Sandra L. McFadden

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.

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.

R-phenylisopropyladenosine attenuates noise-induced hearing loss in the chinchilla

Reactive oxygen species, which are cytotoxic to living tissues, are thought to be partly responsible for noise-induced hearing loss. In this study R-phenylisopropyladenosine (R-PIA), a stable non-hydrolyzable adenosine analogue which has been found effective in upregulating antioxidant enzyme activity levels, was topologically applied to the round window of the right ears of chinchillas. Physiological saline was applied to the round window of the left ears (control). The animals were then exposed to a 4 kHz octave band noise at 105 dB SPL for 4 h. Inferior colliculus evoked potential thresholds and distortion product otoacoustic emissions (DPOAE) were measured and hair cell damage was documented. The mean threshold shifts immediately after the noise exposure were 70-90 dB at frequencies between 2 and 16 kHz. There were no significant differences in threshold shifts at this point between the R-PIA-treated and control ears. By 4 days after noise exposure, however, the R-PIA-treated ears showed 20-30 dB more recovery than saline-treated ears at frequencies between 4 and 16 kHz. More importantly, threshold measurements made 20 days after noise exposure showed 10-15 dB less permanent threshold shifts in R-PIA-treated ears. The amplitudes of DPOAE also recovered to a greater extent and outer hair cell losses were less severe in the R-PIA-treated ears. The results suggest that administration of R-PIA facilitates the recovery process of the outer hair cell after noise exposure.

The Role of Antioxidants in Protection from Impulse Noise

ABSTRACT: The hearing loss from exposure to noise and ototoxic drugs share a number of audiological and pathological similarities. Recent research has shown that reactive oxygen species (ROS) may be a common factor in both noise‐ and drug‐induced hearing loss. This review describes three experiments that point to ROS as a causative factor in both noise‐ and drug‐induced hearing loss and antioxidants as a protective agent. In the first experiment, the ears of chinchillas were treated with R‐N6‐phenylisopropyladenoisine (R‐PIA) and exposed to 150‐dB impulse noise. The treated ears developed substantially less permanent hearing loss (PTS) and hair cell loss than the untreated ears. One interpretation of this experiment is that R‐PIA increases the availability of glutathione (GSH). In the second experiment, the role of GSH was specifically examined. The ears of chinchillas were treated with glutathione monoethylester (GEE), a pro‐GSH drug that has been shown to readily cross cell membranes and increase GSH levels. The GEE‐treated ears had significantly less PTS and hair‐cell loss than the nontreated ear. Previous research has shown that moderate levels of noise exposure can increase a subjects resistance to noise, and also increase the availability of antioxidant enzymes in the cochlea. In the third experiment, chinchillas were given a series of “toughening” exposures (i.e., 6 h of a 0.5‐kHz OB noise at 95 dB for 10 days). After the series of “toughening” exposures, the subjects were treated with carboplatin, a drug that causes massive inner‐hair‐cell lesions in the chinchilla. The animals receiving the 10‐day toughening exposure developed less PTS and hair‐cell loss than the control animals.

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.

Noise-induced hearing loss in chinchillas pre-treated with glutathione monoethylester and R-PIA.

The protective effects of glutathione monoethylester (GEE) and GEE in combination with R-N6-phenylisopropyladenosine (R-PIA) were evaluated in the chinchilla when exposed to impulse (145 dB pSPL) or continuous (105 dB SPL, 4 kHz OB) noise. Six groups of 10 chinchillas were used as subjects. Before exposure to noise, the subjects were anesthetized, a 30 microl drop of drug was placed on the round window (GEE [50, 100, 150 mM], GEE 50 mM and R-PIA). Forty minutes later the subject was exposed to either impulse or continuous noise. The 50 mM treatment provided significant protection from impulse noise, but not from continuous noise exposure. The combination provided significant protection from both the continuous and impulse noise. In a separate set of experiments, glutathione (GSH) levels were measured in the perilymph. All the drug treatments elevated GSH levels. The results are discussed in terms of antioxidant treatments as a prophylactic measure against noise-induced hearing loss.

M40403, a superoxide dismutase mimetic, protects cochlear hair cells from gentamicin, but not cisplatin toxicity.

Gentamicin, an aminoglycoside antibiotic, and cisplatin, a platinum-based anticancer drug, are two commonly used clinical drugs with ototoxic side effects. The ototoxicity of gentamicin and cisplatin has been linked to the production of reactive oxygen species (ROS), although the specific ROS pathways have not been identified. One ROS that might play a role in ototoxicity is the superoxide radical, which is enzymatically dismutated to molecular oxygen and hydrogen peroxide by endogenous superoxide dismutase (SOD) enzymes. M40403, a manganese-based nonpeptidyl molecule that mimics the activity of SOD, was tested for its ability to protect against gentamicin and cisplatin toxicity in cochlear organotypic cultures from neonatal C57BL/10J mice. Cultures were treated with gentamicin or cisplatin alone or in combination with M40403. M40403 alone had no effect on outer hair cell (OHC) or inner hair cell (IHC) survival at doses of 1, 5, and 10 microM, but a high dose of 30 microM reduced hair cell numbers by approximately 30%. Gentamicin alone and cisplatin alone killed OHCs and IHCs in a dose-dependent manner. The addition of M40403 to gentamicin-treated cultures significantly increased OHC and IHC survival in a dose-dependent manner, whereas M40403 failed to protect hair cells in cisplatin-treated cultures at any dose. The results suggest that the toxicity of gentamicin and cisplatin to cochlear hair cells are mediated by different pathways. Clinically, increased levels of SOD or SOD mimetics might provide significant protection against aminoglycoside ototoxicity.

