噪声暴露后耳蜗凋亡与坏死毛细胞琥珀酸脱氢酶活性的变化

目的揭示噪声暴露后耳蜗凋亡、坏死毛细胞线粒体功能的变化。方法将灰鼠暴露于155dBSPL的脉冲噪声75次，噪声暴露后5h解剖取双侧耳蜗。采用琥珀酸脱氢酶（SuccinateDehydrogenase，SDH）染色法进行耳蜗基底膜细胞线粒体染色，细胞核DNA荧光染料碘化丙啶（propidiumiodide，PI）双重染色耳蜗基底膜，以未受噪声暴露动物为对照，显微镜下观察噪声暴露后耳蜗基底膜核固缩和核肿胀毛细胞琥珀酸脱氢酶染色的变化。结果噪声暴露损伤区的耳蜗外毛细胞SDH着色变浅．蓝色颗粒物存在于PI标记固缩的耳蜗毛细胞核周围，而肿胀的毛细胞核周围缺少SDH阳性染色物。结论强脉冲噪声暴露后，凋亡的耳蜗外毛细胞依然存在不同程度的线粒体功能，而坏死的外毛细胞线粒体功能受损严重。     

噪声损伤引起耳蜗外毛细胞凋亡时细胞核内单链DNA的产生及EndoG的转移

目的观察噪声损伤后耳蜗外毛细胞内单链DNA和EndoG的变化，探讨耳蜗外毛细胞的死亡机制。方法豚鼠随机分为噪声暴露组、MNNG耳蜗灌流组和对照组（每组各12只）；小鼠随机分为噪声暴露组和对照组（每组12只）。分离解剖耳蜗后，用碘化丙啶（PI）染色细胞核、Pholloidin染色F—actin，免疫荧光抗体分别染色单链DNA（ssDNA）、核酸内切酶G（Endonuclease G EndoG）和凋亡诱导因子（Apoptosis inducing factors，AIF），制备耳蜗铺片，激光共聚焦显微镜下观察凋亡和坏死毛细胞内的荧光信号变化。结果（1）暴露于120dB SPL的白噪声环境中每天4小时，连续2天后引起豚鼠和小鼠耳蜗外毛细胞凋亡时，其细胞核内产生ssDNA，而在正常细胞内没有三ssDNA；（2）在正常情况下，EndoG分布于耳蜗毛细胞的细胞核外，在暴露于上述噪声后发生凋亡和坏死的豚鼠耳蜗外毛细胞中，EndoG从细胞核外转移到细胞核内，细胞核中的EndoG显著增加；（3）豚鼠耳蜗外淋巴灌流烷化剂MNNG后发生耳蜗外毛细胞凋亡和坏死，在凋亡和坏死的耳蜗外毛细胞中，AIF自线粒体转移到细胞核，其变化与噪声损伤引起耳蜗外毛细胞凋亡和坏死时一致。结论噪声刺激或烷化剂MNNG灌流后，造成耳蜗外毛细胞DNA损伤，产生ssDNA，引起AIF和EndoG自线粒体释放，激活Caspase-3，AIF和EndoG进一步向细胞核转移，最终使细胞核内的DNA降解，导致耳蜗毛细胞的死亡。     

葛根素对庆大霉素所致小鼠耳蜗毛细胞损伤的拮抗效应

目的观察葛根素对庆大霉素所致小鼠耳蜗毛细胞损伤的拮抗效应。方法完整分离小鼠耳蜗基底膜，无血清培养液常规培养24小时，然后同时加入0．3ml庆大霉素和不同浓度葛根素（1mg／ml，2mg／ml，4mg／m1），共同培养24h，并设立平行对照。标本置荧光显微镜下观察，行全耳蜗毛细胞计数，应用全耳蜗毛细胞定量分析软件自动分析，根据耳蜗毛细胞损失率的变化确定葛根素对庆大霉素所致耳蜗毛细胞损伤效应的影响。结果不同浓度组葛根素均表现小鼠耳蜗毛细胞损失率显著降低（P〈O．05-0．01），但以4mg／ml浓度效果最好，呈现剂量依赖性特点。结论葛根素对庆大霉素所致的小鼠耳蜗毛细胞损伤有明显拮抗效应。     

