Dalian Ding

Auditory plasticity and hyperactivity following cochlear damage.

This paper will review some of the functional changes that occur in the central auditory pathway after the cochlea is damaged by acoustic overstimulation or by carboplatin, an ototoxic drug that selectively destroys inner hair cells (IHCs) in the chinchilla. Acoustic trauma typically impairs the sensitivity and tuning of auditory nerve fibers and reduces the neural output of the cochlea. Surprisingly, our results show that restricted cochlear damage enhances neural activity in the central auditory pathway. Despite a reduction in the auditory-nerve compound action potential (CAP), the local field potential from the inferior colliculus (IC) increases at a faster than normal rate and its maximum amplitude is enhanced at frequencies below the region of hearing loss. To determine if this enhancement was due to loss of sideband inhibition, we recorded from single neurons in the IC and dorsal cochlear nucleus before and after presenting a traumatizing above the units characteristic frequency (CF). Following the exposure, some neurons showed substantial broadening of tuning below CF, less inhibition, and a significant increase in discharge rate, consistent with a model involving loss of sideband inhibition. The central auditory system of the chinchilla can be deprived of some of its cochlear inputs by selectively destroying IHCs with carboplatin. Selective IHC loss reduces the amplitude of the CAP without affecting the threshold and tuning of the remaining auditory nerve fibers. Although the output of the cochlea is reduced in proportion to the amount of IHC loss, the IC response shows only a modest amplitude reduction, and remarkably, the response of the auditory cortex is enhanced. These results suggest that the gain of the central auditory pathway can be up- or down regulated to compensate for the amount of neural activity from the cochlea.

Targeted deletion of the cytosolic Cu/Zn-superoxide dismutase gene (Sod1) increases susceptibility to noise-induced hearing loss.

Reactive oxygen species (ROS) such as superoxide, peroxide and hydroxyl radicals are generated during normal cellular metabolism and are increased in acute injury and in many chronic disease states. When their production is inadequately regulated, ROS accumulate and irreversibly damage cell components, causing impaired cellular function and death. Antioxidant enzymes such as superoxide dismutase (SOD) play a vital role in minimizing ROS levels and ROS-mediated damage. The cytosolic form of Cu/Zn-SOD appears specialized to remove superoxide produced as a result of injury. ‘Knockout’ mice with targeted deletion of Sod1, the gene that codes for Cu/Zn-SOD, develop normally but show enhanced susceptibility to central nervous system injury. Since loud noise is injurious to the cochlea and is associated with elevated cochlear ROS, we hypothesized that Sod1 knockout mice would be more susceptible to noise-induced permanent threshold shifts (PTS) than wild-type and heterozygous control mice. Fifty-nine mice (15 knockout, 29 heterozygous and 15 wild type for Sod1) were exposed to broad-band noise (4.0–45.0 kHz) at 110 dB SPL for 1 h. Hearing sensitivity was evaluated at 5, 10, 20 and 40 kHz using auditory brainstem responses before exposure and 1, 14 and 28 days afterward. Cu/Zn-SOD deficiency led to minor (0–7 dB) threshold elevations prior to noise exposure, and about 10 dB of additional noise-induced PTS at all test frequencies, compared to controls. The distribution of thresholds at 10 and 20 kHz at 28 days following exposure contained three modes, each showing an effect of Cu/Zn-SOD deficiency. Thus another factor, possibly an additional unlinked gene, may account for the majority of the observed PTS. Our results indicate that genes involved in ROS regulation can impact the vulnerability of the cochlea to noise-induced hearing loss.

Reduction of noise-induced hearing loss using L-NAC and salicylate in the chinchilla

The effects of a combination of two antioxidant compounds were studied in a chinchilla model of noise-induced hearing loss. After obtaining baseline hearing thresholds using inferior colliculus evoked potentials, chinchillas were exposed for 6 h to octave band noise centered at 4 kHz (105 dB SPL). Post-noise thresholds were obtained 1 h after the noise exposure, and then animals received either saline or salicylate and N-L-acetylcysteine combination. Another group received antioxidant treatment 1 h prior to noise. Hearing was tested at 1, 2 and 3 weeks post-noise. Subsequently, the cochleae were harvested, and cytocochleograms were prepared. There was a 20-40 dB SPL threshold shift at 3 weeks for tested controls. Permanent threshold shifts (PTS) were significantly reduced (P<0.05) to approximately 10 dB for the pre-treatment group at week 3. The PTS for the post-treatment group at week 3 was similar to the pre-treatment group at 1 and 2 kHz (0-10 dB) but was intermediate between the control and pre-treatment groups at 4 and 8 kHz (23 dB). Animals pre-treated with antioxidant had a significant reduction in hair cell loss but those post-treated with antioxidant had no protection from hair cell loss. These findings demonstrate the feasibility of reduction of noise-induced hearing loss using clinically available antioxidant compounds.

