Gingko biloba extracts protect auditory hair cells from cisplatin-induced ototoxicity by inhibiting perturbation of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) may play an important role in the hearing process. Cisplatin is an anticancer drug that causes hearing loss and Gingko biloba extracts (EGb 761) have been used as an antioxidant and enhancer for GJIC. The purpose of this study was to examine the efficiency of EGb 761 in protecting against cisplatin-induced apoptosis and disturbance of GJIC. House Ear Institute-Organ of Corti 1 auditory cells were cultured and treated with cisplatin (50 µM) and EGb (300 µg/ml) for 24h, and then analyzed by immunocytochemistry (Annexin V/propidium iodide) and Western blots. GJIC was evaluated by scrape-loading dye transfer (SLDT). Basal turn organ of Corti (oC) explants from neonatal (p3) rats were exposed to cisplatin (1-10 µM) and EGb (50-400 µg/ml). The number of intact hair cells was counted by co-labeling with phalloidin and MyoVIIa. EGb prevented cisplatin-induced apoptosis in immunostaining and decreased caspase 3 and poly-ADP-ribose polymerase bands, which were increased in cisplatin-treated cells in Western blots. EGb prevented abnormal intracellular locations of connexin (Cx) 26, 30, 31, and 43 in cells treated with cisplatin and increased quantities of Cx bands. EGb also prevented cisplatin-induced disturbance of GJIC in SLDT. In oC explants, EGb significantly prevented hair cell damage induced by cisplatin. In animal studies, EGb significantly prevented cisplatin-induced hearing loss across 16 and 32 kHz. These results show that cisplatin induces ototoxicity including hearing loss as well as down-regulation of GJIC and inhibition of Cxs in auditory cells. EGb prevents hearing loss in cisplatin-treated rats by inhibiting down-regulation of Cx expression and GJIC. The disturbance of GJIC or Cx expression may be one of the important mechanisms of cisplatin-induced ototoxicity.

A combination of cilostazol and Ginkgo biloba extract protects against cisplatin-induced Cochleo-vestibular dysfunction by inhibiting the mitochondrial apoptotic and ERK pathways.

Cisplatin (cis-diammine-dichloroplatinum; CDDP) is an anticancer drug that induces significant hearing loss and balance dysfunction as side effects. Cilostazol (CS, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3, 4-dihydro-2-(1H)-quinolinone) has neuroprotective and antioxidant effects, whereas Ginkgo biloba extract (GbE) has preventive effects on CDDP-induced hearing loss in rats, and GbE enhances the antiatherogenic effect of CS by inhibiting the generation of reactive oxygen species (ROS). The purpose of this study was to investigate the effects of renexin (RXN), which contains GbE and CS, against CDDP-induced cochleo-vestibular dysfunction in rats and to elucidate the mechanism underlying the protective effects of RXN on auditory cells. Rats intraperitoneally injected with CDDP exhibited an increase in hearing threshold and vestibular dysfunction, which agreed with hair cell damage in the Organ of Corti and otoliths. However, these impairments were significantly prevented in a dose-dependent manner by pre- and co-treatment with RXN, and these preventive effects in RXN-treated rats were more prominent than those in GbE-treated rats. In a CDDP pharmacokinetic study, platinum concentration was very similar between CDDP-only treated and RXN+CDDP cotreated rats. RXN markedly attenuated CDDP-induced intracellular ROS and significantly reduced CDDP-activated expression of p-extracellular regulated kinase (ERK), BAX, cytochrome c, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, but increased BCL-XL expression. These results show that RXN may have a synergistic effect by strongly protecting hearing and vestibular dysfunction induced by CDDP by inhibiting ROS production, mitochondrial pathways and the ERK pathway, without interfering with CDDP pharmacokinetics. Therefore, RXN could potentially be used to reduce CDDP-related hearing loss and dizziness.

Pravastatin attenuates noise-induced cochlear injury in mice.

