Haiyan Jiang

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 trauma impairs neurogenesis in the rat hippocampus

The hippocampus, a major site of neurogenesis in the adult brain, plays an important role in memory. Based on earlier observations where exposure to high-intensity noise not only caused hearing loss but also impaired memory function, it is conceivably that noise exposure may suppress hippocampal neurogenesis. To evaluate this possibility, nine rats were unilaterally exposed for 2 h to a high-intensity, narrow band of noise centered at 12 kHz at 126 dB SPL. The rats were also screened for noise-induced tinnitus, a potential stressor which may suppress neurogenesis. Five rats developed persistent tinnitus-like behavior while the other four rats showed no signs of tinnitus. Age-matched sham controls showed no signs of hearing loss or tinnitus. The inner ear and hippocampus were evaluated for sensory hair cell loss and neurogenesis 10 weeks post-exposure. All noise exposed rats showed severe loss of sensory hair cells in the noise-exposed ear, but essentially no damage in the unexposed ear. Frontal sections from the hippocampus were immunolabeled for doublecortin to identify neuronal precursor cells, or Ki67 to label proliferating cells. Noise-exposed rats showed a significant reduction of neuronal precursors and fewer dividing cells as compared to sham controls. However, we could not detect any difference between rats with behavioral evidence of tinnitus versus rats without tinnitus. These results show for the first time that high intensity noise exposure not only damages the cochlea but also causes a significant and persistent decrease in hippocampal neurogenesis that may contribute to functional deficits in memory.

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.

Cell death after co-administration of cisplatin and ethacrynic acid

Ethacrynic acid (EA) significantly enhances the ototoxic effects of cisplatin. To gain insights into the mechanisms underlying Cis/EA ototoxicity, cochleas were labeled with several apoptotic markers. Cis/EA treatment caused extensive outer hair cell (OHC) and inner hair cell (IHC) damage; OHC lesions decreased from the base towards apex of the cochlea whereas the IHC lesion was relatively constant (25-60%) along the length of the cochlea. Propidium iodide labeled OHC nuclei appeared relatively normal at 6h post-treatment, were condensed and fragmented at 12h post-treatment and were frequently missing 48 h post-treatment. Initiator caspase 8, associated with membrane death receptors, and TRADD, a protein that recruits caspase 8, were present in OHC at 6h post-treatment. Caspase 8 labeling increased from 6 to 24h, but was largely absent at 48 h post-treatment. Executioner caspase 3 and caspase 6, which lie downstream of caspase 8, were expressed in OHC 12-24h post-treatment. Initiator caspase 9, associated with mitochondrial damage, was only expressed at low levels at 48 h post-treatment. These results suggest that the rapid onset of Cis/EA induced programmed cell death is initiated by membrane death receptors associated with TRADD and caspase 8.

Differential expression of apoptosis-related genes in the cochlea of noise-exposed rats

Exposure to intense noise induces apoptosis in hair cells in the cochlea. To identify the molecular changes associated with noise-induced apoptosis, we used quantitative real-time PCR to evaluate the changes in 84 apoptosis-related genes in cochlear samples from the sensory epithelium and lateral wall. Sprague-Dawley rats exposed to a continuous noise at 115 dB SPL for 2 h. The exposure caused a 40-60 dB threshold shift 4 h post-exposure that decreased to 20-30 dB 7 days post-exposure. These functional changes were associated with apoptotic markers including nuclear condensation and fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Immediately after the noise exposure, 12 genes were downregulated, whereas only one gene (Traf4) was upregulated. At 4 h post-exposure, eight genes were upregulated; three (Tnrsf1a, Tnfrsf1b, Tnfrst5) belonged to the Tnfrsf family, three (Bir3, Mcl1 and Prok2) have anti-apoptotic properties and one (Gadd45a) is a target of p53. At 7 days post-exposure, all the upregulated genes returned to pre-noise levels. Interestingly, the normal control cochlea had high constitutive levels of several apoptosis-related genes. These constitutively expressed genes, together with the inducible genes, may participate in the induction of cochlear apoptotic activity.

Mechanisms of rapid sensory hair-cell death following co-administration of gentamicin and ethacrynic acid

Concurrent administration of a high dose of gentamicin (GM; 125mg/kg IM) and ethacrynic acid (EA; 40mg/kg IV) results in rapid destruction of virtually all cochlear hair cells; however, the cell death signaling pathways underlying this rapid form of hair-cell degeneration are unclear. To elucidate the mechanisms underlying GM/EA-mediated cell death, several key cell death markers were assessed in the chinchilla cochlea during the early stages of degeneration. In the middle and basal turns of the cochlea, massive hair-cell loss including destruction of the stereocilia and cuticular plate occurred 12h after GM/EA treatment. Condensation and fragmentation of outer hair-cell nuclei, morphological features of apoptosis, were first observed 5-6h post-treatment in the basal turn of the cochlea. Metabolic function, reflected by succinate dehydrogenase histochemistry and mitochondrial staining, decreased significantly in the basal turn 4h following GM/EA treatment; these early changes were accompanied by the release of cytochrome c from the mitochondria into the cytosol and intense expression of initiator caspase-9 and effector caspase-3. GM/EA failed to induce expression of extrinsic initiator caspase-8. These results suggest that the rapid loss of hair cells following GM/EA treatment involves cell death pathways mediated by mitochondrial dysfunction leading to the release of cytochrome c, activation of initiator caspase-9 and effector caspase-3.

