Apoptotic Pattern of Cochlear Outer Hair Cells and Frequency-specific Hearing Threshold Shift in Noise-exposed BALB/c Mice

OBJECTIVES: Apoptosis of outer hair cell (OHC) can be identified through nuclear staining by specific nuclear changes. The change of filamentous actin (F-actin) is also involved in early cell death process. The study was designed to investigate OHC death along the whole length of the organ of Corti. METHODS: BALB/c hybrid mice were used in this study. The noise group was exposed to white noise of 120 dB SPL for 3 hr per day for 3 consecutive days. The tone burst auditory brainstem response (ABR) test was conducted and cochleas from each group were obtained for the immunostaining of FITC phalloidin for F-actin and propidium iodide (PI) for nuclei. RESULTS: ABR threshold of the noise group significantly increased after noise exposure (P<0.001). No threshold shift was found in the control group. Threshold shift of the noise group constantly increased from 4 to 16 kHz, but threshold shifts at 16 kHz and 32 kHz were similar. Patterns of OHC staining were subclassified as FITC+PI- cells, FITC+ PI+ cells, FITC-PI+ cells and missing cells. Proportion of normal live OHCs (FITC+PI-) rapidly decreased from the apex to the base. In the basal turn, FITC-PI+ cells and vacancy OHC (missing cells) were observed easily. Apoptotic and missing cells were most abundant at 60% of the whole length of the Corti organ. CONCLUSION: We could subclassify morphologic changes in OHC death after noise exposure. Quantitative changes in OHCs along the whole Corti organ showed a plateau pattern similar to that of a frequency-specific threshold shift.

The Effect of Pretreatment of CoCl2 on the Prevention of Noise-Induced Hearing Loss / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The study was designed to investigate the changes in the expression of hypoxia inducible factor-1alpha (HIF-1alpha) according to time after being exposed to noise trauma and find out the effect of HIF-1 alpha in the prevention of noiseinduced hearing loss by pre-treatment with cobalt chloride (CoCl2). SUBJECTS AND METHOD: BALB/c hybrid mice with 25 dB HL or less ABR were used in this study. In the study group, subjects were exposed to 120 dB SPL broad white band noise for 3 hours per day for 3 days. The changes in their hearing were documented before and after 1, 2, 3, 4, and 9 days of the first noise exposure. CoCl2 was injected into the peritoneum 2 hours prior to each noise exposure to see the effect of induced HIF-1alpha on noise-induced hearing loss. For the control, injection with distilled water was performed and hearing thresholds were measured in the same manner. Cochlea from each group was obtained in order to observe the morphological changes in the inner ear and the expression of the HIF-1alpha using immunohistochemial staining and immuno-fluorescein staining along with quantification of the hair cell loss. RESULTS: Mice exposed to the noise for 3 days, showed permanent threshold shift and the expression of HIF-1alpha was increased. When HIF-1alpha was induced by pre-treatment of CoCl2 prior to the noise exposure, however, hearing recovery was observed to some degree. And hair cell survival rate was also higher when treated with CoCl2 compared to the distilled water treated group. CONCLUSION: When pre-treated with CoCl2, inducing HIF-1alpha before the noise trauma, it allowed for a less stereocilia loss in the hair cells in the organ of Corti. HIF-1alpha may play an important role in the prevention of noise-induced hearing loss.

The Effect of Halothane on Hearing Loss and Apoptosis in Noise Exposing Mouse / 대한마취과학회지

BACKGROUND: In large clinical series, noise induced hearing loss (NIHL) following middle ear surgery has been demonstrated in 1.2% to 4.5% of patients and it is associated with a lower incidence than expected. The aim of the present work was to analyze the effect of halothane anesthesia on NIHL and hair cell morphological change. METHODS: We used 40 BALB/c mice with normal Preyer's reflex to investigate the effect of halothane on the NIHL. Control (n = 20) and halothane group (n = 20, respectively) were exposed to 120 dB SPL (sound pressure level), broad band white noise 3 hours daily for 3 consecutive days. The halothane group was anesthetized with halothane while exposed to noise. Hearing thresholds were determined with the auditory brainstem response (ABR). On day 7 post-noise, mice were sacrificed and the cochlea were collected for the histological study. RESULTS: ABR thresholds in the halothane group were less elevated after noise exposure than in the control group and then gradually recovered. In control group, the damage to the outer hair cell and supporting cell was noticeable, but not in halothane group. The expression of Bcl-2 protein was detected in halothane group, the expression of Bax protein was seen in control group. As a result in TUNEL stain, the result is positive in the control group but negative in the halothane group. CONCLUSIONS: The occurrence of NIHL decreased and the tissue damage was suppressed while anesthetized by halothane. And the noise-induced cell death of hair cell was also suppressed during anesthesia.

