[Protecting Effects of Jian'erji on Age-related Hearing Loss of C57BL/6J Mice].

Objective To observe decreased hearing in aged C57BL/6J mice, and to study pro- tective effects of Jian' erji ( JEJ ) for age-related hearing loss (AHL) and its possible mechanism. Methods Totally 36 C57BL/6J mice were randomly divided into four groups, i.e., the normal control group (n =6) , the AHL control group (n =12) , the high dose JEJ group (n =12) , the low dose JEJ group (n =6). Mice in the normal control group drank tap water from ablactation till 2 months old. Mice in the AHL control group drank tap water from ablactation till 7 months old. Mice in high and low dose JEJ groups drank JEJ at the daily dose of 3. 65 g/kg and 0. 91 g/kg respectively from ablactation till 7 months old. Six mice were selected from each group for auditory brainstem response (ABR) using brainstem evoked potentiometer on the day of ending the test. The cochlear tissue, auditory cortex, and liver were immediately collected from 6 mice of the high dose JEJ group and 6 of the AHL control group at the same ages. Contents of malondialdehyde (MDA) , end product of lipid peroxidation were detected by UV spectrophotometer using MDA coomassie blue kit. Results ABR thresholds evoked by short-pure tone from 4 to 48 KHz were in the normal range of 2 months old mice in the normal control group. Compared with 2 months old mice in the normal control group, ABR thresholds were significantly elevated in 7 months old mice of the AHL control group (P <0. 05). Significant differences also existed in ABR thresholds from 8 to 48 KHz in the high dose JEJ group (P <0. 05). Compared with 7 months old mice of the AHL control group, MDA contents in cochlear tissue, auditory cortex, and liver were obviously reduced in the high dose JEJ group (P <0. 01). Conclusions C57BL/6J mice showed significant symptoms of AHL in high frequency range at 7 months old. Daily drinking of high dose JEJ could significantly delay the occurrence and progress of AHL. Its protection might be related to antioxidant effects JEJ contained.

Bilirubin induces auditory neuropathy in neonatal guinea pigs via auditory nerve fiber damage.

Bilirubin can cause temporary or permanent sensorineural deafness in newborn babies with hyperbilirubinemia. However, the underlying targets and physiological effects of bilirubin-induced damage in the peripheral auditory system are unclear. Using cochlear functional assays and electron microscopy imaging of the inner ear in neonatal guinea pigs, we show here that bilirubin exposure resulted in threshold elevation in both compound action potential (CAP) and auditory brainstem response (ABR), which was apparent at 1 hr and peaked 8 hr after drug administration. The threshold elevation was associated with delayed wave latencies and elongated interwave intervals in ABR and CAP. At 72 hr postinjection, these measures returned to control levels, except for the CAP amplitude. Cochlear microphonics remained unchanged during the experiment. Morphological abnormalities were consistent with the electrophysiological dysfunction, revealing fewer auditory nerve fibers (ANFs) in the basal turn, myelin sheath lesions of spiral ganglion neurons (SGNs) and ANFs, and loss of type 1 afferent endings beneath inner hair cells (IHCs) without loss of hair cells at 8 hr posttreatment. Similar to the electrophysiological findings, morphological changes were mostly reversed 10 days after treatment, except for the ANF reduction in the basal turn. These results suggest that hyperbilirubinemia in neonatal guinea pigs impaired auditory peripheral neuromechanisms that targeted mainly the IHC synapses and the myelin sheath of SGNs and their fibers. Our observations indicate a potential connection between hyperbilirubinemia and auditory neuropathy.

Ototoxic destruction by co-administration of kanamycin and ethacrynic acid in rats.

It is well known that ethacrynic acid (EA) can potentiate the ototoxicity of aminoglycoside antibiotics (AmAn) such as kanamycin (KM), if they were applied at the same time. Currently, to create the model of EA-KM-induced cochlear lesion in rats, adult rats received a single injection of EA (75 mg/kg, intravenous injection), or followed immediately by KM (500 mg/kg, intramuscular injection). The hearing function was assessed by auditory brainstem response (ABR) measurement in response to click and/or tone bursts at 4, 8, 12, 16, 20, 24, and 32 kHz. The static microcirculation status in the stria vascularis after a single EA injection was evaluated with eosin staining. The pathological changes in cochlear and vestibular hair cells were also quantified after co-administration of EA and KM. After a single EA injection, blood flow in vessels supplying the stria vascularis rapidly diminished. However, the blood supply to the cochlear lateral wall partially recovered 5 h after EA treatment. Threshold changes in ABR were basically parallel to the microcirculation changes in stria vascularis after single EA treatment. Importantly, disposable co-administration of EA and KM resulted in a permanent hearing loss and severe damage to the cochlear hair cells, but spared the vestibular hair cells. Since the cochlear lateral wall is the important part of the blood-cochlea barrier, EA-induced anoxic damage to the epithelium of stria vascularis may enhance the entry of KM to the cochlea. Thus, experimental animal model of selective cochlear damage with normal vestibular systems can be reliably created through co-administration of EA and KM.

