OTOTOXIC MODEL OF OXALIPLATIN AND PROTECTION FROM NICOTINAMIDE ADENINE DINUCLEOTIDE

Oxaliplatin,an anticancer drug commonly used to treat colorectal cancer and other tumors,has a number of serious side effects,most notably neuropathy and ototoxicity.To gain insights into its ototoxic profile,oxaliplatin was applied to rat cochlear organ cultures.Consistent with it neurotoxic propensity,oxaliplatin selectively damaged nerve fibers at a very low dose 1μM.In contrast,the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher(50μM).Oxailiplatin-induced cochlear lesions initially increased with dose,but unexpectedly decreased at very high doses.This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms.Previous studies have demonstrated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide(NAD+).To determine if NAD+would protect spiral ganglion axons and the hair cells from oxaliplatin damage,cochlear cultures were treated with oxaliplatin alone at doses of 10μM or 50μM respectively as controls or combined with 20 mM NAD+.Treatment with 10μM oxaliplatin for 48 hours resulted in minor damage to auditory nerve fibers,but spared cochlear hair cells.However,when cochlear cultures were treated with 10μM oxaliplatin plus 20 mM NAD+,most auditory nerve fibers were intact.50μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apoptotic characteristics of cell fragmentations.However,50μM oxaliplatin plus 20 mM NAD+treatment greatly reduced neuronal degenerations and hair cell missing.The results suggested that NAD+provides significant protection against oxaliplatin-induced neurotoxicity and ototoxicity,which may be due to its actions of antioxidant,antiapoptosis,and energy supply.     

Cochlear structure and function after round window application of ototoxins

Topical round window application of ototoxic agents has been a useful method for studying ototoxicity and hearing loss in the mammalian cochlea. For example, species-specific differences in cochlear susceptibility to damage have been documented using this technique. Carboplatin has been characterized in the literature as a selective inner hair cell (IHC) toxin in chinchillas, while cisplatin has been characterized as a selective outer hair cell (OHC) toxin. The present experiment quantified dose-dependent damage to cochlear hair cells in the chinchilla after a single direct round window application of either cisplatin or carboplatin. Detailed cytocochleograms were obtained for the entire cochlear duct, for a range of doses, along with auditory brainstem response thresholds. In agreement with the literature, although there was variability, at the lowest concentrations tested (2 and 3 mg/ml), carboplatin produced substantial IHC damage with no OHC damage. In contrast, the effects of cisplatin were more variable, and contrary to published reports, across the range of doses producing OHC damage, IHC damage was always observed. Limitations of direct round window ototoxin treatments are discussed, in addition to their potential application in the study of tinnitus.     

Vestibular destruction by slow infusion of gentamicin into semicircular canals

Intratympanic or round window application of gentamicin is often used to alleviate disabling vertigo arising from unilateral Meniere's disease; however, treatment is often accompanied by hearing loss because the drug initially enters the cochlea before diffusing to the vestibular system. In order to enhance vestibular damage and reduce the risk of hearing loss, gentamicin was infused directly into the vestibular system. An osmotic pump containing 50, 100, 200 or 400 microg/ml of gentamicin was infused into the superior semicircular canal of the chinchilla for 7 days. Afterwards, vestibular damage was evaluated by measuring the decline in hair cell density in the utricle, saccule and superior semicircular canals. Auditory damage was assessed with distortion product otoacoustic emissions (DPOAE) and outer hair cell (OHC) and inner hair cell (IHC) loss. Infusion with the two lowest gentamicin concentrations resulted in significant hair cell loss and reduced duration of the nystagmus response, but had little or no effect on OHC or DPOAE. Higher doses of gentamicin damaged cochlear hair cells and reduced the DPOAE. In conclusion, slow infusion of a low dose of gentamicin into the semicircular canals mainly damages the vestibular hair cells and inactivates the nystagmus response without damaging cochlear hair cells or DPOAE.     