Selective loss of inner hair cells and type-I ganglion neurons in carboplatin-treated chinchillas. Mechanisms of damage and protection.

Abstract: Carboplatin preferentially destroys inner hair cells (IHCs) and type‐I spiral ganglion neurons while sparing outer hair cells (OHCs). Loss of IHCs and type‐I ganglion cells is associated with a significant reduction of the compound action potential (CAP). However, the cochlear microphonic (CM) potential and distortion product otoacoustic emissions (DPOAEs) remain normal, indicating that the OHCs are functionally intact. In the vestibular system, carboplatin selectively destroys type‐I hair cells and their afferent neurons. Damage of type‐I vestibular hair cells and their afferent terminals is associated with significant depression of nystagmus induced by cold, caloric stimulation. Histochemical studies revealed a rapid decrease in succinate dehydrogenase (SDH) staining in IHCs soon after carboplatin treatment, and staining intensity remained depressed in surviving IHCs for at least 1 month after carboplatin treatment. These results suggest that carboplatin depresses the metabolic function in surviving IHCs. Several lines of evidence suggest that free radicals may contribute to carboplatin‐induced sensory cell damage. Intracochlear infusion of l‐buthionine‐[S,R]‐sulfoximine (BSO), which depletes intracellular glutathione (GSH), increases IHC and OHC loss. Previous in vitro studies have shown that neurotrophin 4/5 (NT‐4/5) promotes the survival of spiral ganglion neurons from cisplatin ototoxicity. In vivo perfusion of NT‐4/5 promoted the survival of spiral ganglion neurons, but did not protect the hair cells.

Time course of efferent fiber and spiral ganglion cell degeneration following complete hair cell loss in the chinchilla

Ethacrynic acid (EA) is known to interact with aminoglycoside antibiotics such as gentamicin (GM). In the chinchilla, co-administration of GM and EA can produce hair cell lesions ranging from a small loss of outer hair cells (OHCs) in the base of the cochlea to complete destruction of all hair cells, depending on dosing parameters. Although hair cell loss has been characterized, little is known about the fate of efferent fibers or spiral ganglion neurons (SGNs) in this model. To study the time course of efferent fiber and SGN loss, chinchillas were injected with GM (125 mg/kg IM) followed immediately by EA (40 mg/kg IV). Estimates of efferent fiber loss and density changes were made after 3 days or 1, 2, 3, or 4 weeks of survival. Estimates of SGN loss and density changes were made after 15 days or 1, 2, 4, or 6 months of survival. Cochlear function was rapidly abolished and all cochlear hair cells were missing within 24 h after treatment. Inner hair cells (IHCs) in the middle turn of the cochlea died earlier than cells in the apex or base, and OHCs in Rows 1 and 2 died earlier than OHCs in Row 3. Degeneration of efferent nerve fibers began 3-7 days post-injection, versus 15-30 days for SGNs, and the loss of efferent fibers was essentially complete within 1 month, versus 2-4 months for SGNs. The rapid time course of efferent fiber and SGN loss in the chinchilla may make it a practical model for studying mechanisms of neural loss and survival in the mammalian inner ear.

Dietary vitamin C supplementation reduces noise-induced hearing loss in guinea pigs

Vitamin C (ascorbate) is a water-soluble, low molecular weight antioxidant that works in conjunction with glutathione and other cellular antioxidants, and is effective against a variety of reactive oxygen species, including superoxide and hydroxyl radicals that have been implicated in the etiology of noise-induced hearing loss (NIHL). Whereas most animals can manufacture their own vitamin C, humans and a few other mammals such as guinea pigs lack the terminal enzyme for vitamin C synthesis and must obtain it from dietary sources. To determine if susceptibility to NIHL could be influenced by manipulating dietary levels of vitamin C, albino guinea pigs were raised for 35 days on a diet with normal, supplemented or deficient levels of ascorbate, then exposed to 4 kHz octave band noise at 114 dB SPL for 6 h to induce permanent threshold shifts (PTS) of the scalp-recorded auditory brainstem response. Animals that received the highest levels of dietary ascorbate developed significantly less PTS for click stimuli and 4, 8, 12, and 16 kHz tones than animals on normal and deficient diets. Outer hair cell loss was minimal in all groups after noise exposure, but permanent damage to stereocilia were observed in noise-exposed ears. The results support the hypothesis that dietary factors influence individual susceptibility to hearing loss, and suggest that high levels of vitamin C may be beneficial in reducing susceptibility to NIHL.