Ototoxicity remains a major dose-limiting side effect of cisplatin. The current studies were carried out to evaluate the effectiveness of a novel Src-protein tyrosine kinase inhibitor in protecting the ear from cisplatin ototoxicity without compromising cisplatin's antitumor effects. The Src inhibitor has been shown to be effective in protecting the ear from noise-induced hearing loss. Three studies were carried out to determine whether this compound has otoprotective activity in rats treated with cisplatin. The first two studies used the Src inhibitor as a cotreatment with single doses of cisplatin in Fischer 344/NHsd rats and nude rats, respectively. Cochlear damage was assessed by auditory brainstem response threshold shifts and outer hair cell loss. The third study was carried out in nude rats with implanted HT-29 tumors, and the Src inhibitor was administered as a cotreatment with a lower dose of cisplatin. Cochlear damage and changes in tumor volume were assessed in the third study. In the first two studies, cotreatment with the Src inhibitor reduced cisplatin-induced hearing loss significantly. In the third study, little hearing loss was induced because of the use of a lower dose of cisplatin. However, cotreatment with the Src inhibitor did not exert a negative effect on cisplatin's slowing of tumor growth in the treated rats. The findings suggest that the Src inhibitor may provide an effective cotreatment with cisplatin to reduce cisplatin's ototoxicity, without compromising its antitumor capability.

Our study was to elucidate the mechanisms whereby BMS-345541 (BMS, a specific IκB kinase β inhibitor) inhibits the repair of DNA double-strand breaks (DSBs) and evaluate whether BMS can sensitize MCF-7 breast cancer cells (MCF-7 cells) to ionizing radiation (IR) in an apoptosis-independent manner. In this study, MCF-7 cells were exposed to IR in vitro and in vivo with or without pretreatment of BMS. The effects of BMS on the repair of IR-induced DSBs by homologous recombination (HR) and non-homologous end-joining (NHEJ) were analyzed by the DR-GFP and EJ5-GFP reporter assays and IR-induced γ-H2AX, 53BP1, Brca1 and Rad51 foci assays. The mechanisms by which BMS inhibits HR were examined by microarray analysis and quantitative reverse transcription PCR. The effects of BMS on the sensitivity of MCF-7 cells to IR were determined by MTT and clonogenic assays in vitro and tumor growth inhibition in vivo in a xenograft mouse model. The results showed that BMS selectively inhibited HR repair of DSBs in MCF-7 cells, most likely by down-regulation of several genes that participate in HR. This resulted in a significant increase in the DNA damage response that sensitizes MCF-7 cells to IR-induced cell death in an apoptosis-independent manner. Furthermore, BMS treatment sensitized MCF-7 xenograft tumors to radiation therapy in vivo in an association with a significant delay in the repair of IR-induced DSBs. These data suggest that BMS is a novel HR inhibitor that has the potential to be used as a radiosensitizer to increase the responsiveness of cancer to radiotherapy.

In the present study, glial cell responses to spiral ganglion neuron (SGN) degeneration were evaluated using a murine model of auditory neuropathy. Ouabain, a well-known Na,K-ATPase inhibitor, has been shown to induce SGN degeneration while sparing hair cell function. In addition to selectively removing type I SGNs, ouabain leads to hyperplasia and hypertrophy of glia-like cells in the injured auditory nerves. As the transcription factor Sox2 is predominantly expressed in proliferating and undifferentiated neural precursors during neurogenesis,we sought to examine Sox2 expression patterns following SGN injury by ouabain. Real-time RT-PCR and Western blot analyses of cochlea indicated a significant increase in Sox2 expression by 3 days posttreatment with ouabain. Cells incorporating bromodeoxyuridine(BrdU) and expressing Sox2 were counted in the auditory nerves of control and ouabain-treated ears. The glial phenotype of Sox2+cells was identified by two neural glial markers: S100 and Sox10. The number of Sox2+ glial cells significantly increased at 3 days post-treatment and reached its maximum level at 7 days post-treatment. Similarly,the number of BrdU+ cells increased at 3 and 7 days post-treatment in the injured nerves. Quantitative analysis with dual-immunostaining procedures indicated that about 70% of BrdU+ cells in the injured nerves were Sox2+ glial cells. These results demonstrate that up-regulation of Sox2 expression is associated with increased cell proliferation in the auditory nerve after injury.