卡铂对灰鼠中枢听觉系统的影响

目的 卡铂选择性破坏灰鼠的内毛细胞和Ⅰ型传入神经末梢已被人们所证实,但是,卡铂是否损害耳蜗核、下丘和听觉皮层还不清楚,本文旨在观察卡铂对灰鼠听觉中枢的毒性作用。方法采用恒低温冷冻连续脑组织切片,以中枢听觉系统神经元的密度来评价卡铂对灰鼠中枢听觉系统的影响。结果发现注射卡铂3和4周后,耳蜗背侧核和腹侧核神经元明显的减少,与正常动物比较有显著性差异。而下丘和听觉皮层神经元的变化与正常灰鼠比较无明显差异。结论 说明注射卡铂3和4周后对耳蜗核有明显的毒性作用,可引起耳蜗核神经元明显的减少,但是,对灰鼠下丘和听皮层未见明显的毒性作用。     

Selective inner hair cell ototoxicity induced by carboplatin

Carboplatin is a second-generation platinum antineoplastic agent. It has biological activity similar to cisplatin and is currently recommended for the treatment of ovarian cancer. In clinical use, carboplatin appears less ototoxic than cisplatin. This paper reports the ototoxic effects of carboplatin, in doses equivalent to the clinical use of the drug, in the chinchilla. Intravenous carboplatin 200 to 400 mg/m² by bolus injection caused significant ototoxicity in this model as revealed by brainstem evoked responses (ABR audiometry). The cochlear pathology as seen by scanning electron microscopy revealed predominantly inner hair cell (IHC) stereocilia damage. Furthermore, the extent (grade) of the morphological lesions appears to be well correlated with the auditory brainstem response pattern of threshold elevation.     

Assessment of ototoxicity of tea tree oil in a chinchilla animal model

ObjectiveThe aim of the present study is to examine the effects of tea tree oil on hearing function and cochlear morphology after intratympanic administration in a chinchilla animal model.MethodsNine chinchillas received intratympanic injection of 3% tea tree oil dissolved in olive oil in one ear, whereas the contralateral control ear received olive oil only. Outcome measures included auditory brainstem responses conducted before treatment and at 10 days and 30 days following the injection. Post-mortem cochlear morphology was assessed using scanning electron microscopy.ResultsAt 10 and 30 days following the injection, there was no significant change in auditory brain response thresholds at 8, 16, 20 or 25 kHz. Scanning electron microscopy imaging showed no damage to auditory hair cells.ConclusionTea tree oil (3%) does not appear to be ototoxic in a chinchilla animal model. Future preclinical and clinical studies are required to establish the effectiveness of TTO in treating otitis.     

卡铂导致毛细胞及其传出神经损害的耳蜗分析图

目的：介绍一种同时评估耳蜗传出神经和毛细胞的简便的组织化学技术。方法：首先应用脱氢酶染色选择性标记毛细胞,再用乙酰胆碱醌酶染色标记传出神经纤维,双重染色的耳蜗铺片样品在光学显微镜下沿着耳蜗基底膜的全长分别对毛细胞和穿越Ｃｏｒｔｉ隧道的传出神经纤维计数,根据卡铂耳中毒灰鼠的毛细胞及其传出神经纤维损伤的百分比制备耳蜗图。结果：耳蜗分析图充分显示;当绝大多数内毛细胞坏死以及部分外毛细胞坏死时,越隧道的传     

D-Methionine attenuates inner hair cell loss in carboplatin-treated chinchillas

Carboplatin, a second-generation platinum-based antineoplastic drug, preferentially destroys inner hair cells (IHCs) in the chinchilla while sparing outer hair cells (OHCs). D-Methionine (D-Met), a sulfur-containing amino acid, has been shown to protect hair cells from cisplatin damage in rats, but its ability to protect IHCs from carboplatin damage has not yet been evaluated in the chinchilla. We tested whether D-Met would protect the hair cells in the chinchilla from carboplatin. Animals were divided into two groups: a control group that only received carboplatin (100 mg/kg, i.p.) and an experimental group that received 300 mg/kg D-Met (i.p.) 30 min before carboplatin treatment. Ototoxicity was assessed by measuring the amount of IHC and OHC loss. Average IHC loss in the group treated with D-Met was 62% compared with 84% in the untreated control group. Thus, D-Met causes a statistically significant reduction in IHC loss induced by carboplatin.     