[Hair cells apoptosis and the activation of P53 protein in intensive impulse noise induced cochlear lesion].

OBJECTIVE: To explore the pattern of hair cell injury and expression of P53 apoptosis protein in intensive impulse noise injured cochlear hair cells in guinea pigs. METHODS: Twelve adult guinea pigs were exposed to a series of 40 pairs of impulse noise (2 second intervals) at the intensity of 168 dB (SPL). Animals were terminated at 3, 6 and 12 hours after noise exposure, respectively. Cochlear surface preparations were performed with a double staining of FITC-conjugated phalloidin and propidium iodide for the observations of the stereocilia and the nucleus. P53 immunochemical staining was also performed 12 hours post-noise exposure to observe if there was expression of p53 protein in injured hair cells. Results Three hours after noise exposure, the outer hair cells at the end of basal turn and beginning of second turn were destroyed first with a character of nuclear condensation. Six hours post-noise exposure, many hair cells in the center of damage region had nuclear fragmentations, and the damaging area expanded towards to basal turn and apical turn. Twelve hours after noise exposure, the nucleus in most outer hair cells and inner hair cells at the region of damage center were missing. The nuclear condensation and fragmentation were appeared in hair cells in both sides of the center region of degeneration. P53 immunoreactive products were also found in damaged hair cells, not only in the central damage area, but also in the basal turn and the third turn. CONCLUSIONS: Intensive impulse noise resulted in apoptosis of cochlear hair cells that initiated between the end of basal turn and the beginning of second turn. Hair cell degeneration spread to basal and third turn along the basilar membrane. P53 may play an important role in impulse noise induced-hair cell apoptosis.

Effects of Erlong Zuoci pill and its disassembled prescriptions on gentamicin-induced ototoxicity model in vitro.

OBJECTIVE: To study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro. METHODS: After the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software. RESULTS: GM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05). CONCLUSIONS: ELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.

[Streptomycin-induced apoptosis of rat cochlear hair cell cultured in vitro].

OBJECTIVE: To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells. METHODS: F344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope. RESULTS: Streptomycin with 1 mmol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated. CONCLUSIONS: Apoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.

[Ototoxic effects of streptomycin in vestibular organotypic cultures].

OBJECTIVE: To investigate the ototoxic effects of streptomycin in vestibular organotypic cultures. METHODS: F344 rats with age at postnatal day three or four were used for this study. The maculae of saccule and utricle were routinely dissected out and cultured with serum-free medium containing various dose of streptomycin for 24 hours. The ciliary turf of vestibular hair cells was stained with fluorescent phalloidin, and its nucleus was stained with to pro-3 DNA probe. The vestibular hair cells were quantitatively counted and photographed under confocal fluorescent microscope. RESULTS: Morphological feature of vestibular hair cells were good in normal control cultures. However, the density of hair cells was decreased in evidence with increase of streptomycin sulfate concentrations. Twenty-four hours after streptomycin cultures, 0.25 mmol/L streptomycin caused a 10% hair cell missing, 50% hair cell loss from 1 mmol/L streptomycin treatment, and more than 75% hair cells gone post-3 mmol/L streptomycin cultures. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in vestibular hair cells, whereas the vestibular supporting cells were normal. CONCLUSION: Streptomycin induced-vestibular hair cells lesion was in a dose dependent manner with nuclear shrinkage and fragmentation. This may suggest that streptomycin leads vestibular hair cell die through apoptosis.

[Correlation between reduction of distortion product otoacoustic emission and percentage of outer hair cell missing in chinchillas].