Age-related hearing loss in C57BL/6J mice is mediated by Bak-dependent mitochondrial apoptosis

Age-related hearing loss (AHL), known as presbycusis, is a universal feature of mammalian aging and is the most common sensory disorder in the elderly population. The molecular mechanisms underlying AHL are unknown, and currently there is no treatment for the disorder. Here we report that C57BL/6J mice with a deletion of the mitochondrial pro-apoptotic gene Bak exhibit reduced age-related apoptotic cell death of spiral ganglion neurons and hair cells in the cochlea, and prevention of AHL. Oxidative stress induces Bak expression in primary cochlear cells, and Bak deficiency prevents apoptotic cell death. Furthermore, a mitochondrially targeted catalase transgene suppresses Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL. Oral supplementation with the mitochondrial antioxidants α-lipoic acid and coenzyme Q10 also suppresses Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL. Thus, induction of a Bak-dependent mitochondrial apoptosis program in response to oxidative stress is a key mechanism of AHL in C57BL/6J mice.

Age-related cochlear hair cell loss is enhanced in mice lacking copper/zinc superoxide dismutase

Age-related hearing loss in humans and many strains of mice is associated with a base-to-apex gradient of cochlear hair cell loss. To determine if copper/zinc superoxide dismutase (Cu/Zn SOD) deficiency influences age-related cochlear pathology, we compared hair cell losses in cochleas obtained from 2-, 7-, and 17- to 19-month-old wild type (WT) mice with normal levels of Cu/Zn SOD and mutant knockout (KO) mice with a targeted deletion of Sod1, the gene that codes for Cu/Zn SOD. WT and KO mice exhibited similar patterns of hair cell loss with age, i.e., a baso-apical progression of hair cell loss, with greater loss of outer hair cells than inner hair cells. Within each age group, the magnitude of loss was much greater in KO mice compared to WT mice. The results indicate that Cu/Zn SOD deficiency potentiates cochlear hair cell degeneration, presumably through metabolic pathways involving the superoxide radical.

Pifithrin-α supresses p53 and protects cochlear and vestibular hair cells from cisplatin-induced apoptosis

Abstract Cisplatin, a commonly used antineoplastic agent, destroys the sensory hair cells in the cochlear and vestibular system leading to irreversible hearing loss and balance problems. Cisplatin-induced hair cell damage presumably occurs by apoptosis. Recent studies suggest that p53 may play an important role initiating cisplatin-induced apoptosis in some cell types. To determine if p53 plays a role in cisplatin-mediated hair cell loss, cochlear and utricular organotypic cultures were prepared from postnatal day 3–4 rats and treated with cisplatin or cisplatin plus pifithrin-α (PFT), a p53 inhibitor. Control cultures were devoid of p53 immunolabeling, caspase-1 and caspase-3 labeling and p53 protein was absent from Western blots. Cisplatin (1–10 μg/ml) caused a dose-dependent loss of hair cells in cochlear and utricular cultures, up-regulated phospho-p53 serine 15 immunolabeling, increased the expression of phospho-p53 serine 15 in Western blots from 6 to 48 h after the onset of cisplatin-treatment, and increased caspase-1 and caspase-3 labeling in cochlear and vestibular cultures. Addition of PFT (20–100 μM) to cisplatin-treated cochlear and utricular cultures resulted in a dose-dependent increase in hair cell survival; suppressed the expression of p53 in Western blots and eliminated caspase-1 and caspase-3 labeling in cultures. These results suggest that the tumor suppressor protein, p53, plays a critical role in initiating apoptosis in cochlear and vestibular hair cells. Temporary suppression of p53 with PFT provides significant protection against cisplatin-induced hair cell loss and offers the potential for reducing the ototoxic, vestibulotoxic and neurotoxic side effects of cisplatin.

Cu/Zn SOD deficiency potentiates hearing loss and cochlear pathology in aged 129,CD-1 mice.

Copper/zinc superoxide dismutase (Cu/Zn SOD) is a first‐line defense against free radical damage in the cochlea and other tissues. To determine whether deficiencies in Cu/Zn SOD increase age‐related hearing loss and cochlear pathology, we collected auditory brainstem responses (ABRs) and determined cochlear hair cell loss in 13‐month‐old 129/CD‐1 mice with (a) no measurable Cu/Zn SOD activity (homozygous knockout mice), (b) 50% reduction of Cu/Zn SOD (heterozygous knockout mice), and (c) normal levels of Cu/Zn SOD (wild‐type mice). ABRs were obtained by using 4‐, 8‐, 16‐, and 32‐kHz tone bursts. Cochleas were harvested immediately after testing, and separate counts were made of inner and outer hair cells. Compared with wild‐type mice, homozygous and heterozygous knockout mice exhibited significant threshold elevations and greater hair cell loss. Phenotypic variability was higher among heterozygous knockout mice than among wild‐type or homozygous knockout mice. Separate groups of wild‐type and homozygous knockout mice were examined for loss of spiral ganglion cells and eighth nerve fibers. At 13 months of age, both wild‐type and knockout mice had significantly fewer nerve fibers than did 2‐month‐old wild‐type mice, with significantly greater loss in aged knockout mice than in aged wild‐type mice. Thirteen‐month‐old knockout mice also had a significant loss of spiral ganglion cells compared with 2‐month‐old wild‐type mice. The results indicate that Cu/Zn SOD deficiencies increase the vulnerability of the cochlea to damage associated with normal aging, presumably through metabolic pathways involving the superoxide radical. J. Comp. Neurol. 413:101–112, 1999.