Sex differences in auditory sensitivity of chinchillas before and after exposure to impulse noise

OBJECTIVE: To determine if chinchillas exhibit sex differences in 1) basic auditory sensitivity and 2) susceptibility to cochlear damage and hearing loss from high-level impulse noise. DESIGN: The auditory sensitivity of 73 chinchillas was assessed by measuring evoked potentials from electrodes implanted in the inferior colliculus (IC-EVPs) and cubic (2f1-f2) distortion product otoacoustic emissions (CDPs). A subgroup of 16 chinchillas were retested after exposure to simulated M16 rifle fire (150 dB pSPL impulse noise). Thresholds and postexposure temporary and permanent threshold shifts were compared as a function of sex and frequency using analysis of variance procedures. Cochleograms, showing the percent of hair cells missing as a function of location on the basilar membrane, were constructed to show inner hair cell (IHC) and outer hair cell (OHC) losses for each group. RESULTS: Female chinchillas had slightly lower high-frequency thresholds, and slightly higher low-frequency thresholds than male chinchillas, but similar IC-EVP and CDP amplitude functions. Significant sex differences were observed after exposure to high-level impulse noise. Overall, female chinchillas developed approximately 10 dB more high-frequency hearing loss, but approximately 5 dB less low-frequency hearing loss than males. Hair cell losses, particularly IHC losses, were substantially less for females as compared with males. CONCLUSIONS: The results point to close similarities between chinchillas and humans with regard to sex/gender differences in basic auditory sensitivity before noise exposure, suggesting that the chinchilla may be a good model for exploring the anatomical and physiological bases of these differences. In addition, the results show significant sex differences in the physiological and anatomical response of the chinchilla cochlea to high-level noise. Similar differences in humans could have important implications with regard to military assignments and hearing conservation programs.     

卡铂对离体培养成年灰鼠前庭毛细胞的损害

目的 观察并建立不同浓度卡铂损害成年灰鼠前庭终器的离体实验模型。方法 应用前庭终器分离取材技术、前庭器官离体培养技术和组织学检查技术，观察不同浓度卡铂对成年灰鼠前庭各终器的损害。结果 卡铂主要损害灰鼠前庭I型毛细胞，这种损害随着卡铂剂量的增加而加重。结论 卡铂选择性破坏离体培养的灰鼠前庭I型毛细胞。     

内毛细胞缺损对噪声引起外毛细胞损害的潜在影响

目的　观察噪声对正常灰鼠和内毛细胞缺损灰鼠的外毛细胞是否具有不同的破坏效应。方法　 6只正常灰鼠和 12只卡铂致聋灰鼠作为受试对象。提前 30天给 12只灰鼠按照 10 0mg/kg的剂量腹腔注射卡铂 ,以达到预先选择性破坏其内毛细胞的目的。将 6只正常灰鼠和 6只经卡铂处理的灰鼠移入噪声室 ,在强度为 12 0dB、中心频率在 4kHz的脉冲噪声环境暴露 6小时 ;另外 6只经卡铂处理的灰鼠不经噪声暴露作为对照组。在噪声暴露后 30天取出耳蜗 ,常规制备耳蜗铺片 ,然后进行全耳蜗毛细胞计数 ,并将各组动物的毛细胞计数结果制备成平均受损情况分布图 ,观察和比较噪声引起的外毛细胞损害水平在具有正常内毛细胞的灰鼠和丧失内毛细胞的灰鼠是否存在差别。结果　单纯注射卡铂的灰鼠内毛细胞平均损失在 90 %左右 ,但外毛细胞无损害。经噪声暴露的正常灰鼠外毛细胞在耳蜗底回中部约相当于 4kHz共振频率区域平均损失 6 0 % ,但是在该区域两侧靠近底回和顶回的外毛细胞损失少于 2 0 %。预先经卡铂破坏 95 %内毛细胞的灰鼠经噪声暴露后 ,外毛细胞在耳蜗底回中部平均损失达到 90 % ,在该区域两侧的外毛细胞损失百分比大于 4 0 %。结论　内毛细胞缺损可以造成更多的外毛细胞在噪声刺激中被破坏 ,提示正常内毛细胞的存在可能有     

