 5 h/day, 10 days). After removing the occlusion, the animals were then subjected bilaterally to intense noise (2-20 kHz, 110 dB SPL, 5 h) 5 days after the last conditioning exposure. Animals without ear canal occlusion were also exposed to the intense sound without conditioning (group II) or following the same conditioning exposure (group III). Intense sound exposure caused significantly greater permanent ABR threshold shifts at all frequencies tested (4, 8, 12, 16 and 20 kHz) in group II than in group III. In group I, the occluded ears showed significantly greater threshold shifts at all frequencies compared to the unoccluded ears. The threshold shifts in the occluded ears in group I were identical to those observed in group II; and the shifts in unoccluded ears in group I were identical to those in group III. Protective effects provided by sound conditioning were almost the same in group III and in the unoccluded ears in group I. The extent of hair cell damage supported the physiological findings. These results indicate that acquired resistance to acoustic trauma provided by sound conditioning is restricted to the cochlea exposed to conditioning sound, suggesting that conditioning protection is mediated primarily by the changes that occur locally within the conditioned cochlea. This animal model, with unilateral external ear canal occlusion during sound conditioning, is useful for studies of the mechanisms of conditioning protection.

Basilar membrane velocity noise.

Basilar membrane (BM) noise, measured as a velocity signal under the quiet acoustic condition, was investigated in the guinea pig. The cochleas of anesthetized young healthy guinea pigs were surgically exposed and a hole was made on the lateral wall of the scala tympani of the first cochlear turn for visualization of the BM and measurement of the BM velocity with a laser interferometer. The amplitude and frequency of the BM velocity noise were analyzed by a spectrum analyzer under different conditions. The spectrum of the BM velocity noise was a band limited function with a peak velocity at the topographic best frequency of the measured location on the BM. The peak velocity ranged to about 8 microm/s and depended on the physiological condition of the cochlea. Saline blockage of the external auditory canal or the middle ear did not change the BM noise. BM noise was much smaller, or was not evident, when the cochlear sensitivity decreased. The suppression tuning curve of the BM velocity noise indicates that the maximum suppression caused by an acoustic pure tone occurred at the best frequency location. A low sound level wide band acoustic noise given to the external ear canal produced a spectrum function having the same frequency and amplitude response as the BM noise. Electrical stimulation of the crossed olivocochlear bundle significantly depresses the BM velocity noise. These data demonstrate that the BM noise is a representation of internal rather than external noise. The amplitude and frequency of the BM noise reflect the usual cochlear sensitivity and frequency selectivity. Since the organ of Corti in the sensitive cochlea is a highly sensitive and tuned mechanical system, the internal (to the animal) noise responsible for the BM noise may originate from mechanical vibrations remote from the cochlea and propagated to the ear, or may be caused by Brownian motion of cellular structures in the cochlea.

Severe vestibular and auditory impairment in three alleles of Ames waltzer (av) mice.

The genetic and physiological characterization of circling, hearing-impaired mouse mutants has greatly facilitated our understanding of non-syndromic sensorineural deafness, the most common form of hereditary human hearing loss. Here we report the first phenotypic characterization of three alleles of Ames waltzer (av). Neither electrical potentials (auditory brainstem response) nor behavioral responses to sound could be evoked in any of the three alleles at any age or frequency. However, the endocochlear potential was found to be normal, indicating that the primary pathology is not in the stria vascularis. To determine the earliest changes and help identify the primary causes of deafness in av, we performed morphological studies in 15-16 day old mutants, just prior to the maturation of the cochlea. Although av(2J) is slightly more affected than the other two alleles, our studies show a high similarity between all three alleles. The first detectable changes are observed in the stereocilia and cytoplasm of hair cells, and in the cellular shape and microvilli of supporting cells. These changes are followed by degeneration of the cochlear and vestibular neuroepithelium.

Glial cell line-derived neurotrophic factor has a dose dependent influence on noise-induced hearing loss in the guinea pig cochlea.

We examined the effectiveness of glial cell line-derived neurotrophic factor (GDNF) to attenuate cochlear damage from intense noise stress. Subjects were exposed to 115 dB SPL one octave band noise centered at 4 kHz for 5 h. They received artificial perilymph with or without GDNF into the left scala tympani at 0.5 microliter/h from 4 days before noise exposure through 8 days following noise exposure. Different concentrations of GDNF (1 ng/ml, 10 ng/ml, 100 ng/ml, and 1 microgram/ml) were applied chronically directly into the guinea pig cochlea via a microcannula and osmotic pump. Noise-induced hearing loss was assessed with pure tone auditory brainstem responses (at 2, 4, 8 and 20 kHz), measured prior to surgery, 1 day before noise exposure, and 7 days following noise exposure. Subjects were killed on day 8 following exposure for histological preparation and quantitative assessment of hair cell (HC) damage. A dose-dependent protective effect of GDNF on both sensory cell preservation and hearing function was found in the treated ears. At 1 ng/ml, GDNF showed no significant protection; at 10 ng/ml, GDNF showed significant HC protection; and at 100ng/ml, it was greater and bilateral. At 1 microgram/ml, GDNF appeared to have a toxic effect under noise stress in some cochleae. These findings indicate that GDNF at certain concentrations can effectively protect the inner ear from noise-induced hearing loss.