With the exception of humans, the somata of type I spiral ganglion neurons (SGNs) of most mammalian species are heavily myelinated. In an earlier study, we used Ly5.1 congenic mice as transplant recipients to investigate the role of hematopoietic stem cells in the adult mouse inner ear. An unanticipated finding was that a large percentage of the SGNs in this strain were unmyelinated. Further characterization of the auditory phenotype of young adult Ly5.1 mice in the present study revealed several unusual characteristics, including 1) large aggregates of unmyelinated SGNs in the apical and middle turns, 2) symmetrical junction‐like contacts between the unmyelinated neurons, 3) abnormal expression patterns for CNPase and connexin 29 in the SGN clusters, 4) reduced SGN density in the basal cochlea without a corresponding loss of sensory hair cells, 5) significantly delayed auditory brainstem response (ABR) wave I latencies at low and middle frequencies compared with control mice with similar ABR threshold, and 6) elevated ABR thresholds and deceased wave I amplitudes at high frequencies. Taken together, these data suggest a defect in Schwann cells that leads to incomplete myelinization of SGNs during cochlear development. The Ly5.1 mouse strain appears to be the only rodent model so far identified with a high degree of the “human‐like” feature of unmyelinated SGNs that aggregate into neural clusters. Thus, this strain may provide a suitable animal platform for modeling human auditory information processing such as synchronous neural activity and other auditory response properties. J. Comp. Neurol. 518:3254–3271, 2010. © 2010 Wiley‐Liss, Inc.

Previous studies reported that exposure to non-traumatic level sounds after traumatic noise exposure reduced the degree of noise-induced hearing loss and hair cell stereocilia damage. The current study investigated the effects of a 3-day post-noise acoustic environment on the degree of noise-induced hearing loss and cochlear damage. Female chinchillas were exposed to traumatic continuous noise (4kHz octave-band noise) at 107dB SPL for 1h and then placed in either an augmented acoustic environment (AAE) or deprived acoustic environment (DAE) for 3days. The AAE group was exposed to a broad-band noise (4–20kHz) at 80dB SPL and the DAE animals were fit with conventional earplugs to minimize the level of acoustic stimulation. Auditory brainstem responses (ABRs) were recorded before and 3days after the traumatic noise exposure. The AAE group showed a significantly lower average threshold shift at the frequencies of 4 and 8kHz (p<0.01). Correspondingly, significantly fewer missing and dying outer hair cells (OHCs) were observed in the AAE group than in the DAE group. Although the cochlear reduced and oxidized glutathione levels (GSH and GSSG, respectively) were essentially the same in two groups at day 3, significant correlations were found between GSSG levels and mean ABR threshold shift (1–16kHz) in the AAE group; as well as GSSG and percentage of total OHC loss in the DAE group. The results suggest that post-noise acoustic environment influenced the degree of hearing loss and OHC deterioration after traumatic noise exposure.

Objectives/Hypothesis: To investigate the effects of an augmented acoustic environment (AAE) on age-related hearing loss (ARHL) and outer hair cell (OHC) pathology in middle-aged Fischer 344/NHsd (F344/NHsd) rats.Methods: Eleven F344/NHsd rats were divided into two groups: 1) the AAE group (n = 5), which was exposed to 4-20 kHz broadband noise at 80 dB SPL for 12 h/d, 5 d/wk for 13 weeks starting from 16 months of age; and 2) the control group (n = 6), which did not receive the AAE during the same time span. Auditory brainstem response thresholds were obtained at different time points, and OHC pathology was examined after 13 weeks of AAE using propidium iodide and antiprestin antibody staining.Results: The AAE-treated rats showed smaller mean threshold shifts (-1 to -3 dB) at 20-40 kHz than the control group (7.5-16.7 dB) at 13 weeks. No significant group differences were observed in the percentage of missing OHCs or abnormal OHC nuclei. However, examination of prestin in a pair of AAE and control rats revealed more uniform prestin staining intensity among OHCs in the AAE-treated cochlea than in the control cochlea.Conclusions: Thirteen-week AAE treatment in the middle-aged F344/NHsd rats slowed progression of ARHL. The AAE did not show a significant effect on OHC degeneration, but it is speculated that the AAE may maintain the integrity of prestin to preserve OHC functionality. However, further study is warranted to understand the protective mechanism of AAE as an intervention against ARHL.