Functional reorganization in chinchilla inferior colliculus associated with chronic and acute cochlear damage

This paper describes some of the unexpected functional changes that occur in the inferior colliculus (IC) following noise- and drug-induced cochlear pathology. A striking example of this is the compensation that is seen in IC responsiveness after drug-induced selective inner hair cell (IHC) loss. Despite a massive reduction in the compound action potential (CAP) caused by partial IHC loss, the evoked potential amplitude from the IC shows little or no reduction. Acoustic trauma, which impairs cochlear sensitivity and tuning, also reduces the CAP amplitude. Despite this reduced neural input, IC amplitude sometimes increases at a faster than normal rate and the response amplitude is enhanced at frequencies below the hearing loss. Single unit recordings suggest the IC enhancement phenomenon may be due to the loss of lateral inhibition. After an acute traumatizing exposure to a tone located above the characteristic frequency (CF), approximately 50% of IC neurons show a significant increase in their spike rate, a significant expansion of the low frequency tail of the tuning curve and a significant improvement in sensitivity in the tail of the tuning curve. These changes suggest that IC neurons receive inhibition from a high frequency side band and that this inhibition is diminished by acoustic trauma above CF. To determine if side band inhibition was locally mediated, specific antagonist(s) to inhibitory neurotransmitters were applied and found to produce effects similar to acoustic trauma. The results suggest that lesioned-induced central auditory plasticity could contribute to several symptoms associated with sensorineural hearing loss such as loudness recruitment, tinnitus and poor speech discrimination in noise.

Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity

Calpains, a family of calcium-activated proteases that breakdown proteins, kinases, phosphatases and transcription factors, can promote cell death. Since leupeptin, a calpain inhibitor, protected against hair cell loss from acoustic overstimulation, we hypothesized that it might protect cochlear and vestibular hair cells against gentamicin (GM) ototoxicity. To test this hypothesis, mouse organotypic cultures from the cochlea, maculae of the utricle and the crista of the semicircular canal (P1-P3) were treated with different doses of GM (0.1-3 mM) alone or in the presence of leupeptin (0.1-3 mM). The percentage of outer hair cells (OHCs) and inner hair cells (IHCs) decreased with increasing doses of GM between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced GM-induced damage to IHCs and OHCs; this protective effect was dose-dependent. GM also significantly reduced hair cell density in the crista and utricle in a dose-dependent manner between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced hair cell loss in the crista and utricle for GM concentrations between 0.1 and 3 mM. These results suggest that one of the early steps in GM ototoxicity may involve calcium-activated proteases that lead to the demise of cochlear and vestibular hair cells.

Effects of selective inner hair cell loss on auditory nerve fiber threshold, tuning and spontaneous and driven discharge rate.

Current theories assume that the outer hair cells (OHC) are responsible for the sharp tuning and exquisite sensitivity of the ear whereas inner hair cells (IHC) are mainly responsible for transmitting acoustic information to the central nervous system. To further evaluate this model, we used a single (38 mg/kg) or double dose (38 mg/kg, 2 times) of carboplatin to produce a moderate (20-28%) or severe (60-95%) IHC loss while sparing a large proportion of the OHCs. The surviving OHCs were functionally intact as indicated by normal cochlear microphonic (CM) potentials and distortion product otoacoustic emissions (DPOAE). Single-unit responses were recorded from auditory nerve fibers to determine the effects of the moderate or severe IHC loss on the output of the surviving IHCs. Most neurons that responded to sound in the single-dose group had normal or near-normal thresholds and normal tuning. Relatively few neurons in the double-dose group responded to sound because of the severe IHC loss. The neurons that did respond to sound had narrow tuning curves. Some neurons in the double-dose group also had thresholds that were within the normal range, but most had thresholds that were elevated a mild-to-moderate degree. These results indicate that intact IHCs can retain relatively normal sensitivity and tuning despite massive IHC loss in surrounding regions of the cochlea. However, the spontaneous and driven discharge rates of neurons in the carboplatin-treated animals were significantly lower than normal. These changes could conceivably be due to sublethal damage to surviving IHCs or to postsynaptic dysfunction in the auditory nerve.