KHRI-3 monoclonal antibody-induced damage to the inner ear: antibody staining of nascent scars.

Intracochlear infusion of the KHRI-3 monoclonal antibody results in in vivo binding to guinea pig inner ear supporting cells, loss of hair cells and hearing loss. To further characterize the basis for KHRI-3-induced hearing loss, antibody was produced in a bioreactor in serum-free medium, affinity purified, and compared to conventionally prepared antibody by infusion into the scala tympani using mini-osmotic pumps. In vivo antibody binding was observed in 10 of 11 guinea pigs. A previously unreported pattern of KHRI-3 antibody binding to cells involved in scar formation was noted in five guinea pigs. All but one of the KHRI-3-infused animals demonstrated a hearing loss of > 10 dB in the treated ear. In five of 11 animals the threshold shift was 30 dB or more, and all had hair cell losses. In one guinea pig infused with 2 mg/ml of antibody, the organ of Corti was absent in the basal turn of the infused ear. This ear had a 45-50 dB threshold shift but, curiously, no detectable antibody binding in the residual organ of Corti. Organ of Corti tissue was fragile in antibody-infused ears. Breaks within the outer hair cell region occurred in 5/11 infused ears. The contralateral ears were normal except for one noise-exposed animal that demonstrated hair cell loss in the uninfused ear. Three animals were exposed to 6 kHz noise (108 dB) for 30 min on day 7. Antibody access to the organ of Corti may be increased in animals exposed to noise, since the strongest in vivo binding was observed in noise-exposed animals. Loss of integrity of the organ of Corti seems to be the primary mechanism of inner ear damage by KHRI-3 antibody. The binding of KHRI-3 antibody in new scars suggests a role of the antigen in scar formation. Antibodies with binding properties similar to KHRI-3 have been detected in 51% of patients diagnosed with autoimmune sensorineural hearing loss; thus, it seems likely that such autoantibodies also may have pathologic effects resulting in hearing loss in humans.

Monoclonal antibody induced hearing loss.

Monoclonal antibodies KHRI-3 and KHRI-5 identify antigens expressed on inner ear supporting cells and auditory hair cells respectively. To determine if these antibodies affect inner ear function groups of syngeneic Balb/c mice were inoculated with hybridomas KHRI-3, KHRI-5 and other Ig-secreting hybridomas. Hybridomas UM-A9, UM-7F11, the non-secreting SP2/0 myeloma and mice with no hybridoma were used as controls. Animals were tested for auditory brainstem responses (ABR) for frequencies of 4, 8, 16 and 24 kHz, before the inoculation of the hybridomas and at intervals of 6 to 10 days thereafter or daily once tumors became palpable. In normal mice there were no changes in ABR thresholds over the course of the experiment. Other control animals showed little change in ABR even when the growth of the hybridoma or myeloma tumors were far advanced. Of the KHRI-5 hybridoma bearing animals only one of seven animals exhibited threshold shifts greater than 15 dB. In contrast, most mice bearing the KHRI-3 hybridoma exhibited high frequency threshold shifts of 40-50 dB that coincided temporally with the growth of the hybridoma, the presence of circulating KHRI-3 antibody, and greatly increased immunoglobulin titers. Ears from KHRI-3-bearing mice that developed high frequency hearing loss also had a novel type of lesion in the basal turn of the cochlea that was characterized by loss of outer hair cells and absence of typical supporting cell scars. Such changes were not found in control hybridoma-bearing mice. These findings suggest that KHRI-3 antibody has an effect on hearing that is secondary to damage to the organ of Corti and loss of outer hair cells. Our results have important implications for antibody-mediated mechanisms of hearing loss and provide an animal model in which to study this phenomenon.

Vestibular morphological analysis of the effects of cisplatin vs. platinum analogs, CBDCA (JM-8) and CHIP (JM-9).