细胞色素C在老年SD大鼠耳蜗不同阶段凋亡坏死毛细胞中表达的变化

目的：观察细胞色素c在老年SD大鼠不同阶段凋亡、坏死毛细胞中表达的变化,探讨老年大鼠耳蜗毛细胞死亡的机制。方法：16只SD大鼠分组：青年组8只,2～3月龄;老年组8只,24～26月龄。应用电位反应测听仪检测不同频率短纯音（4、8、16、32和40kHz）诱发的青年组和老年组大鼠双侧ABR。听觉功能测试后,解剖取出双耳听泡,分离耳蜗基膜。分别用细胞核DNA染料碘化丙锭染色不同月龄大鼠耳蜗基膜细胞核,细胞色素C免疫荧光染料标记大鼠耳蜗基膜细胞的细胞色素c蛋白,荧光显微镜下观察不同月龄大鼠耳蜗基膜细胞荧光染色的变化。结果：老年组大鼠的不同频率ABR阈值均高于青年组（P〈0．01）。微弱拘细胞色素c荧光标记物存在于青年组大鼠耳蜗基膜毛细胞质（细胞膜的内侧,线粒体存在部位）,增强的细胞色素C免疫反应物存在于老年组大鼠耳蜗基膜细胞。细胞色素c阳性反应见于：①细胞核型不变;②碘化丙锭染色略有加深,细胞核型不变;③细胞核轻微核固缩;④核肿胀的毛细胞。相反,部分核固缩≤3／4核的毛细胞细胞色素C阳性反应物减少或消失,核缺失区域的毛细胞未见细胞色素c阳性反应物。老年大鼠耳蜗基膜凋亡毛细胞线粒体释放的细胞色素c存在核内转移现象。结论：细胞色素c免疫荧光染色为老年耳蜗基膜毛细胞退行性变早期的细胞生物学标记之一。细胞色素C向细胞核内的转移,可能与老年大鼠耳蜗凋亡毛细胞染色质凝聚相关。     

TUNEL与碘化丙啶双染检测耳蜗毛细胞的损伤

目的应用TUNEL与PI（碘化丙啶）双染法检测耳蜗毛细胞损伤。方法听力正常豚鼠经庆大霉素致聋后，做耳蜗基底膜铺片，用TUNEL与PI双染检测耳蜗毛细胞损伤的情况。结果在核固缩、核碎裂及部分核肿胀的细胞中TUNEL染色为阳性．细胞核正常及部分核肿胀的细胞中TUNEL染色为阴性。所有有核细胞均能被PI染色。结论通过TUNEL与PI双染可以确定TUNEL染色为阳性的细胞为凋亡的细胞，而核肿胀的细胞中TUNEL染色为阴性者可能为胀亡细胞。     

庆大霉素致豚鼠耳蜗毛细胞凋亡时Caspase-3的表达

目的探讨庆大霉素所致耳蜗外毛细胞的损伤特点及耳毒性机制。方法豚鼠随机分3个组,分别腹腔注射庆大霉素、庆大霉素加速尿、生理盐水各连续6天,比较用药前后各组豚鼠畸变产物耳声发射（distortion product otoacoustic emissions,DPOAE）变化、耳蜗铺片、毛细胞凋亡情况及Caspase-3在毛细胞的表达。结果注射庆大霉素和庆大霉素加速尿2个组,用药后DPOAE明显下降,耳蜗铺片见毛细胞损伤明显,细胞凋亡发生及Caspase-3免疫复合物阳性表达。结论庆大霉素可致豚鼠耳蜗毛细胞损伤并加速凋亡,Caspase-3在毛细胞凋亡过程中起重要作用。     

