强脉冲噪声导致的豚鼠耳蜗毛细胞凋亡及P53蛋白的表达

目的 探讨强脉冲噪声暴露引起的豚鼠毛细胞损害模式和P53凋亡蛋白在损伤毛细胞中的表达.方法 12只成年豚鼠,声压级168 dB的强脉冲噪声连续暴露80次,每次间隔时间2 s,分别在噪声暴露后3 h、6 h及12 h各处死4只动物,应用异硫氰酸荧光素(flourescein iso-thiocyanate,FITC)标记的鬼笔环肽(phalloidin)和细胞核DNA荧光染料碘化丙啶(propidium iodide,PI)来施行双重染色,在激光共聚焦显微镜下观察耳蜗毛细胞静纤毛和细胞核的形态学变化;将噪声暴露后12 h的耳蜗基底膜样品进行P53免疫荧光染色,观察受损毛细胞中是否存在P53蛋白的表达;耳蜗琥珀酸脱氢酶染色,制备基底膜铺片,400倍显微镜下行毛细胞计数并绘制耳蜗图.结果 强脉冲噪声暴露后3 h,耳蜗底回末端和第二回起始端已经发生明显的外毛细胞结构破坏,同时伴有细胞核的浓缩;暴露后6 h耳蜗毛细胞的破坏范围扩大,在毛细胞损害的中心区域可见外毛细胞的核碎片;毛细胞损害中心区域在噪声暴露后12 h,多数外毛细胞的细胞核消失,而周边区域的毛细胞可见核浓缩或核碎片.耳蜗图显示,噪声暴露后3、6及12 h,耳蜗毛细胞的破坏范围不断扩大.免疫荧光显示耳蜗第二回外毛细胞损伤的中心部位出现明显的P53阳性表达,在耳蜗底回和第三回外毛细胞中亦出现P53的阳性表达.结论 强脉冲噪声暴露引起耳蜗毛细胞死亡的主要方式是凋亡,损伤的中心部位在耳蜗第二回,并逐渐向耳蜗底回和第三回扩散;P53蛋白在此过程中可能扮演了重要角色.

Abstract：

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 12hours 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.     

The Cochlear Ribbon Synaptic Response to Aminogiycoside Ototoxicity in C57BL/6J Mice

Objective To investigate the early change of cochlear ribbon synapses on inner hair cells in response to aminoglycoside ototoxicity. Methods C57BL/6J mice received intraperitoneal injection of gentamicin (100 mg/kg/day), and the apical coil organ of Corti was examined on the 4th, 7th and 10th day (n=10). Litter-mates without gentamicin treatment served as controls (n=10). RIBEYE on the presynaptic membrane and AMPA receptors on the postsynaptic membrane were labeled with CtBP2 or GluR2/3 respectively. Three di-mension reconstruction was conducted using the 3DS MAX 8.0 software. Results There were no disruptions of outer or inner hair cells in all groups. However, the number of ribbon synapses on cochlear inner hair cells increased significantly within 7 days after gentamicin exposure (P<0.01), followed by a significant de-crease after 7 days.Conclusion During the early stage of aminoglycoside ototoxicity, increased population of cochlear ribbon synapses may indicate a significant down-regulation of synaptic function.     