Noise-induced hearing loss (NIHL) is one of the most common forms of sensorineural hearing loss and a well-known contributor to presbycusis. Based on the generation of reactive oxygen species (ROS) in the pathogenesis of NIHL, augmentation of the antioxidative defense system is a major target for pharmacological prevention. In this study, we assessed whether administration of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of cholesterol synthesis, before noise exposure protects against cochlear injury in BALB/c mice. Noise exposure produced both compound threshold shift (CTS) and permanent threshold shift (PTS) over 40 dB at 16 and 32 kHz. Pretreatment with pravastatin (25 mg/kg) for 5 days significantly decreased both CTS and PTS. Pravastatin also reduced hair cell death after noise exposure in the cochlea, which was identified by surface preparation and scanning electron microscopy (SEM). It also reduced the formation of noise-induced 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation. Activation of Rac1, one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, which is a major superoxide generator in the cell membrane, was inhibited by the administration of pravastatin. These findings suggest that pravastatin can protect against cochlear acoustic injury by lowering ROS generation via inhibition of the formation of the NADPH oxidase complex. This study will be helpful for the development of new therapeutic strategies for NIHL and other hearing loss-related diseases caused by ROS overproduction.

α-Synuclein deficiency and efferent nerve degeneration in the mouse cochlea: a possible cause of early-onset presbycusis.

OBJECTIVES/HYPOTHESIS: Efferent nerves under the outer hair cells (OHCs) play a role in the protection of these cells from loud stimuli. Previously, we showed that cochlear α-synuclein expression is localized to efferent auditory synapses at the base of the OHCs. To prove our hypothesis that α-synuclein deficiency and efferent auditory deficit might be a cause of hearing loss, we compared the morphology of efferent nerve endings and α-synuclein expression within the cochleae of two mouse models of presbycusis. STUDY DESIGN: Comparative animal study of presbycusis. METHODS: The C57BL/6J(C57) mouse strain, a well-known model of early-onset hearing loss, and the CBA mouse strain, a model of relatively late-onset hearing loss, were examined. Auditory brainstem responses and distortion product otoacoustic emissions were recorded, and cochlear morphology with efferent nerve ending was compared. Western blotting was used to examine α-synuclein expression in the cochlea. RESULTS: Compared with CBA mice, C57 mice showed earlier onset high-frequency hearing loss and decreased function in OHCs, especially within high-frequency regions. C57 mice demonstrated more severe pathologic changes within the cochlea, particularly within the basal turn, than CBA mice of the same age. Weaker α-synuclein and synaptophysin expression in the efferent nerve endings and cochlear homogenates in C57 mice was observed. CONCLUSIONS: Our results support the hypothesis that efferent nerve degeneration, possibly due to differential α-synuclein expression, is a potential cause of early-onset presbycusis. Further studies at the cellular level are necessary to verify our results.

Epicatechin protects the auditory organ by attenuating cisplatin-induced ototoxicity through inhibition of ERK.

Cisplatin, a widely used chemotherapeutic drug, causes ototoxicity in a large percentage of patients. The purpose of this study was to determine the efficacy of epicatechin (EC) as an otoprotective agent to prevent cisplatin toxicity and to investigate the molecular mechanism of EC. The effects of EC on cisplatin-induced ototoxicity were investigated in a cochlear organ of Corti-derived cell line, HEI-OC1 and in a rat model. In addition, signaling mechanisms were investigated, specifically those involving MAP kinase. Cisplatin induced apoptosis and demonstrated, conjugation of annexin V/PI in FACS, and an increase of subG1 in HEI-OC1. EC protected HEI-OC1 against cisplatin and showed inhibition of cisplatin-induced apoptosis of the HEI-OC1 by transmission electron microscopy. Intratympanic administration of EC protected against cisplatin-induced ototoxicity in the rat model, as determined by auditory brainstem responses. EC inhibited activation of JNK, ERK, cytochrome-c and caspase-3 by cisplatin. An ERK Inhibitor, cisplatin-induced ototoxicity in a dose dependent manner but a JNK inhibitor did not. The results of this study suggest that EC may provide a mechanism by which ototoxicity caused by the administration of cisplatin can be reduced through the inhibition of ERK. EC may have clinical use as a chemopreventive agent that prevents cisplatin ototoxicity.