活性氧诱导离体大鼠耳蜗Corti器毛细胞凋亡的观察

目的探讨氧自由基中毒后耳蜗毛细胞有无凋亡及其变化规律。方法选用新生1～5d龄SD大鼠24只,随机分为4组。每组6只（12耳）：①无血清培养液组（H2O2浓度为零）;②0．05mmol／LH2O2组;③0．1mmol／LH2O2组;①0．5mmol／LH2O2组。分离Cord器,并用尖刀按顶回、中回、底回将其分为3段,分别放人相应浓度的H2O2培养液中培养．培养结束后用丫啶橙／碘化丙啶双重染色技术检测并计数凋亡的毛细胞。结果不同浓度H2O2组均检测到凋亡毛细胞,外毛细胞（OHC）是H2O2攻击的主要靶细胞,而支持细胞无凋亡。底回毛细胞损伤明显重于顶回和中回,各回外毛细胞损伤明显高于内毛细胞;随H2O2浓度增加,各回凋亡细胞增加。结论本实验条件下,外源性H2O2可直接诱导离体培养大鼠耳蜗Corti器毛细胞凋亡。支持细胞无凋亡。     

过氧化氢对豚鼠耳蜗功能及形态的影响

目的 在体观察过氧化氢(H2O2)对豚鼠耳蜗功能及形态的影响。方法 实验动物分为4组，分别灌流人工外淋巴液(artificial perilymph,APL)，50μMH2O2，100μMH2O2和200μMH2O2(H2O2均溶于APL中)，并用Pl和H033342双染色方法观察H2O2引起的内耳细胞损伤情况。结果 所有H2O2灌流组复合动作电位((CAP)阈移和耳蜗微音电位(CM)幅度变化与人工外淋巴液组比较差异有显著性，且各H2O2组的变化呈现出浓度依赖性；Pl和H033342双染色方法发现外毛细胞是H2O2攻击的主要靶细胞，而Hensen细胞未见任何损伤痕迹。结论 H2O2可导致耳蜗功能下降及耳蜗毛细胞损伤：Hensen细胞较毛细胞可能具有更强的抗氧化能力。     

模拟失重条件下飞船内噪声对豚鼠耳蜗形态与功能的影响

目的探讨模拟失重条件下,飞船内稳态噪声对豚鼠耳蜗形态与功能的影响。方法32只豚鼠随机分为单纯失重组16只、失重＋稳态噪声组16只。后肢悬吊法模拟失重,暴露于模拟飞船内在天飞行段的噪声环境,共5天。实验前、实验结束后即刻和实验结束后3天测试脑干诱发电位（ABR）阈值,取耳蜗标本行扫描电镜和透射电镜观察。结果实验组实验结束后即刻ABR阈值较实验前及实验结束后3天均增高（P〈0.01）;实验结束后3天ABR阈值较实验前高（P〈0.05）;实验结束后即刻及实验结束后3天失重＋稳态噪声组ABR阈值均较单纯失重组高（P〈0.01）。扫描电镜观察实验组实验结束后即刻耳蜗内、外毛细胞均受损。实验结束后3天,单纯失重组少数耳蜗各回内、外毛细胞的损伤程度比实验结束即刻加重;失重＋稳态噪声组耳蜗各回内毛细胞损伤较实验结束即刻重,外毛细胞损伤较实验结束即刻轻。实验组各时间段内毛细胞的损伤均重于外毛细胞,自第一回至第四回毛细胞损伤逐渐加重。透射电镜观察实验组耳蜗毛细胞及神经节细胞均可见空泡样改变,线粒体分布减少,细胞核固缩,可见细胞凋亡和细胞坏死两种细胞死亡现象。结论失重及失重＋稳态噪声均可造成豚鼠耳蜗形态和功能损伤,后者造成的损伤更重。失重对耳蜗毛细胞损伤以内毛细胞为重,损伤从底回至顶回逐渐加重。实验结束后3天较实验后即刻的听功能有所恢复但内毛细胞损伤加重。     