As previously reported [Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2006. Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats. Neurobiol. Aging 27, 490–500; Buckiova, D., Popelar, J., Syka, J., 2007. Aging cochleas in the F344 rat: morphological and functional changes. Exp. Gerontol. 42, 629–638; Bielefeld, E.C., Coling, D., Chen, G.D., Li, M.N., Tanaka, C., Hu, B.H., Henderson, D., 2008. Age-related hearing loss in the Fischer 344/NHsd rat substrain. Hear. Res. 241, 26–33], aged Fischer 344 (F344) rats with severe hearing loss retain many outer hair cells (OHCs) especially in the middle turn of the cochlea. The current study confirmed the previous findings showing that aged OHCs were present, but dysfunctional. Distortion product otoacoustic emissions (DPOAE), which are believed to reflect in vivo OHC motility, were absent in the aged rats while the majority of OHCs (>80%) were present and morphologically intact. There was no detectable injury of OHC stereocilia as assessed by actin-staining and examination under the light microscope. Cochlear microphonics (CM) at 12 kHz, recorded from the middle turn, only showed a slight age-related reduction, indicating a normal mechanoelectrical transduction apparatus in the remaining OHCs in the cochlear regions with 10–20% OHC loss. Activities of succinate dehydrogenase (SDH), an enzyme shared by the citric acid cycle and the mitochondrial electron transport chain (METC), were also at normal levels in aged OHCs. Importantly, aged OHCs showed reduced levels of prestin immunolabeling compared to young controls. Together with our previous finding showing that the stria vascularis and endocochlear potential were essentially normal in aged F344 rats [Bielefeld, E.C., Coling, D., Chen, G.D., Li, M.N., Tanaka, C., Hu, B.H., Henderson, D., 2008. Age-related hearing loss in the Fischer 344/NHsd rat substrain. Hear. Res. 241, 26–33], the results suggest that disruption of prestin is the major cause of DPOAE loss and loss of cochlear sensitivity.

Apoptotic cell death has been implicated in cochlear degeneration during aging. To better understand the impact and the biological process of outer hair cell (OHC) apoptosis, we investigated the contribution of apoptotic cell death to the formation of the OHC lesions, and observed the temporal patterns of the occurrence of apoptotic events associated with the mitochondrial pathway in Fischer 344/NHsd rats, with ages ranging from 20 to 27 months. The results showed that the ratio of apoptotic to necrotic OHCs was 8:1. During the process of cell degeneration, the onset of Bax expression, cytochrome c release, and nuclear DNA fragmentation preceded the onset of nuclear condensation. In contrast, the activation of caspases-3 and -9, as well as the degradation of F-actin, took place after the onset of nuclear condensation. The results of this study suggest that the initiation of nuclear degradation is a caspase-3-independent process. Moreover, the study revealed that OHCs with Bax expression or cytochrome c release could enter either the apoptotic or necrotic pathway, suggesting the presence of a regulatory mechanism that guides degenerating OHCs to die via either the apoptotic or necrotic pathway.

Studies of the F344 rat have shown a variety of age-related auditory anatomy and physiology changes. The current study was undertaken to clarify the ARHL in the F344 rat, by examining the auditory pathway of the F344/NHsd substrain that is distributed by Harlan Laboratories for research in the United States. The F344/NHsd rat begins to lose its hearing at about 12 months, and by 24 months, there are 50–60dB auditory brainstem response threshold shifts at 20 and 40kHz and 20dB losses at 5–10kHz. Distortion product otoacoustic emissions (DPOAE) amplitudes at 1.8–12kHz stimuli were depressed in the older (18–24 months) rats. Amplitude input–output functions of the compound action potential (CAP) were also depressed across frequency. The endocochlear potential (EP) was 90–100mV in the 3month old rats. All but one of the 24month old rats’ EPs were in the +75–85mV range. Tympanometry revealed no differences in middle ear function between the young and older rats. Collectively, these findings suggest damage to the outer hair cells, but anatomical examination of the outer hair cells revealed a relative lack of cell loss compared to the magnitude of the hearing and DPOAE loss.