Synthetic second generation chemotherapeutic platinum analogs are presently being tested to identify an analog with greater antitumor activity, but less ototoxicity and nephrotoxicity than cisplatin. The objective of this study was to analyze potential vestibular effects of cisplatin and of the two platinum analogs, CBDCA (cis-diammine 1,1-cyclobutane dicarboxylato [2]-0,0(1) platinum or JM-8) and CHIP (cis-dichlorotrans-dihydroxy-bis(isopropylamine) platinum [IV] or JM-9) using scanning and transmission electron microscopy of vestibular neuroepithelium from the albino guinea pig. Vestibular neuroepithelial damage was not demonstrated in either cisplatin- or the analog-treated animals when administered at equitoxic doses.

Amelioration of cisplatin-induced ototoxicity by fosfomycin.

The continued chemotherapeutic application of cisplatin (cis-diamminedichloroplatinum [II]) necessitates reduction of its dose-limiting toxicity without decreasing its tumoricidal effect. This research project evaluated the efficacy of fosfomycin, a phosphonic acid antibiotic, in decreasing or ameliorating the ototoxicity (high frequency sensorineural hearing loss) and nephrotoxicity (renal tubular necrosis and interstitial nephritis) of cisplatin. Experimentally, fosfomycin effectively inhibits aminoglycoside-induced ototoxicity and nephrotoxicity in animals and humans. The efficacy of fosfomycin in blocking platinum-induced toxicity in the guinea pig was evaluated histologically and functionally using cytocochleography and light microscopy of the organ of Corti and the auditory brain stem evoked response (ABR), and light microscopy of renal corticomedullary tissues, small bowel, liver, lung, and peripheral nerve. The results demonstrate that fosfomycin ameliorates the acute renal tubular necrosis and interstitial nephritis and markedly inhibits the elevation of ABR thresholds and simultaneous outer hair cell loss that can result from cisplatinum administration. Fosfomycin should be considered a potential antidote for the dose-limiting ototoxicity and nephrotoxicity of cisplatin chemotherapy.

Experimental model of immune-mediated hearing loss using cross-species immunization.

The presence of immune-mediated hearing loss was investigated in an animal model. Eight guinea pigs and four mice underwent immunizations with a preparation of chick or guinea pig cochlear tissue and Freund's adjuvant. Hearing thresholds were monitored by auditory brainstem response (ABR) testing over a 5-week period after immunization. The serum and temporal bones of test and control animals were then examined using an enzyme-linked immunosorbent assay (ELISA), immunocytochemical, and histological techniques. Hearing loss of 20 dB or greater occurred in eight animals. ELISA demonstrated antibodies to cochlear antigens in the sera of all test animals. Immunocytochemistry revealed immunostaining of hair cell stereocilia in the organ of Corti and saccule. Endolymphatic hydrops, and organ of Corti degeneration was observed in the temporal bones of three animals. This study provides evidence to suggest that cross-species immunization with cochlear antigens might produce a humoral response that can be associated with inner ear pathologic change and sensorineural hearing loss.

Age-related differences in cochlear microcirculation and auditory brain stem response.

OBJECTIVE: To examine possible age-related differences in auditory sensitivity and cochlear vascular properties. DESIGN: This study is designed to provide information regarding cochlear function using physiological and audiological measures. Each animal underwent intravital microscopic evaluation of red blood cell velocity, vessel diameter, and vascular permeability in the second turn of the cochlear lateral wall. Auditory brain stem responses were used to determine hearing sensitivity. SUBJECTS: Four age ranges of male Fischer rats were studied: young, 2 to 4 months (n = 9); mid-young, 9 to 11 months (n = 8); mid-old, 18 to 20 months (n = 6); and old, 30 to 34 months (n = 10). RESULTS: Auditory brain stem response testing showed an age-related decrease in auditory sensitivity. Intravital microscopic analysis showed age-related statistically significant decreases in red blood cell velocity and increased vascular permeability with a trend for reduced capillary diameters. CONCLUSIONS: The process of aging is associated with many biochemical and physiological changes that include decrease in cellular water concentration, ionic changes, and decreased elasticity of cellular membranes. One contributing factor to this process may be altered vascular characteristics, such as reduced flow and vascular plasticity, as well as increased vascular permeability. These age-related changes may result in reductions in oxygen and nutrient delivery, and also waste elimination. Our results suggest that progressive age-associated vascular compromise may be a contributing factor in presbycusis.

Ototoxicity of cisplatin vs. platinum analogs CBDCA (JM-8) and CHIP (JM-9).