Histone deacetylase inhibition enhances adenoviral vector transduction in inner ear tissue.

Adenovirus vectors (AdVs) are efficient tools for gene therapy in many tissues. Several studies have demonstrated successful transgene transduction with AdVs in the inner ear of rodents [Kawamoto K, Ishimoto SI, Minoda R, Brough DE, Raphael Y (2003) J Neurosci 23:4395-4400]. However, toxicity of AdVs [Morral N, O'Neal WK, Rice K, Leland MM, Piedra PA, Aguilar-Cordova E, Carey KD, Beaudet AL, Langston C (2002) Hum Gene Ther 13:143-154.] or lack of tropism to important cell types such as hair cells [Shou J, Zheng JL, Gao WQ (2003) Mol Cell Neurosci 23:169-179] appears to limit their experimental and potential clinical utility. Histone deacetylase inhibitors (HDIs) are known to enhance AdV-mediated transgene expression in various organs [Dion LD, Goldsmith KT, Tang DC, Engler JA, Yoshida M, Garver RI Jr (1997) Virology 231:201-209], but their effects in the inner ear have not been documented. We investigated the ability of one HDI, trichostatin A (TSA), to enhance AdV-mediated transgene expression in inner ear tissue. We cultured neonatal rat macular and cochlear explants, and transduced them with an AdV encoding green fluorescent protein (Ad-GFP) under the control of a constitutive promoter for 24 h. In the absence of TSA, GFP expression was limited, and very few hair cells were transduced. TSA did not enhance transduction when applied at the onset of Ad-GFP transduction. However, administration of TSA during or just after Ad-GFP application increased GFP expression in supporting cells approximately fourfold. Moreover, vestibular hair cell transduction was enhanced approximately sixfold, and that of inner hair cells by more than 17-fold. These results suggest that TSA increases AdV-mediated transgene expression in the inner ear, including the successful transduction of hair cells. HDIs, some of which are currently under clinical trials (Sandor et al., 2002), could be useful tools in overcoming current limitations of gene therapy in the inner ear using Ad-GFP.

Three-dimensional anatomy of the temporal bone in normal mice.

This study was performed to determine the three-dimensional (3D) structure of the murine temporal bone and to provide a survey atlas of the temporal bone structures in mice. The temporal bones of adult BALB/c mice were examined and 3D high-resolution reconstructions of the temporal bone were obtained using a micro-CT system. Using the system described here, the bony labyrinth and membranous labyrinth could be investigated in a non-destructive manner. The turning rate of the cochlea was two (human rate: two and a half). The shapes of the superior and posterior semicircular canals were more flexed than those in humans. The malleus manubrium was directed anteriorly and was thin and fan-shaped like a Persian sword. The size of the incus relative to the malleus was smaller than that in the human ossicles. The 3D reconstruction of murine temporal bone described in this study provides anatomical information that will be useful in future studies using mouse model.

Generation of highly-reactive oxygen species is closely related to hair cell damage in rat organ of Corti treated with gentamicin.