庆大霉素和窄带白噪声对鸡耳蜗损害后毛细胞再生形式研究

本文报告用庆大霉素（GM）和窄带白噪声（NB）联合作用对鸡耳蜗损害后观察毛细胞再生的组织形态。GM＋NB损害鸡内耳6天后，可见鸡耳蜗基底乳头（BP）基部至中上部有广泛的毛细胞消失，病理改变有三种形式：（1）空洞变形；（2）毛细胞崩溃消失；（3）支持细胞转化。损害后一周再生毛细胞发育成熟、数目增多；两周后再生毛细胞已生长旺盛，完全成熟，说明鸡耳蜗毛细胸有较强的再生能力。     

人神经营养因子3和绿色荧光蛋白基因共表达腺病毒载体对耳蜗组织的转染

目的:构建一个神经营养因子3(NT3)和绿色荧光蛋白(EGFP)基因共表达重组腺病毒载体,确定Ad/NT-3对离体耳蜗的转染效率和NT3对螺旋神经元存活的作用.方法:使用pAdeasy-1和pAdTrack CMV腺病毒载体系统,产生带绿色荧光标记的NT3重组腺病毒.转染培养新生的大鼠耳蜗基底膜,观察病毒的转染效率.对培养15 d的耳蜗进行神经丝蛋白200免疫荧光染色,计数螺旋神经元.观察NT3对神经存活的影响.结果:重组NT3病毒能够转染整个耳蜗组织中的各型细胞.以外沟细胞最高.约为49%,其次为齿间细胞,为27%.仅有少量的毛细胞和螺旋神经元被转染.Ad/NT-3重组腺病毒处理15 d的耳蜗螺旋神经元存活的数目多于Ad/EGFP腺病毒转染的耳蜗.结论:构建的Ad/NT-3重组腺病毒能够同时在耳蜗组织中表达EGFP和NT3蛋白,主要表达于外沟细胞和齿问细胞,这些细胞释放NT3能够维持耳蜗神经元存活.     

内毛细胞传入神经突触病变和遗传性听功能障碍

近年来的研究表明,耳蜗内毛细胞传入神经突触（cochlear ribbon synapse）在听觉系统中发挥着十分关键的作用。本文介绍了OTOF,SLC17A8,Diaphanous homolog 3（DIAPH3）和SMAD4等目前研究比较深入的几个基因缺陷所导致的遗传性听功能障碍得分子病理机制。上述基因缺陷导致耳聋的一个共同机制是基因缺陷首先破坏了耳蜗内毛细胞传入神经突触的结构和功能。在表型上,这些基因缺陷动物共同表型出了听神经病的临床特征：脑干诱发电位（auditory brainstem respons,ABR）不能引出,畸变产物耳声发射（distortion product acoustic emission,DPOAE）和耳蜗电位（cochlear microphonics,CM）正常或基本正常。形态学研究证明了功能检测的结构及动物耳蜗毛细胞和突触后听神经的形态结构都是正常或基本正常的。对这些基因缺陷致聋机制的研究具有重要的临床价值,特别是对今后开展针对上述基因缺陷耳聋儿童的人工耳蜗植入治疗具有指导意义。     

N-acetylcysteine and N-nitroarginine methyl ester attenuate Carboplatin-induced ototoxicity in dissociated spiral ganglion neuron cultures

Objectives

Carboplatin, a platinum-containing anti-cancer drug used to treat a variety of cancers, induces ototoxicity. Since, reactive oxygen species (ROS) and nitric oxide (NO) seem to be responsible for this toxicity, the antioxidant, N-acetyl-L-cysteine (L-NAC), and NO synthetase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) were predicted to have protective effects against carboplatin ototoxicity. The aim of this study was to test for the protective effects of L-NAC and L-NAME on cochlear hair cells and spiral ganglion neurons (SGNs).