Relationship between noise-induced hearing-loss, persistent tinnitus and growth-associated protein-43 expression in the rat cochlear nucleus: does synaptic plasticity in ventral cochlear nucleus suppress tinnitus?

Aberrant, lesion-induced neuroplastic changes in the auditory pathway are believed to give rise to the phantom sound of tinnitus. Noise-induced cochlear damage can induce extensive fiber growth and synaptogenesis in the cochlear nucleus, but it is currently unclear if these changes are linked to tinnitus. To address this issue, we unilaterally exposed nine rats to narrow-band noise centered at 12 kHz at 126 dB sound pressure level (SPL) for 2 h and sacrificed them 10 weeks later for evaluation of synaptic plasticity (growth-associated protein 43 [GAP-43] expression) in the cochlear nucleus. Noise-exposed rats along with three age-matched controls were screened for tinnitus-like behavior with gap prepulse inhibition of the acoustic startle (GPIAS) before, 1-10 days after, and 8-10 weeks after the noise exposure. All nine noise-exposed rats showed similar patterns of severe hair cell loss at high- and mid-frequency regions in the exposed ear. Eight of the nine showed strong up-regulation of GAP-43 in auditory nerve fibers and pronounced shrinkage of the ventral cochlear nucleus (VCN) on the noise-exposed side, and strong up-regulation of GAP-43 in the medial ventral VCN, but not in the lateral VCN or the dorsal cochlear nucleus. GAP-43 up-regulation in VCN was significantly greater in Noise-No-Tinnitus rats than in Noise-Tinnitus rats. One Noise-No-Tinnitus rat showed no up-regulation of GAP-43 in auditory nerve fibers and only little VCN shrinkage, suggesting that auditory nerve degeneration plays a role in tinnitus generation. Our results suggest that noise-induced tinnitus is suppressed by strong up-regulation of GAP-43 in the medial VCN. GAP-43 up-regulation most likely originates from medial olivocochlear neurons. Their increased excitatory input on inhibitory neurons in VCN may possibly reduce central hyperactivity and tinnitus.

Addition of exogenous NAD+ prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.

Background Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects. Principal Findings In this study, we show that the coenzyme NAD+, known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD+ protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD+ reduced the levels of these oxidative stress and apoptosis markers. Conclusions/Significance Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD+ suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.

Separate and combined effects of Sod1 and Cdh23 mutations on age-related hearing loss and cochlear pathology in C57BL/6J mice

Both the ahl allele of Cdh23 and the null mutation of Sod1 have been shown to contribute to age-related hearing loss (AHL) in mice, but mixed strain backgrounds have confounded analyses of their individual and combined effects. To test for the effects of Sod1 deficiency independently from those of Cdh23(ahl), we produced mice with four digenic genotypes: Sod1(+/+)Cdh23(ahl)(/ahl), Sod1(+/+)Cdh23(+/+), Sod1(-/-)Cdh23(ahl)(/ahl), and Sod1(-/-)Cdh23(+/+), all on a uniform C57BL(/)6J strain background. We assessed hearing loss by ABR threshold measurements and evaluated cochlear pathologies in age-matched mice of each digenic combination. ABR analysis showed that Sod1(+/+)Cdh23(+/+) mice retain normal hearing up to 15 months of age and that hearing loss of Sod1(+/+)Cdh23(ahl)(/ahl) mice is more age and frequency dependent than that of Sod1(-/-)Cdh23(+/+) mice. ABR results also showed that mice with both gene mutations (Sod1(-/-)Cdh23(ahl)(/ahl)) exhibit the earliest onset and most severe hearing loss, greater than predicted for strictly additive effects. Histological analysis of cochleas showed that hair cell lesions are most severe in Sod1(-)(/-)Cdh23(ahl)(/ahl) mice followed closely by Sod1(+)(/+)Cdh23(ahl)(/ahl) mice and much smaller in Sod1(-)(/-)Cdh23(+)(/+) and Sod1(+)(/+)Cdh23(+)(/+) mice. Despite extensive damage to cochlear hair cells, vestibular hair cells appeared remarkably normal in all strains. Although both Sod1(-/-) and Cdh23(ahl)(/ahl) genotypes had strong effects on hearing loss, the Cdh23(ahl/ahl) genotype was primarily responsible for the increase in hair cell loss, suggesting that the two mutations have different underlying mechanisms of pathology.

Ouabain-induced apoptosis in cochlear hair cells and spiral ganglion neurons in vitro.

Ouabain is a common tool to explore the pathophysiological changes in adult mammalian cochlea in vivo. In prior studies, locally administering ouabain via round window membrane demonstrated that the ototoxic effects of ouabain in vivo varied among mammalian species. Little is known about the ototoxic effects in vitro. Thus, we prepared cochlear organotypic cultures from postnatal day-3 rats and treated these cultures with ouabain at 50, 500, and 1000 μM for different time to elucidate the ototoxic effects of ouabain in vitro and to provide insights that could explain the comparative ototoxic effects of ouabain in vivo. Degeneration of cochlear hair cells and spiral ganglion neurons was evaluated by hair-cell staining and neurofilament labeling, respectively. Annexin V staining was used to detect apoptotic cells. A quantitative RT-PCR apoptosis-focused gene array determined changes in apoptosis-related genes. The results showed that ouabain-induced damage in vitro was dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway. The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.