Reactive oxygen species (ROS) have been suggested to play a major role in aminoglycoside-induced hair cell (HC) loss, but are difficult to detect. Moreover, ROS can occur normally in cells where they have roles in metabolism, cell signaling and other processes. Two new probes, aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) are dyes which selectively detect highly-reactive oxygen species (hROS), those most associated with cellular damage. We assessed the presence of hROS in the neonatal rat organ of Corti during chronic exposure to 50 microM gentamicin in vitro, to examine the relationship between cell damage and hROS across HC type and across the three cochlear turns. hROS were initially detected at 48 hours (h), with an increase at 72 h and persistence until at least 96 h. At 48 h, hROS were restricted to outer HCs and occurred prior to loss of stereocilia. At 72 h, outer HCs showed both hROS and stereocilia loss, and hROS were noted in a few inner HCs. Basal turn HCs showed more hROS than middle turn HCs. Very little hROS accumulation or stereocilia loss was observed in the apical turn, even at 72 h. First row outer HCs were most vulnerable to gentamicin-induced hROS, followed by second and then third row outer HCs. Inner HCs behaved similarly to third row outer HCs. By 96 h stereocilia damage was extensive, but surviving HCs showed persisting fluorescence. APF consistently showed more fluorescence than HPF. The results suggest that hROS accumulation is an important initial step in gentamicin-induced HC damage, and that the differential sensitivity of HCs in the organ of Corti is closely related to differences in hROS accumulation.

Rare cases of Ménière's disease in children.

Classical Ménière's disease is rarely found in children and literature regarding it is scarce. In general, the frequency of Ménière's disease in children is only 0.4-7.0 per cent of that in adults. The progression pattern of Ménière's disease in children is not known well. Here, we report three cases of Ménière's disease in children less than 15 years old, treated over nine years. The three cases comprise 14- and 13-year-old boys and a nine-year-old girl. Two of the three patients initially complained only of recurrent bouts of vertigo, without any tinnitus, ear fullness or hearing impairment. In all three cases, the early pure tone audiograms showed only high tone frequency loss, regardless of subjective hearing loss, and the decrease in the hearing threshold was observed one to eight years after the dizziness attacks began. The hearing threshold was usually decreased to a level of mild or moderate hearing impairment. After diuretic treatment, vertigo was generally well controlled, and some cases showed improvement in hearing. Of the total number of patients with Ménière's disease who visited our department over nine years, 2.6 per cent (3/114) were children, and the overall incidence of Ménière's disease in children with vertigo was 2.0 per cent (3/147). In conclusion, Ménière's disease in children rarely develops and may have characteristics of high tone loss in initial audiograms.

Simple compressive method for treatment of auricular haematoma using dental silicone material.

Most of the previous treatment methods for auricular haematoma are inconvenient for both patients and doctors because they are time-consuming and complex and must be performed under sterile conditions. The purpose of this study was to evaluate the effectiveness of a simple compressive method using a dental (silicone) impression material and comparing it with other methods for treatment of auricular haematomas. The authors aspirated a haematoma and then placed a mixed base and catalyst of silicone putty material on the anterior and posterior surfaces of the auricle in the shape of an inverted U for seven days. From the 24 cases managed with this method, 23 cases (95.8 per cent) were successfully healed. Eight patients were treated with a collodion-cotton wool cast and 16 of 19 patients were successfully treated with dental cotton-wool rolls. The average number of those visiting the hospital was 2.7 for the collodion-cotton wool cast, 6.9 for the dental cotton-wool roll, and 3.1 for dental silicone. The mean treatment durations were 8.1 days for the collodion-cotton wool cast, 13.8 days for the dental cotton-wool roll, and 8.6 days for dental silicone. The authors believe that this compressive method using dental silicone material is simple and appropriate for the treatment of auricular haematoma.

Vascularized bone flap for access to the maxillary sinus.

PURPOSE: This article describes a vascularized bony window for access to the maxillary sinus and reports the clinical results. PATIENTS AND METHODS: A bony U-shaped window in the anterior sinus wall was pedicled on the surrounding soft tissue and periosteum. After the described sinus was cleared of disease, the window was repositioned in its original site either using resorbable sutures or not. The method was used in 47 maxillary sinus operations in 45 patients. Twenty-four patients were followed-up for more than 48 months. RESULTS: The vascularized bony window technique showed uneventful healing in all patients and none of the 24 patients reported any problems. CONCLUSIONS: The vascularized bony window technique provides a large antrostomy, which gives good access and visibility and results in satisfactory postoperative healing.