Methods

Cochlear organotypic cultures and dissociated spiral ganglion neuron cultures, from mice postnatal day 5 cultures were used in this study. The cultures were treated with carboplatin alone or in combination with L-NAC or L-NAME, and carboplatin-induced damage was monitored.

Results

Treatment with carboplatin induced a significant loss of outer hair cells, while inner hair cells were preserved in the cochlear organotypic cultures. Addition of L-NAC or L-NAME reduced the amount of carboplatin-induced hair cell damage; the differences did not reach statistical significance. However, carboplatin significantly decreased the number of surviving SGNs in dissociated cultures. The toxic effects were significantly reduced by addition of L-NAC or L-NAME. In addition, carboplatin induced the loss of neurites from the SGN somata, and this was not blocked with L-NAC or L-NAME.

Conclusion

The results of this study suggest that ROS and NO are involved in carboplatin-induced damage to hair cells and SGNs, and administration of L-NAC/L-NAME can be used to attenuate the toxicity.     

Adenovirus-mediated expression of brain-derived neurotrophic factor protects spiral ganglion neurons from ototoxic damage

Hair cell loss, the most common cause of deafness, is often associated with auditory nerve degeneration. Our goal was to determine the influence of combined ciliary-derived neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) gene therapy on the survival of spiral ganglion neurons (SGNs) after elimination of inner hair cells in the mature guinea pig ear. Seven days after bilateral deafening, a 5-microl suspension of CNTF and/or BDNF adenovirus vectors was injected into the left scala tympani through the round window. Animals were sacrificed 28 days after deafening, and their inner ears were prepared for SGN counts. The SGN counts revealed that BDNF alone and the combined CNTF and BDNF treatment significantly enhanced SGN survival. CNTF did not significantly enhance the protective effect of BDNF. These data present possible strategies for enhancing SGN survival in cochlear implant procedures.     

Structural and Ultrastructural Changes to Type I Spiral Ganglion Neurons and Schwann Cells in the Deafened Guinea Pig Cochlea

Sensorineural hearing loss is commonly caused by damage to cochlear sensory hair cells. Coinciding with hair cell degeneration, the peripheral fibres of type I spiral ganglion neurons (SGNs) that normally form synaptic connections with the inner hair cell gradually degenerate. We examined the time course of these degenerative changes in type I SGNs and their satellite Schwann cells at the ultrastructural level in guinea pigs at 2, 6, and 12 weeks following aminoglycoside-induced hearing loss. Degeneration of the peripheral fibres occurred prior to the degeneration of the type I SGN soma and was characterised by shrinkage of the fibre followed by retraction of the axoplasm, often leaving a normal myelin lumen devoid of axoplasmic content. A statistically significant reduction in the cross-sectional area of peripheral fibres was evident as early as 2 weeks following deafening (p < 0.001, ANOVA). This was followed by a decrease in type I SGN density within Rosenthal’s canal that was statistically significant 6 weeks following deafening (p < 0.001, ANOVA). At any time point examined, few type I SGN soma were observed undergoing degeneration, implying that once initiated, soma degeneration was rapid. While there was a significant reduction in soma area as well as changes to the morphology of the soma, the ultrastructure of surviving type I SGN soma appeared relatively normal over the 12-week period following deafening. Satellite Schwann cells exhibited greater survival traits than their type I SGN; however, on loss of neural contact, they reverted to a non-myelinating phenotype, exhibiting an astrocyte-like morphology with the formation of processes that appeared to be searching for new neural targets. In 6- and 12-week deafened cochlea, we observed cellular interaction between Schwann cell processes and residual SGNs that distorted the morphology of the SGN soma. Understanding the response of SGNs, Schwann cells, and the complex relationship between them following aminoglycoside deafening is important if we are to develop effective therapeutic techniques designed to rescue SGNs.     