OBJECTIVE: To explore the quantitative relationship between the reduction of distortion product otoacoustic emission (DPOAE) and the percentage of outer hair cell loss. METHODS: Coadministration of cisplatin (0.2 mg/kg) and ethacrynic acid (40 mg/kg) were used to establish a cochlear lesion model in chinchillas. DPOAE was measured before and 1 week, 2 weeks, and 3 weeks later respectively after cisplatin and ethacrynic acid treatment. Animals were terminated 3 weeks after the treatment. Cochlear surface preparations were performed, and the cochlear hair cells were counted through entire length of the cochlea. The correlation between DPOAE reduction and outer hair cell missing was analyzed using Pearson correlation analysis. RESULTS: Cisplatin and ethacrynic acid treatment induced cochlear hair cell lesion that the outer hair cell loss in the cochlea developed in a stereotypic pattern; damage began in the base of the cochlea and progressed towards the apex. Reduction of DPOAE was relatively consistent with outer hair cells loss. On the average, 1% outer hair cells loss may result in 0.24 dB reduction in DPOAE levels. Pearson analysis showed a positive correlation between the reduction in DPOAE and missing of outer hair cells (r = 0.796, P < 0.05). CONCLUSIONS: It may be helpful to evaluate missing percentage of outer hair cells from reduction in DPOAE levels.

Effects of exposing C57BL/6J mice to high- and low-frequency augmented acoustic environments: auditory brainstem response thresholds, cytocochleograms, anterior cochlear nucleus morphology and the role of gonadal hormones.

Gonadectomized and intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to an augmented acoustic environment (AAE): repetitive bursts of a 70dB SPL noise band. The high-frequency AAE (HAAE) was a half-octave band centered at 20kHz; the low-frequency AAE (LAAE) was a 2-8kHz band. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (a trait of B6 mice) were evaluated by obtaining auditory brainstem response (ABR) thresholds at ages 3-, 6-, and 9-months. At 9-months of age, hair cell counts (cytocochleograms) were obtained, and morphometric measures of the anteroventral cochlear nucleus (AVCN) were obtained. LAAE treatment caused elevation in ABR thresholds (8-24kHz), with the highest thresholds occurring in intact females. LAAE treatment caused some loss of outer hair cells in the basal half of the cochlea (in addition to losses normally occurring in B6 mice), with intact females losing more cells than intact males. The loss of AVCN neurons and shrinkage of tissue volume that typically occur in 9-month-old B6 mice was lessened by LAAE treatment in intact (but not gonadectomized) male mice, whereas the degenerative changes were exacerbated in intact (but not gonadectomized) females. These LAAE effects were prominent in, but not restricted to, the tonotopic low-frequency (ventral) AVCN. HAAE treatment resulted in some loss of neurons in the high-frequency (dorsal) AVCN. In general, LAAE treatment plus male gonadal hormones (intact males) had an ameliorative effect whereas HAAE or LAAE treatment plus ovarian hormones (intact females) had a negative effect on age-related changes in the B6 auditory system.

Physiological effects of auditory nerve myelinopathy in chinchillas.

The goals were to study the physiological effects of auditory nerve myelinopathy in chinchillas and to test the hypothesis that myelin abnormalities could account for auditory neuropathy, a hearing disorder characterized by absent auditory brainstem responses (ABRs) with preserved outer hair cell function. Doxorubicin, a cytotoxic drug used as an experimental demyelinating agent, was injected into the auditory nerve bundle of 18 chinchillas; six other chinchillas were injected with vehicle alone. Cochlear microphonics, compound action potentials (CAPs), inferior colliculus evoked potentials (IC-EVPs), cubic distortion product otoacoustic emissions and ABRs were recorded before and up to 2 months after injection. Cochleograms showed no hair cell loss in any of the animals and measures of outer hair cell function were normal (cubic distortion product otoacoustic emissions) or enhanced (cochlear microphonics) after injection. ABR was present in animals with mild myelin damage (n = 10) and absent in animals with severe myelin damage that included the myelin surrounding spiral ganglion cell bodies and fibers in Rosenthal's canal (n = 8). Animals with mild damage had reduced response amplitudes at 1 day, followed by recovery of CAP and enhancement of the IC-EVP. In animals with severe damage, CAP and IC-EVP thresholds were elevated, amplitudes were reduced, and latencies were prolonged at 1 day and thereafter. CAPs deteriorated over time, whereas IC-EVPs partially recovered; latencies remained consistently prolonged despite changes in amplitudes. The results support auditory nerve myelinopathy as a possible pathomechanism of auditory neuropathy but indicate that myelinopathy must be severe before physiological measures are affected.

Effects of exposing gonadectomized and intact C57BL/6J mice to a high-frequency augmented acoustic environment: Auditory brainstem response thresholds and cytocochleograms.