OTOTOXIC EFFECTS OF CARBOPLATIN IN ORGANOTYPIC CULTURES IN CHINCHILLAS AND RATS

Carboplatin, a second-generation platinum chemotherapeutic drug, is considerably less ototoxic than cisplatin. While common laboratory species such as mice, guinea pigs and rats are highly resistant to carboplatin ototoxicity, the chinchilla stands out as highly susceptible. Moreover, carboplatin causes an unusual gradient of cell death in chinchillas. Moderate doses selectively damage type I spiral ganglion neurons (SGN) and inner hair cells (IHC) and the lesion tends to be relatively uniform along the length of the cochlea. Higher doses eventually damage outer hair cells (OHC), but the lesion follows the traditional gradient in which damage is more severe in the base than the apex. While carboplatin ototoxicity has been well documented in adult animals in vivo, little is known about its in vitro toxicity. To elucidate the ototoxic effects of carboplatin in vitro, we prepared cochlear and vestibular organotypic cultures from postnatal day 3 rats and adult chinchillas. Chinchilla cochlear and vestibular cultures were treated with carboplatin concentrations ranging from 50 μM to 10 mM for 48 h. Consistent with in vivo data, carboplatin selectively damaged IHC at low concentrations (50-100 μM). Surprisingly, IHC loss decreased at higher doses and IHC were intact at doses exceeding 500 μM. The mechanisms underlying this nonlinear response are unclear but could be related to a decrease in carboplatin uptake via active transport mechanisms (e.g., copper). Unlike the cochlea, the carboplatin dose-response function increased with dose with the highest dose destroying all chinchilla vestibular hair cells. Cochlear hair cells and auditory nerve fibers in rat cochlear organotypic cultures were unaffected by carboplatin concentrations <10 μM; however, the damage in OHC were more severe than IHC once the dose reached 100 μM. A dose at 500 μM destroyed all the cochlear hair cells, but hair cell loss decreased at high concentrations and nearly all the cochlear hair cells were present at the highest dose, 5 mM. Unlike the nonlinear dose-response seen with cochlear hair cells, rat auditory nerve fiber and spiral ganglion losses increased with doses above 50 μM with the highest dose destroying virtually all SGN. The remarkable species differences seen in vitro suggest that chinchilla IHC and type I SGN posse some unique biological mechanism that makes them especially vulnerable to carboplatin toxicity.     

离体培养时bFGF对顺铂耳蜗毒性的拮抗作用

目的 研究不同浓度的顺铂对耳蜗基底膜毛细胞的损伤、不同浓度的bFGF对顺铂耳蜗基底膜毛细胞损伤的保护作用以及caspase-9的表达。方法 对耳蜗基 底膜进行离体培养,用荧光染色方法对毛细胞数量和caspase-9的表达进行观察。结果 在不同浓度中,15μg/ml顺铂对基底膜毛细胞损伤最大;bFGF浓度≥100ng/ml时,对顺铂所致的基底膜毛细胞损伤具有良好的拮抗作用。结论 顺铂对耳蜗基底膜上毛细胞的损伤具有剂量依赖性; bFGF拮抗顺铂所致的耳蜗基底膜毛细胞的损伤;caspase-9的表达与bFGF对顺铂耳蜗毒性的拮抗作用相关。     

A Method for Establishing an Animal Model of Like-Auditary Neuropathy

Objective: To establish an animal model of like-auditory neuropathy in neonatal rat. Methods The ani-mals were injected with phenylhydrazine hydrochloride or saline at 7-day of age. ABR and DPOAE were performed to assess the auditory function. The cochlea basilar membrane stretched preparation and cochlear frozen sections were prepared for immunohistochemical staining to examine the morphological change of hair cells and spiral ganglion cells (SGNs). Results At 7-day age the ABR waveI, III, V, latencies andI-III,I-V IWIs in the experimental group were significantly prolonged compared with those in the control group. The ABR thresholds were also elevated in the experimental group. We found there is no significant differ-ence in DPOAE in phenylhydrazine hydrochloride exposure group compare to control group. The cochlear hair cells showed no signs of loss in both group, but the total number of neurofilaments positive cells in SGNs were significantly reduced in the phenylhydrazine treated animals. Conclusion Our study suggests that phenylhydrazine hydrochloride can change the auditory function and induce peripheral nerve pathology by targeted mainly the SGNs in neonatal rat.     

COMBINATIONAL ADMINISTRATION OF AMINOGLYCOSIDES AND LOOP DIURETICS AS AN EFFICIENT STRATEGY TO ESTABLISH DEAFNESS MODELS IN RATS

It is known that aminoglycoside antibiotics can damage the vestibular and auditory sensory epithelia, and the loop diuretics can enhance the ototoxic effect of aminoglycosides. Previous studies on the synergistic effect of these two types of drugs have used mice, guinea pigs and cats, but not rats. The aim of this study was to determine this synergistic effects in rat cochleae. Rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. Au- ditory brainstem response （ABR）, scanning electron microscopy （SEM） and immunocytochemistry were used to determine the effects of drug administration. In the group receiving combined administration of furosemide and kanamycin, the ABR thresh- old showed significant elevation 3 days after drug administration, greater than single drug administration. The hair cells showed various degrees of injury from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. Neuron fibers of the hair cells showed significant loss 7 days after the drug administration, but the number of spiral ganglia did not decrease and supporting cells showed no signs of injury. Our study suggest that combined administration of fu- rosemide and kanamycin has an synergistic ototoxic effect, and can result in hair cell loss and hearing loss in rats.     