High frequency radial movements of the reticular lamina induced by outer hair cell motility

Recently, it was shown in cochlear explants from the guinea pig cochlea that electrokinetic motile responses of outer hair cells can induce radial and transverse motion of the reticular lamina. Here we demonstrate, that the radial component of these motions can be measured up to high frequencies (15 kHz). Cochlear explants were taken from guinea pig inner ears and exposed to a sinusoidal electric field. A double photodiode was used as a linear position detector with high spatial and temporatl resolution to detect radial movements in the plane of the reticular lamina. The organ of Corti of the second, third and fourth cochlear turns was stimulated with frequencies of the electrical field between 0.5 Hz and 20 kHz. Sinusoidal movements of up to 15 kHz were recorded. At higher frequencies the signal-to-noise ratio became too small. The largest responses were measured at the three rows of outer hair cells. If the strength of the electrical field was 2 kV/m, into which the cochlear explants were placed, the amplitudes of outer hair cell movements were around 1 μm at 1 Hz and 10 nm at 10 kH. Uncoupling of the outer hair cells from the tunnel of Corti and from the inner hair cells decreased the oscillations of inner hair cells but did not affect outer hair cells. The movements showed frequency dependent amplitudes like a complex low-pass filter but no best frequency was observed.     

The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection

A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the level of transfection was remarkably lower following longer durations of deafness. There was a significant increase in SGN survival in the entire basal turn for cochleae that received NT gene therapy compared to the untreated contralateral control cochleae for the one week deaf group. In the four week deaf group significant SGN survival was observed in the lower basal turn only. There was no increase in SGN survival for the eight week deaf group in any cochlear region. These findings indicated that the efficacy of NT gene therapy diminished with increasing durations of deafness leading to reduced benefits in terms of SGN protection. Clinically, there remains a window of opportunity in which NT gene therapy can provide ongoing trophic support for SGNs.     

lead neurotoxicity in rat cochlear organotypic cultures

Lead is a major environmental toxicant throughout the world.Lead can induce severe neurotoxicity including irreversible hearing impairment.Many in vivo studies have shown that lead damages the auditory...     

Does cochlear implantation and electrical stimulation affect residual hair cells and spiral ganglion neurons?

Increasing numbers of cochlear implant subjects have some level of residual hearing at the time of implantation. The present study examined whether (i) hair cells that have survived one pathological insult (aminoglycoside deafening), can survive and function following long-term cochlear implantation and electrical stimulation (ES); and (ii) chronic ES in these cochleae results in greater trophic support of spiral ganglion neurons (SGNs) compared with cochleae devoid of hair cells. Eight cats, with either partial (n=4) or severe (n=4) sensorineural hearing loss, were bilaterally implanted with scala tympani electrode arrays 2 months after deafening, and received unilateral ES using charge balanced biphasic current pulses for periods of up to 235 days. Frequency-specific compound action potentials and click-evoked auditory brainstem responses (ABRs) were recorded periodically to monitor the residual acoustic hearing. Electrically evoked ABRs (EABRs) were recorded to confirm the stimulus levels were 3-6 dB above the EABR threshold. On completion of the ES program the cochleae were examined histologically. Partially deafened animals showed no significant increase in acoustic thresholds over the implantation period. Moreover, chronic ES of an electrode array located in the base of the cochlea did not adversely affect hair cells in the middle or apical turns. There was evidence of a small but statistically significant rescue of SGNs in the middle and apical turns of stimulated cochleae in animals with partial hearing. Chronic ES did not, however, prevent a reduction in SGN density for the severely deaf cohort, although SGNs adjacent to the stimulating electrodes did exhibit a significant increase in soma area (p<0.01). In sum, chronic ES in partial hearing animals does not adversely affect functioning residual hair cells apical to the electrode array. Moreover, while there is an increase in the soma area of SGNs close to the stimulating electrodes in severely deaf cochleae, this trophic effect does not result in increased SGN survival.     

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

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