Gonadectomized and surgically intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to a high-frequency augmented acoustic environment (AAE): repetitive bursts of a half-octave noise band centered at 20 kHz, 70 dB SPL. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (exhibited by B6 mice) were evaluated by obtaining auditory brainstem response thresholds at ages 3-, 6-, and 9-months; hair cell counts (cytocochleograms) were obtained at 9 months. A sex difference in the rate of genetic progressive hearing loss in B6 mice (observed by earlier studies) was confirmed, with females exhibiting a faster rate of threshold elevations and more severe loss of hair cells at age 9 months. Gonadectomy had no consistent effects on the rate or severity of hearing loss in non-exposed mice of either sex. An unexpected finding was that the high-frequency AAE treatment caused additional ABR threshold elevations and hair cell loss. In an earlier study, the same high-frequency AAE treatment on DBA/2J mice ameliorated hearing loss. The most severe AAE-induced losses occurred in surgically intact females, suggesting a potentiating effect of ovarian hormone(s).

Effects of exposing DBA/2J mice to a high-frequency augmented acoustic environment on the cochlea and anteroventral cochlear nucleus.

DBA/2J (D2) mice, which exhibit very early progressive sensorineural hearing loss, were treated for 12h nightly with an augmented acoustic environment (AAE) initiated before the onset of hearing. The AAE consisted of repetitive bursts of a 70 dB sound pressure level, half-octave noise band centered at 20 kHz (i.e. low frequencies were excluded). At 55 days of age, AAE-treated mice, compared to control mice, exhibited less elevation of auditory brainstem response thresholds for tone frequencies from 16 to 32 kHz and fewer missing outer hair cells in the high-frequency tonotopic region of the cochlea. The dorsal region of their anteroventral cochlear nucleus (most strongly stimulated by the AAE) was larger, had more surviving neurons, and larger neurons than those of untreated control mice. These and previous findings using an AAE band containing lower frequencies indicate that AAE treatment effects are frequency-related. The findings provide support for the hypothesis that the beneficial effects of AAE treatment on the cochlea are associated with increased physiological activity evoked by the AAE, and the central AAE effects result from increased AAE-evoked neural activity and a healthier cochlea providing the auditory input.

Amino acid concentrations in chinchilla cochlear nucleus at different times after carboplatin treatment.

Amino acid concentrations were measured in the cochlear nucleus for a group of 20 chinchillas: four each of control and 4, 8, 29, and 85 days after treatment with the ototoxic anti-tumor drug carboplatin (100 mg/kg, i.p.). The treated chinchillas showed various extents of inner hair cell loss, generally more complete at longer survival times, but little loss of outer hair cells. Aspartate concentration in rostral anteroventral cochlear nucleus (AVCN) showed a decline to 28% less than the control value at 29 and 85 days after treatment, whereas glutamate concentration showed little change through 29 days, then dropped by 22% at 85 days after treatment. In caudal posteroventral cochlear nucleus (PVCN), the aspartate concentration decreased by 32% at 29 days, in animals with significant inner hair cell loss, and 48% at 85 days after treatment, while the glutamate concentration showed no decrease through 29 days and 40% decrease at 85 days. The concentration of gamma-aminobutyrate (GABA) was about 18% lower than control in caudal PVCN at all survival times. Significant correlations were found between the proportion of inner hair cells remaining and glutamate and aspartate concentrations in PVCN and AVCN, but not GABA or other amino acids.

Substance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons.

Substance P (SP), a member of the tachykinin family of neurotransmitters and neuromodulators, has been identified on spiral ganglion neurons (SGNs) in the inner ear; however, its high affinity receptor, neurokinin-1 (NK1), has not been identified and the physiological effects of SP on SGNs are not well understood. To address these issues, immunolabeling, RT-PCR, Western blots and whole-cell patch-clamp recordings were made from SGNs in P0-P5 mouse cochlear organotypic cultures. The NK1 receptor was detected on SGNs by immunocytochemistry, the protein was detected in cochlear tissues by Western blots, and the mRNA for the NK1 receptor was also found in cochlear tissues of postnatal mice (P2) by RT-PCR. Application of SP (1 to 25 microM) significantly increased the latency of SGN action potentials (APs) (mean increase 7.8 +/- 4 ms; 25 microM of SP), prolonged the duration of the action potential and made the resting potential (RP) more positive (mean 9.0 +/- 7 mV) relative to normal values (-54 +/- 6 mV). SP (1 to 25 microM) also suppressed voltage-activated potassium currents (IK+) and calcium currents (ICa2+). Puffing 25 microM of SP onto SGNs suppressed IK+ by 43 +/- 9% (n = 7) and ICa2+ by 40.6 +/- 5.6% (n = 7); both currents recovered when SP was washed out. A SP antagonist blocked the SP-induced suppression of IK+ and ICa2+. These results indicate that SP acting through NK1 receptors can have direct neuromodulatory effects on SGNs.