Effect of inner and outer hair cell lesions on electrically evoked otoacoustic emissions

When the cochlea is stimulated by a sinusoidal current, the inner ear emits an acoustic signal at the stimulus frequency, termed the electrically evoked otoacoustic emission (EEOAE). Recent studies have found EEOAEs in birds lacking outer hair cells (OHCs), raising the possibility that other types of hair cells, including inner hair cells (IHCs), may generate EEOAEs. To determine the relative contribution of IHCs and OHCs to the generation of the EEOAE, we measured the amplitude of EEOAEs, distortion product otoacoustic emissions (DPOAEs), the cochlear microphonic (CM) and the compound action potential (CAP) in normal chinchillas and chinchillas with IHC lesions or IHC plus OHC lesions induced by carboplatin. Selective IHC loss had little or no effect on CM amplitude and caused a slight reduction in mean DPOAE amplitude. However, IHC loss resulted in a massive reduction in CAP amplitude. Importantly, selective IHC lesions did not reduce EEOAE amplitude, but instead, EEOAE amplitude increased at high frequencies. When both IHCs and OHCs were destroyed, the amplitude of the CM, DPOAE and EEOAE all decreased. The increase in EEOAE amplitude seen with IHC loss may be due to (1) loss of tonic efferent activity to the OHCs, (2) change in the mechanical properties of the cochlea or (3) elimination of EEOAEs produced by IHCs in phase opposition to those from OHCs.     

Paraquat-induced hair cell damage and protection with the superoxide dismutase mimetic m40403

Some forms of ototoxicity appear to be mediated primarily by the superoxide radical; however, the exact role the superoxide radical plays in cochlear damage is not well understood because most ototoxic drugs produce multiple reactive oxygen species. To characterize the role of the superoxide radical in cochlear damage and the protective effect of compounds that inactivate superoxide, we treated mouse cochlear organotypic cultures for 24 h with paraquat, an herbicide that produces high levels of superoxide. M40403, a highly specific, nonpeptidyl mimetic of superoxide dismutase, was added to some cultures to inactivate the superoxide radical generated by paraquat. The number of outer hair cells (OHC) and inner hair cells (IHC) systematically decreased with increasing concentration of paraquat (0.01-10 mM). M40403 (10 muM) significantly increased OHC and IHC survival in cultures treated with 0.01-1.0 mM of paraquat. These results suggest that excess production of superoxide radical is a sufficient condition for hair cell loss.     

Induction of spontaneous otoacoustic emissions in chinchillas from carboplatin-induced inner hair cell loss

Fifteen chinchillas were evaluated for spontaneous otoacoustic emissions (SOAEs) before and after administering carboplatin (126–200 mg/kg), an anti-neoplastic drug that selectively destroys inner hair cells (IHCs) in this species. SOAEs were absent from all animals prior to carboplatin treatment, but at 1 week post-treatment, 47% of the animals and 30% of the ears had developed SOAEs. SOAE frequencies were clustered between 5 and 10 kHz and SOAE intensity ranged from 10 to 32 dB SPL. All of the ears with SOAEs had IHC lesions exceeding 60% throughout most of the cochlea and two ears had outer hair cell lesions of 25–60% at a cochlear place associated with the frequency of the SOAE. Thus, high doses of carboplatin that cause IHC loss can be used to create an animal model with SOAEs.     