The progenitors of inner ear hair cells and their regulating genes

Hair cells in the mammalian inner ear are very fragile and are often injured as a result of acoustic trauma or exposure to ototoxic drugs (cisplatin, aminoglycosides, etc)[1]. In amphibians and birds, spontaneous post-injury regeneration of all inner ear sensory hair cell occurs, while in the mammalian cochlea, such hearing loss is usually permanent as there are currently no treatments that can lead to post-injury hair cell regeneration.     

THE COCHLEAR AMPLIFIER： IS IT HAIR BUNDLE MOTION OF OUTER HAIR CELLS？

Cochlear outer hair cells （OHCs） are involved in a mechanical feedback loop in which the fast somatic motility of OHCs is required for cochlear amplification. Alternatively, amplification is thought to arise from active hair bundle movements ob- served in non-mammalian hair cells. We measured the voltage-evoked hair bundle motions in the gerbil cochlea to determine if such movements are also present in mammalian OHCs. The OHCs displayed a large hair bundle movement that was not based on mechanotransducer channels but based on somatic motility. Significantly, bundle movements were able to generate radial motion of the rectorial membrane in situ. This result implies that the motility-associated hair bundle motion may be part of the cochlear amplifier.     

灰鼠畸变产物耳声发射改变和外毛细胞缺失程度的相关分析

目的 探讨灰鼠畸变产物耳声发射(DPOAE)幅值的降低与外毛细胞缺失率之间的关系.方法 联合应用顺铂和利尿酸钠,建立灰鼠耳蜗毛细胞损伤模型.12只灰鼠在静脉注射利尿酸钠(40 mg/kg)同时,腹腔注射顺铂(0.2 mg/kg),分别于用药前和用药后的1周、2周及3周在清醒状态下检测动物DPOAE,并在施行最后1次DPOAE检测后处死动物.常规制备灰鼠耳蜗基底膜铺片并进行全耳蜗毛细胞计数.计算用药前和处死前DPOAE幅值的改变程度,通过Pearson相关分析对DPOAE幅值的降低与外毛细胞缺失程度之间的相关性进行分析.结果 顺铂和利尿酸钠引起灰鼠耳蜗外毛细胞缺失的特点是病变始发于基底膜的底回并逐渐向顶回发展,外毛细胞的缺损程度沿着基底膜从底回向顶回逐渐减轻,而不同频率DPOAE幅值降低的程度亦遵循从高频向低频发展的规律,各频率DPOAE在注射顺铂和利尿酸钠后的幅值改变与基底膜上相应部位的外毛细胞缺损程度存在一定的对应关系(r=0.796,P<0.05).外毛细胞每损失1%,可引起约0.24 dB的DPOAE幅值降低.结论 在灰鼠耳蜗损伤模型中,从DPOAE幅值的变化可以大致评估耳蜗外毛细胞的缺失程度.     

Ultrastructural damage in cochleas used for studies of basilar membrane mechanics

Cat cochleas used for interferometric studies of basilar membrane mechanics were examined with the electron microscope. The structures most severely damaged in the experimental cochleas are the outer hair cells and the radial afferent fibers to the inner hair cells. Since the basilar membrane and other supporting structures appear to be normal, mechanical changes observed in the experimental cochleas are most probably due to outer hair cell damage. Individual animals with varying degrees of damage showed large differences in the frequency of basilar membrane resonance at the same place in the cochlea. Shifts in tuning of this magnitude could occur as a consequence of hair cell damage only if the stiffness of the stereocilia and associated structures was greater initially than the stiffness of the basilar membrane and gradually decreased with damage. The present series of observations, therefore, suggest that the stiffness of the outer hair cell stereocilia determines basilar membrane tuning.     

顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶表达的影响

目的：观察顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶（calpain ）表达的影响,探讨顺铂致耳蜗毛细胞凋亡的机制。方法取出生后3 d的昆明小鼠300只（600耳）,分离出耳蜗基底膜600条,体外培养24 h后,随机分为对照组和4、8、16μg／ml顺铂组,每组150条;对照组加入2ml新鲜培养基,顺铂组分别加入2 ml含不同浓度顺铂（4、8、16μg／ml）的新鲜培养基,再继续培养24 h后,应用Hoechst 33258荧光染色观察耳蜗毛细胞凋亡情况,并应用免疫荧光染色和免疫印迹（Western blot）技术检测calpain 1（μ－calpain）和calpain 2（m －calpain）在耳蜗毛细胞的表达。结果4、8、16μg／m l顺铂组耳蜗毛细胞凋亡率分别为15．63％±0．20％、38．40％±2．64％和64．24％±0．05％,均高于对照组（5．55％±0．12％）,呈现明显的量效关系;不同浓度顺铂组μ－calpain和m －cal‐pain的表达均较对照组明显增强（ P＜0．01）,且m －calpain的表达随顺铂浓度的增高而明显增强（ P＜0．01）。结论顺铂可通过calpain通路诱导小鼠耳蜗毛细胞凋亡,从而发挥毒性效应。     

全耳蜗毛细胞定量分析系统

目的 介绍一种计算机辅助下的全耳蜗毛细胞定量分析系统。方法 选取氨基糖甙类药物损伤、噪声性损伤以及老年性聋动物模型的耳蜗制备全耳蜗基底膜铺片，从蜗尖向蜗底逐个视野依次进行内外毛细胞计数，将采集的数据输入到计算机并用毛细胞定量分析系统制备成耳蜗图。结果 氨基糖甙类药物耳蜗损害模型的耳蜗图显示毛细胞缺损自底回向顶回发展，外毛细胞的损伤比内毛细胞严重；强噪声引起的毛细胞破坏局限在与刺激声频率相对应的基底膜区域；6月龄C57BL／6J小鼠的耳蜗图显示老年性聋的早期损害起始于耳蜗底回的起始端，其中外毛细胞的破坏比内毛细胞严重。结论 耳蜗毛细胞定量分析系统可清晰显示全耳蜗毛细胞在基底膜上不同部位的损失程度和破坏范围，并可对应到基底膜上各个频率敏感部位。将传统的耳蜗铺片与计算机技术相结合制备的耳蜗图，具有全面可靠、简便精确、规范等优点。     

Cisplatin-induced increases in spontaneous neural activity in the dorsal cochlear nucleus and associated outer hair cell loss

Tinnitus is one of the consequences of cisplatin chemotherapy, but its underlying mechanisms are not well understood. Since it has been shown that cisplatin causes outer hair cell loss, it is possible that loss of these cells might induce tinnitus by increasing spontaneous activity in the central auditory system. To test this possibility, the present study examined the effects of cisplatin treatment on cochlear hair cells and on spontaneous neural activity in the dorsal cochlear nucleus of hamsters. Recordings, carried out approximately 1 month after cisplatin treatment, demonstrated significant increases in spontaneous activity across broad regions of the dorsal cochlear nucleus relative to levels in saline-treated controls. Histological results showed that cisplatin-treated animals also displayed dramatic loss of outer hair cells over most of the basal turn of the cochlea. Inner hair cells remained intact, although some evidence of damage to their stereocilia was evident. These findings indicate that cisplatin treatment causes abnormalities in spontaneous activity in the dorsal cochlear nucleus that are associated with widespread damage to outer hair cells. However, since some damage to inner hair cells was also observed, the role of inner hair cell injury in contributing to higher spontaneous activity cannot be ruled out.     

Basic fibroblast growth factor protects auditory neurons and hair cells from noise exposure and glutamate neurotoxicity

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Neurosci,2000, 16; 6125 - 6134. ［31］Ornitz, D.M.,Xu, J., Colvin, J.S., McEwen, D.G., MacArthur,C. A., Coulier, F., Gao, G., Goldfarb, M. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem, 1996, 271: 15292 -15297.     

cis-diamminedichloroplatinum cochlear toxicity

Twenty-nine guinea pigs were divided into three groups. The first group received distilled water 2 ml/kg ip daily; the second group received DDP 2 mg/kg ip daily; and the third group DDP 4 mg/kg ip daily. ABR hearing threshold was tested before injection. Twenty-four hours after systemic administration, ABR hearing threshold was tested again. The animals were killed and the cochlear specimens were observed under light, scanning and transmission electron microscopes. No abnormality regarding ABR hearing threshold and cochlear morphology was observed in the first group. In the second group, ABR hearing threshold was elevated, inner and outer hair cells, supporting cells, striae vascularis and spiral ganglions were damaged. In the third group, ABR hearing threshold elevated markedly. All above-mentioned cells were severely damaged. The experiment showed that the cochlear damage induced by DDP was dose related. The severest damage was seen in the second turn of the cochlea. The damage to the Deiters' cells was earliest and severest. The outer hair cells were more vulnerable to the damage than the inner hair cells did.     