Chick hair cells do not exhibit voltage-dependent somatic motility.

It is generally believed that mechanical amplification by cochlear hair cells is necessary to enhance the sensitivity and frequency selectivity of hearing. In the mammalian ear, the basis of cochlear amplification is believed to be the voltage-dependent electromotility of outer hair cells (OHCs). The avian basilar papilla contains tall and short hair cells, with the former being comparable to inner hair cells, and the latter comparable to OHCs, based on their innervation patterns. In this study, we sought evidence for somatic electromotility by direct measurements of voltage-dependent length changes in both tall and short hair cells at nanometre resolution. Microchamber and whole-cell voltage-clamp techniques were used. Motility was measured with a photodiode-based measurement system. Non-linear capacitance, an electrical signature of somatic motility, was also measured to complement motility measurement. Significantly, chick hair cells did not exhibit somatic motility nor express non-linear capacitance. The lack of somatic motility suggests that in avian hair cells the active process resides elsewhere, most likely in the hair cell stereocilia.

Effects of carboplatin on amino acid chemistry in chinchilla cochlear nucleus.

Carboplatin, a drug widely used against solid head and neck tumors, selectively destroys cochlear inner hair cells and type I auditory nerve fibers in chinchilla. This should affect neurotransmitter chemistry, involving amino acids, where the type I auditory nerve fibers terminate in the cochlear nucleus. Using microdissection combined with high-performance liquid chromatography, amino acid concentrations were mapped in the cochlear nuclei of chinchillas injected intraperitoneally 6-8 weeks earlier with 100 mg/kg carboplatin and in those of control animals. Glutamate concentrations were 23% lower in the anteroventral cochlear nucleus (AVCN) and 40% lower in the posteroventral cochlear nucleus (PVCN) of carboplatin-injected chinchillas as compared to controls, while aspartate concentrations were 18% lower in AVCN and 27% lower in PVCN. Using a fluorometric assay, activities of glutaminase, an enzyme which catalyzes glutamate synthesis, were 30% lower in AVCN and 38% lower in PVCN of carboplatin-injected chinchillas. Concentrations of glutamine, gamma-aminobutyrate, and glycine were also lower in some ventral and dorsal cochlear nucleus regions of treated animals. These changes probably result mainly from both primary and later effects of reduced type I auditory nerve fiber input to the cochlear nucleus.

Gene Expression Changes in Chinchilla Cochlea from Noise-Induced Temporary Threshold Shift.

Acoustic overstimulation produces many anatomical, biochemical and physiological changes in the inner ear. However, the changes in gene expression that underlie these biological changes are poorly understood. Our approach to investigating this problem is to use gene microarrays to measure the changes in gene expression in the chinchilla inner ear following a 3 h or 6 h noise exposure (95 dB SPL, 707-1414 Hz). This noise exposure causes a temporary threshold shift (~40 dB) and a temporary reduction in distortion product otoacoustic emissions (DPOAE), but no permanent hearing loss or hair cell loss. Here, we present data showing (1) the suitability of mouse and human complementary DNA (cDNA) clones for detecting chinchilla cochlear gene transcripts, and (2) the change in cochlear gene transcripts in noise exposed chinchillas. Chinchilla cochlear transcript probes exhibited strong and discrete signals on both mouse and human cDNA filter arrays. Since the strongest hybridization occurred with mouse clones, mouse cDNA microarrays were used to study noise-induced changes in gene expression. Chinchilla cDNA probes were differentially labelled with Cy3 (control) or Cy5 (noise exposed) by random primed synthesis, hybridized to 8750 mouse cDNAs arrayed on microscope slides and analysed by laser fluorescent microscopy. Several classes of genes exhibited time-dependent up regulation of transcription, including those involved in protein synthesis, metabolism, cytoskeletal proteins, and calcium binding proteins. The results are discussed in relationship to previous studies showing noise-induced changes in structural proteins, calcium binding proteins, metabolic enzymes and membrane bound vesicles.