Auditory nerve activity and cochlear morphology after noise exposure.

Four chinchillas were exposed for 5 days to an octave band of noise centered at 4 kHz and having an SPL of 86 dB. After a recovery period of approximately 6 months, behavioral audiograms were obtained and auditory nerve fiber activity was recorded. The animals were killed and the cochleas embedded in plastic to obtain a surface preparation and 1 mu radial sections of the organ of Corti. Behavioral threshold shifts ranged from 5 to 20 dB at frequencies between 4 and 11 kHz. Auditory nerve fiber thresholds were elevated up to 70 dB for units with characteristic frequencies between 4 and 14 kHz. Units with higher and lower characteristic frequencies had normal thresholds. Cochleagrams showed narrow lesions of inner and/or outer hair cells over approximately a 1 mm distance. A comparison of the three realms of data revealed the following: (1) The greatest threshold shifts from the noise exposure were seen in the single nerve fiber thresholds while the smallest shifts were seen in the behavioral thresholds, (2) the greatest behavioral and neural threshold shifts and greatest cochlear damage occurred 1 octave above the center frequency of the noise exposure, and (3) based on the frequency-place map of the chinchilla cochlea, the range of fibers with elevated thresholds exceeded the extent of the OHC lesion. A number of anatomical changes were seen that effectively increased the extent of the damage found in the chochleagram. These changes included: distortions in the surface topography of the organ of Corti affecting the orientation of IHC; missing pillar cells in the presence of normal OHC and/or IHC and protrusion of the IHC cuticular plate into the subtectorial space.     

Rapid Hair Cell Loss: A Mouse Model for Cochlear Lesions

In comparison to other mammals, mice have proved extremely resistant to aminoglycoside-induced hair cell ablation in vivo. In this paper we examine the pattern and extent of cochlear lesions rapidly induced with a combination of a single dose of aminoglycoside (kanamycin) followed by a loop diuretic (bumetanide). With this protocol, the vestibular system was unaffected, but in the cochlea, there was extensive loss of outer hair cells (OHC) that commenced in the basal coil and progressed apically so that, by 48 h, OHC loss was almost complete. TUNEL-positive nuclei and activated caspase-3 labeling demonstrated that most OHC died via a classical apoptotic pathway. However, scattered debris within the OHC region suggested that many apoptotic cells ruptured prior to completion of apoptosis. Following lesion repair, supporting cells retained characteristics of differentiated cells but positional shift occurred. In comparison to OHC loss, inner hair cell (IHC) death was delayed and only observed in 50% of all cochleae examined even after extensive reorganization of the tissue. The coadmininstration of diuretic with FM1-43, used as a tracer for aminoglycoside uptake, indicated entry into IHC as readily as OHC, suggesting that the differential response to aminoglycoside was not due to differential uptake. Where IHC death was ongoing, there were indications of different modes of cell death: cells with morphological features of autophagy, necrosis, and apoptosis were apparent. In addition to damage to the organ of Corti, there was a significant and progressive decrease in strial thickness beginning as early as 7 days posttreatment. This was due predominantly to degeneration of marginal cells. The strial pathology resembled that reported after noise damage and with aging. This in vivo protocol provides a robust model in which to obtain extensive OHC loss in the mature cochleae of mice and is a means with which to examine different aspects of cochlear pathology in transgenic or mutant strains.     

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells

IntroductionInner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells.ObjectiveTo investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation.MethodsCochlear neural progenitor cells were cultured and induced in DMEM/F12 + RA (10^?6 M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu.ResultsThe nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p < 0.05). The ratios of Brdu⁺-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased.ConclusionRetinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.     