Cis-diamminedichloroplatinum (cisplatin), a divalent platinum compound and cell-cycle nonspecific chemotherapeutic agent, produces a permanent high-frequency sensorineural hearing loss and a dose-related cumulative renal insufficiency with tubular necrosis and interstitial nephritis. Synthetic platinum analogs are presently being tested to identify an analog with greater antitumor activity, but less ototoxicity and nephrotoxicity than cisplatin. The objectives of this study were to analyze the potential cochlear and nephrotoxic effects of two synthetic platinum analogs presently in phases I and II of clinical trials, CBDCA [JM-8 or cis-diammine, 1,1-cyclobutane dicarboxylato (2)-0,0(1)-platinum (NSC-241240)] and CHIP [JM-9 or cis-dichloro-trans-dihydroxybisisopropylamine platinum IV (NSC-256927)]. Cytocochleography, auditory brain-stem evoked response (ABR), double-blind light microscopy of renal tissues, and gamma emission analysis of 195mpt localization in viscera and inner ear were employed in the evaluation of cisplatin and platinum analogs (JM-8 and JM-9) in adult guinea pigs. Final results indicate that the investigational chemotherapeutic analogs CBDCA (JM-8) and CHIP (JM-9) do not produce the ototoxicity and nephrotoxicity characteristic of cisplatin. Furthermore, these findings demonstrate 195mpt localization in the vestibular labyrinth and confirm previous platinum distribution studies in the organ of Corti and stria vascularis tissues.

The mechanism and site of action of lidocaine hydrochloride in guinea pig inner ear.

Lidocaine was applied to the round window (RW) in order to localize its site of action in the cochlea. Cochlear microphonic (CM), summating potential (SP), and compound action potential (CAP) input/output functions were measured to a 16 kHz tone burst to assess the functional changes of the cochlea. In separate experiments, the effect of lidocaine on the whole cell current of isolated outer hair cells (OHC) was studied. A dose of 2 microliters of 40 mM lidocaine in saline solution, when applied to the RW, caused a small change in all measured variables, indicating a passage of the drug through the RW membrane to sites of action. However, 160 mM of lidocaine further decreased CM, SP, and CAP by a total of 40% from the control. A partial recovery occurred for CM during the 30 min follow-up period. CAP and SP continued to decline. In isolated OHCs, lidocaine decreased the whole cell current in a dose-dependent fashion. The KD for lidocaine effect on OHCs was 7 mM. Our in vivo results indicate that lidocaine affects OHCs and reduces CM, causing a subsequent reduction in SP and CAP. The increased effect of lidocaine on CAP and SP, while CM is recovering, suggests an additional specific effect of lidocaine on the cochlear nerve and/or on inner hair cells. Considering that lidocaine alters OHC current (in isolated hair cells) and that lidocaine does not affect endocochlear potential [Laurikainen et al. Acta Otolaryngol (Stockh) 1991: 112: 800-9], the observed CM changes are most likely due to an in vivo effect on OHCs. Thus, the early effect of lidocaine on the cochlea appears to be due to a significant change in organ of Corti function, rather than to direct anesthesia of the cochlear nerve. Later, an independent effect of the drug may occur on neural tissues in the inner ear.

Pathways for protection from noise induced hearing loss.

There is increasing evidence that at least one function of both the medial and the lateral olivocochlear efferent systems is to provide adjustment of the set point of activity in their postsynaptic target, the outer hair cells and afferent processes, respectively. New results, summarized in this review, suggest that both efferent systems can provide protection from noise through this mechanism. There are also intracellular pathways that can provide protection from noise-induced cellular damage in the cochlea. This review also summarizes new results on the pathways that regulate and react to levels of reactive oxygen species in the cochlea as well as the role of stress pathways for the heat shock proteins and for neurotrophic factors in protection, recovery and repair.

Impaired sensorimotor gating in Fmr1 knock out and Fragile X premutation model mice.

Fragile X syndrome (FXS) is a common inherited cause of intellectual disability that results from a CGG repeat expansion in the FMR1 gene. Large repeat expansions trigger both transcriptional and translational suppression of Fragile X protein (FMRP) production. Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is an allelic neurodegenerative disease caused by smaller "pre-mutation" CGG repeat expansions that enhance FMR1 transcription but lead to translational inefficiency and reduced FMRP expression in animal models. Sensorimotor gating as measured by pre-pulse inhibition (PPI) is altered in both FXS patients and Fmr1 knock out (KO) mice. Similarly, FXTAS patients have demonstrated PPI deficits. Recent work suggests there may be overlapping synaptic defects between Fmr1 KO and CGG knock-in premutation mouse models (CGG KI). We therefore sought to interrogate PPI in CGG KI mice. Using a quiet PPI protocol more akin to human testing conditions, we find that Fmr1 KO animals have significantly impaired PPI. Using this same protocol, we find CGG KI mice demonstrate an age-dependent impairment in PPI compared to wild type (WT) controls. This study describes a novel phenotype in CGG KI mice that can be used in future therapeutic development targeting premutation associated symptoms.