In vitro morphological study of cochlear epithelium.

1. The mammalian cochlear epithelium was successfully kept alive in long-term tissue cultures. The Na and K concentration of the culture medium was similar to that of perilymph. 2. The most vulnerable cells in the culture epithelium were the outer and inner hair cells followed by the inner zones cells and the interdental cells of the spiral limbus. The difference in disintegration time between the outer and inner hair cells was about 48 hrs under the same culture conditions. The same phenomenon was noted between the rows of outer hair cells. 3. When the outer hair cells were not supported by Deiters' cells or when the cochlear epithelium was separated between Deiters' cells and Hensens cells, the outer hair cells degenerated quickly. 4. The interdental cells appeared to be relatively highly differentiated and showed much activity. No evidence of secretory function of interdental cells could be obtained. 5. The inner sulcus cells, Hensens cells, Claudius' cells and Reissners membrane cells were flattened and arranged like covering epithelium.     

特异性损伤内耳螺旋神经元小鼠模型的建立

目的：建立小鼠内耳螺旋神经元损伤的耳聋模型,为研究干细胞移植治疗感音神经性聋奠定基础。方法成年雌性SPF级CBA／J小鼠60只,随机分为实验组和生理盐水组,每组30只,实验组动物经圆窗渗透给予10μl哇巴因（ouabain）,生理盐水组同法给予等量生理盐水。每组于给药前及给药后7、14及30天分别检测小鼠听性脑干反应（ABR）和畸变产物耳声发射（DPOAE）,并用免疫组织荧光技术和基底膜铺片技术分别观察耳蜗螺旋神经元（spiral ganglion neurons ,SGNs）细胞及内耳毛细胞（hair cells ,HCs）的变化。结果①与生理盐水组比较,实验组给药后各时间点 ABR反应阈均明显升高,波Ⅰ潜伏期延长,振幅明显降低,差异有统计学意义（ P＜0．05）。②实验组及生理盐水组小鼠DPOAE均可正常引出。③与生理盐水组相比,实验组耳蜗各回螺旋神经元的数量及密度显著降低,差异有统计学意义（P＜0．05）。④实验组在给药后不同时间点,耳蜗各回内、外毛细胞均排列整齐、形态完整、未见明显损伤或丢失。结论哇巴因经圆窗渗透给药可特异性损伤CBA／J小鼠内耳螺旋神经元及其功能,而不损伤内、外毛细胞,是一种理想的研究干细胞移植治疗感音神经性聋的动物模型。     

Potentiation of inner ear damage following electron beam irradiation with CDDP administration

The study was designed to examine the combined ototoxic effect of CDDP (Cisplatin, Cis-diammine dichloroplatinum) and electron beam irradiation, using guinea pigs. One group received physiological saline solution of 4 ml/kg/day, and another group received CDDP of 2 mg/kg/day for five days. And following the injection of saline or CDDP, the electron beam of 14Gy/day was applied to the both groups to the right ear for five days. Animals were sacrificed after 21 days, and temporal bones of these animals were removed for the inner ear histopathology. Temporal bones were classified into four groups (control, electron beam irradiation, CDDP administration, and combined administration group), and the inner ears were observed by the surface preparation technique with a phase contrast microscope, and by scanning and transmission electron microscopy, and by temporal bone study of serial sectioned slides. The main pathologic findings of the inner ear are as follows: Electron beam irradiation group showed no hair cell damage. CDDP group induced slight damage to the outer hair cells. Combined administration group (CDDP + electron beam irradiation) showed severe outer hair cell damage. Stria vascularis was degenerated moderately in the combined administration group and slightly in some animals of electron beam irradiation group. And Reissner's membrane and Hensen's cells, were damaged in the basal turn of cochlea of the combined administration group. All other cochlear structures (spiral ganglion, spiral lamina, basilar membrane, spiral prominence, tectorial membrane, blood vessels of the cochlea) and vestibular organs were lack of significant changes in all groups by a light microscopic observation. This study was clarified that combined administration of CDDP and electron beam irradiation showed severe ototoxic potentiation. Therefore, it is important that we must pay attention to the inner ear damage caused by combined therapy of CDDP and electron beam irradiation involving inner ear for the head and neck tumor.