Auditory plasticity and hyperactivity following cochlear damage

This paper will review some of the functional changes that occur in the central auditory pathway after the cochlea is damaged by acoustic overstimulation or by carboplatin, an ototoxic drug that selectively destroys inner hair cells (IHCs) in the chinchilla. Acoustic trauma typically impairs the sensitivity and tuning of auditory nerve fibers and reduces the neural output of the cochlea. Surprisingly, our results show that restricted cochlear damage enhances neural activity in the central auditory pathway. Despite a reduction in the auditory-nerve compound action potential (CAP), the local field potential from the inferior colliculus (IC) increases at a faster than normal rate and its maximum amplitude is enhanced at frequencies below the region of hearing loss. To determine if this enhancement was due to loss of sideband inhibition, we recorded from single neurons in the IC and dorsal cochlear nucleus before and after presenting a traumatizing above the unit's characteristic frequency (CF). Following the exposure, some neurons showed substantial broadening of tuning below CF, less inhibition, and a significant increase in discharge rate, consistent with a model involving loss of sideband inhibition. The central auditory system of the chinchilla can be deprived of some of its cochlear inputs by selectively destroying IHCs with carboplatin. Selective IHC loss reduces the amplitude of the CAP without affecting the threshold and tuning of the remaining auditory nerve fibers. Although the output of the cochlea is reduced in proportion to the amount of IHC loss, the IC response shows only a modest amplitude reduction, and remarkably, the response of the auditory cortex is enhanced. These results suggest that the gain of the central auditory pathway can be up- or down regulated to compensate for the amount of neural activity from the cochlea.     

Auditory nerve activity and cochlear morphology after noise exposure

Summary Four chinchillas were exposed for 5 days to an octave band of noise centered at 4 kHz and having an SPL of 86 dB. After a recovery period of approximately 6 months, behavioral audiograms were obtained and auditory nerve fiber activity was recorded. The animals were killed and the cochleas embedded in plastic to obtain a surface preparation and 1 radial sections of the organ of Corti. Behavioral threshold shifts ranged from 5 to 20 dB at frequencies between 4 and 11 kHz. Auditory nerve fiber thresholds were elevated up to 70 dB for units with characteristic frequencies between 4 and 14 kHz. Units with higher and lower characteristic frequencies had normal thresholds. Cochleagrams showed narrow lesions of inner and/or outer hair cells over approximately a 1 mm distance. A comparison of the three realms of data revealed the following: (1) The greatest threshold shifts from the noise exposure were seen in the single nerve fiber thresholds while the smallest shifts were seen in the behavioral thresholds, (2) the greatest behavioral and neural threshold shifts and greatest cochlear damage occurred 1 octave above the center frequency of the noise exposure, and (3) based on the frequency-place map of the chinchilla cochlea, the range of fibers with elevated thresholds exceeded the extent of the OHC lesion. A number of anatomical changes were seen that effectively increased the extent of the damage found in the chochleagram. These changes included: distortions in the surface topography of the organ of Corti affecting the orientation of IHC; missing pillar cells in the presence of normal OHC and/or IHC and protrusion of the IHC cuticular plate into the subtectorial space.This research was supported in part by grants from: Deafness Research Foundation; NSF BNS-76-18913; NIEHS 5 K04-ES-00011; and NIOSH 2-R01-04-00364     

Cisplatin-induced vestibular hair cell lesion-less damage at high doses

Cisplatin, a widely used anticancer drug, damages hair cells in cochlear organotypic cultures at low doses, but paradoxically causes little damage at high doses resulting in a U-shaped dose-response function. To determine if the cisplatin dose-response function for vestibular hair cells follows a similar pattern, we treated vestibular organotypic cultures with doses of cisplatin ranging from 10 to 1000?μM. Vestibular hair cell lesions progressively increased as the dose of cisplatin increased with maximum damage occurring around 50–100?μM, but the lesions progressively decreased at higher doses resulting in little hair cell loss at 1000?μM. The U-shaped dose-response function for cisplatin-treated vestibular hair cells in culture appears to be regulated by copper transporters, Ctr1, ATP7A and ATP7B, that dose-dependently regulate the uptake, sequestration and extrusion of cisplatin.