Morphological Change of Mouse Inner Ear Hair Cells after Noise Exposure / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Mechanism of inner ear hair cell distortion after noise exposure has been well described. The present study was designed to determine the response to the auditory system of a genetically well-defined laboratory mouse in preparation for examining the effect of noise on mice with specific genetic mutations. So it is important to recognize the relationship between noise exposure duration and hair cell morphological changes. We try to reveal the hearing loss and inner ear hair cell morphological changes after applying the noise protocol. SUBJECTS AND METHOD: The mice were BALB/c hybrids and aged 8 weeks. Six mice served as non-noise-exposed controls and 8 mice were exposed for 3 hours per day to white band noise with a center frequency from 0.2 kHz to 70 kHz and a sound pressure level of 120 dB. And we divided the noise exposure group into 3 subgroups(1 day, 3 day, 5 day noise exposure group). We checked the photographs of FITC phalloidin stain and scanning electron microscopy of cochlea after noise exposure. RESULTS: The hearing level of mice decreased after noise exposure. We could see the stereocilia damage in cochlea after FITC phalloidin stain in cochlea and sterocilia loss was more severe in basal turn. In scanning electron microscopy, morphological changes of stereocilia were observed to be more severe in the cochlear basal turn than other area. Significant hair cell loss in the cochlear basal turn could be calculated using cochleocytogram. CONCLUSION: 120dB broad white band noise can damage the hair cell of cochlea in mice. These changes were especially severe in the cochlear basal turn. Noise exposure duration is the other important factor in damaging cochlear hair cells. Therefore, we can guess that harmful noise level and noise exposure duration are the main risk factors that injure the inner ear hair cell.

Expression of Hypoxia-Inducible Factor in the Inner Ear of Noise-Exposed Mouse / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Investigations regarding the mechanisms of noise-induced hearing loss have suggested that noise exposure induces tissue ischemia, and damages the inner ear. The hypoxia-inducible factor (HIF), a helix-loop-helix PAS transcription factor, is known to prevent hypoxic cell damage in the brain in cases of ischemic attacks. Like the role it plays in the brain, there is a possibility that HIF could be found in the inner ear playing the similar role. MATERIALS AND METHOD: We used 23 (46 ears) BALB/c mice with normal Preyer's reflex to investigate the effect of the noise-induced hearing loss. Hearing threshold was measured by ABR before and after exposing mice to 120 dB SPL broad band white noise 3 hours daily for 5 consecutive days. And after 1 month, the hearing threshold was rechecked to assure the permanent threshold shift. Cochlea were collected for the histological study and the expression of HIF was observed. RESULTS: Control mice showed hearing level of 27.5+/-1.7 dB (mean+/-SD), which was changed to 80.5+/-2.1 dB immediately after 5-day of exposure to noise, and 79.5+/- 2.5 dB after 1 month of noise exposure. For all noise exposed mice, the damage to the outer hair cell and the Organ of Corti was noticeable. HIF was expressed in the spiral limbus, few supporting cells, stria vascularis and spiral ganglion cells of the noise exposed mouse. CONCLUSION: Noise, a form of acoustic trauma to the inner ear, induced damage to the outer hair cells in mice. At the same time, HIF were expressed in all of the studied animals. The possibility of HIF playing a role of protecting the inner ear from the acoustic trauma as it does in the brain should be investigated further.

The Change of Hearing Threshold and Endocochlear Potential by Bafilomycin Delivered to Round Window in Guinea Pigs / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Vacuolar type H+-ATPase (V-H+-ATPase) has a role in the regulation of endolymphatic pH and certain cells (including strial marginal cells, inner hair cells and epithelial cells of the endolymphatic sac) may be specialized for this regulation. Bafilomycin is a specific V-H+-ATPase inhibitor affecting inner ear function by controlling the intracytolic pH decrease. We designed the study to analyze the effect of bafilomycin delivered to the inner ear on the hearing threshold measured by auditory brainstem response (ABR) and endolymphatic potential (EP). MATERIALS AND METHOD: For measuring the hearing threshold change, 13 guinea pigs with normal Preyer's reflex and normal ABR were used. Guinea pigs were randomly divided into control group (n=3, 6 ears) and study groups which were subdivided into the following ;1 mM bafilomycin group (n=3, 6 ears), 5 mM bafilomycin group (n=3, 6 ears) and 10 mM bafilomycin group (n=4, 8 ears). The mastoid cavity was opened to expose the round window and HBSS buffer (300 osm) was applied for the control group and bafilomycin with different concentrations were also applied to the round windows of studied guinea pigs. The hearing threshold was measured using ABR before and after the application of appropriate solutions. For measuring of EP, the cochlea helix and round window of guinea pig with normal hearing were defined and a tungsten micro needle was inserted into the endolymphatic space at 2nd turn of guinea pig's cochlea. EP was measured after application of HBSS buffer as control, 1 mM, 5 mM, and 10 mM bafilomycin. RESULTS: In the control group, the hearing threshold was 21.6+/-2.8 dB (mean+/-SD) initially both before and after mastoidectomy and stayed that way all throughout the study. The hearing threshold increased as bafilomycin was applied. For 1 mM of bafilomycin application, the threshold changed from initial 30.0+/-5 dB to 33.3+/-5.7 dB after 2 hours. For 5 mM of bafilomycin application, the threshold changed after 2 hours from initial 30.0+/-5 dB to 50.0+/-0 dB. With 10 mM of bafilomycin application, the threshold changed after 2 hours from initial 27.5+/-6.4 dB to 52.5+/-8.6 dB. For 5 mM and 10 mM bafilomycin group, there was a significant statistical change of hearing threshold (p<0.05). However, there was no meaningful difference between 5 mM and 10 mM group (p=0.88). Initial EP was 85+/-10 mV and was significantly decreased in 5 mM (35 mV) and 10 mM (19.8 mV) bafilomycin groups, but such was not observed in 1 mM bafilomycin and control group. CONCLUSION: We could observe the elevation of hearing threshold and decrease EP after applying bafilomycin on the round windows of guinea pigs and also observed that this change reached a critical point when the concentration of bafilomycin was 5 mM or higher. From these results, we can conclude that bafilomycin does affect the hearing threshold through its mechanism and the degree of damage has a critical point dependent on the concentration of bafilomycin.

The Change of Hearing Threshold and Perilymphatic Lactate Concentration in Guinea Pigs with Induced Hypoxia / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Authors aimed to observe the change of hearing threshold and the concentration of perilymphatic lactate in guinea pigs with hypoxia. MATERIALS AND METHOD: Healthy guinea pigs were anesthetized with xylazine and ketamine and perfomed thracheotomy with ventilatory care. After shaving around vertex and mastoid area, the minimum ABR threshold was checked until the wave I was normally traced. Hypoxic condition was induced by the increase of N2 and the decreased O2 saturation was confirmed via monitor, and then the ABR threshold was rechecked. Then, the mastoid cavity was opened and round window was identified under microscopic view. Labyrinthine perilymphatic fluid was collected from a round window by Hamilton's syringe and the lactate level was calculated. Three guinea pigs were used as controls and the perilymphatic fluids were collected. RESULTS: In the guinea pig with normal Preyer's reflex, the initial hearing threshold was 29.5+/-3.6 dB SPL (mean+/-SD) and the average ABR threshold of guinea pig with hypoxic state was 71.0+/-11.4 dB SPL. The average perilymphatic lactate level was 5.339+/-0.07 mg/dL in the guinea pig of normal hearing and 60.024+/-1.66 mg/dL in the guinea pig of hypoxic state. CONCLUSION: Authors observed that hearing threshold was decreased and perilympahatic lactate level was elevated via induced hypoxia. Through these findings, authors can draw the conclusion that lactate may be one of the possible factors explaning the mechanism of the change in the hearing threshold by tissue ischemia.