双醋酸荧光素－碘化丙啶双染色法鉴别豚鼠外毛细胞活性

为提供一种准确判断单离细胞活性的方法，采用双醋酸荧光素（ＦＤＡ）－碘化丙啶（ＰＩ）双染色，荧光显微镜观察，鉴别了豚鼠耳蜗单个外毛细胞的活性。在激发光下染色后的活细胞显绿色，失活细胞核呈红色。在室温下计数５只豚鼠（１０侧听泡）在断头１、２、３、５和７ｈ后的单离细胞平均存活百分率分别为８５．０％、７８．０％、７０．０％、２３．５％和０。本方法为一种鉴别细胞活性的客观可靠的方法。     

Cochlear neuroactive substances

Summary We have reviewed the experiments done in our laboratory concerning various cochlear neuroactive substances. Data using chemical neuroanatomy and neurochemical techniques are described. They allow the identification and localization of neuroactive substances which could act as neurotransmitters and/ or neuromodulators at the different types of synapses in the organ of Corti. Three hypotheses are presented: (1) the inner hair cells use glutamate as a neurotransmitter, but in addition to its excitatory properties, glutamate may also be involved in pathophysiological events affecting afferent auditory dendrites; (2) subpopulations of both the lateral and medial olivocochlear efferent systems can be differentiated by the neuroactive substances they may use; (3) the base and the apex of the cochlea can be distinguished on the basis of neurochemical data.Presented at the First European Congress of Oto-Rhino-Laryngology and Cervico-Facial Surgery, Paris, 26–29 September 1988     

卡那霉素在耳蜗基底膜细胞中的积聚部位

为研究氨基甙类抗生素破坏毛细胞的机理，应用耳蜗基底膜分离技术、组织匀浆分级分离技术和放射性测定技术，发现耳蜗灌流０．２５％卡那霉素外淋巴液后，一个细胞核中含有３．１４ｆｇ卡那霉素，一个线粒体中含有７．６１ｆｇ卡那霉素，一个溶酶体中含有２１．１６ｆｇ卡那霉素，说明卡那霉素进入细胞后，药物主要结合在线粒体，同时也被溶酶体大量吞噬。依据卡那霉素杀菌机理为特异性抑制细菌的蛋白质合成和线粒体与细菌具有相同的蛋白质合成方式，认为线粒体可被卡那霉素特异结合，而溶酶体中的药物可能与其吞噬功能有关。     

Three-Dimensional Distribution of Cochlear Macrophages in the Lateral Wall of Cleared Cochlea

Objectives Resident macrophages are well known to be present in the cochlea, but the exact patterns thereof in spiral ligaments have not been discussed in previous studies. We sought to document the distribution of macrophages in intact cochleae using three-dimensional imaging.Methods Cochleae were obtained from C-X3-C motif chemokine receptor 1^+/GFP mice, and organ clearing was performed. Three-dimensional images of cleared intact cochleae were reconstructed using two-photon microscopy. The locations of individual macrophages were investigated using 100-μm stacked images to reduce bias. Cochlear inflammation was then induced by lipopolysaccharide (LPS) inoculation into the middle ear through the tympanic membrane. Four days after inoculation, three-dimensional images were obtained.Results Macrophages were scarce in areas adjacent to the stria vascularis, particularly the area just beneath it even though many have suspected macrophages to be abundant in this area. This finding remained consistent upon LPS-induced cochlear inflammation, despite a significant increase in the number of macrophages, compared to non-treated cochlea.Conclusion Resident macrophages in spiral ligaments are scarce in areas adjacent to the stria vascularis.     

Cochlear Events and Whole-Nerve Action Potentials

For proper interpretation of electrocochleographic recordings, it is essential to know how the activity of primary auditory neurons is reflected by the whole-nerve action potential (AP). It is easy to show theoretically that for stimulation with tone bursts the latency of the AP decreases with increasing stimulus intensity as a result of cochlear frequency selectivity. It is less easy to show that the phenomenon of saturation as shown by primary auditory neurons is almost equally important in this respect. The proof centers around a peculiar property: theoretically, latency effects will be absent when the input-output relation of auditory neurons can be described by a power law. APs evoked by clicks also show latency changes as a result of variations in stimulus level. These changes can also be interpreted as the result of frequency selectivity and saturation. Here the main factor is that the whole-nerve AP reflects the activity of neurons sensitive to high frequencies far better than those sensitive to low frequencies.     

新生大鼠耳蜗K?lliker器在体凋亡的研究

目的：了解不同天龄新生大鼠耳蜗K?lliker器的形态变化,研究凋亡相关因子的mRNA及蛋白的表达水平,探讨K?lliker器在听觉功能发育过程中凋亡的机制。方法选取出生后不同天龄的Sprague－Dawley大鼠共192只,其中出生后1天（P1）、5天（P5）、12天（P12）各6只大鼠耳蜗冰冻切片后,通过苏木精－伊红染色及免疫荧光染色等方法,观察耳蜗K?lliker器的形态结构变化;取 P1大鼠6只行耳蜗基底膜免疫荧光观察;提取出生后1、3、5、7、10、12及14天（P1、P3、P5、P7、P10、P12和P14）大鼠耳蜗基底膜mRNA（各6只）及蛋白（各18只）,运用real－time PCR及蛋白质印迹的方法,观察出生后各天龄组大鼠耳蜗基底膜 K?lliker 器凋亡过程中 bcl－2、caspase－3、caspase－8及caspase－9的表达规律。结果出生后大鼠听力出现之前其耳蜗K?lliker器支持细胞的形态自顶回向底回从高柱状向矮柱状变化,同时支持细胞的数量逐渐减少。出生后不同天龄大鼠耳蜗基底膜的caspase－3、caspase－8、caspase－9及bcl－2的mRNA和蛋白的表达水平均呈明显的时间依赖性。结论在大鼠出生后、听力出现前耳蜗发育的整个阶段,K?lliker器自底回向顶回逐渐发生退化;caspase－3、caspase－8、caspase－9及bcl－2的mRNA和蛋白的表达表现为时间依赖性。     

耳蜗外侧壁微区微循环损伤对耳蜗内淋巴电位的影响

为了解某些内耳疾病引起的感音神经性聋的区域频率听力下降与耳蜗局部血流改变的关系，采用光化学方法造成耳蜗外侧壁微区微循环血管损伤，应用活体耳蜗微循环与内淋巴电位（ＥＰ）同时观察记录的方法，观察了不同损伤面积的微循环改变对内淋巴电位的影响。实验应用血卟啉单甲醚作为光敏剂，超高压汞灯作为激发光。实验发现相对较小面积的耳蜗外侧壁微循环损伤组（０．２ｍｍ×０．４ｍｍ）ＥＰ无明显变化；相对较大面积的损伤组（０．２ｍｍ×０．８ｍｍ）ＥＰ轻度下降，但这种下降与正常对照组ＥＰ变化相比差异无显著性。结果提示ＥＰ虽产生于血管纹，但小面积血管纹微循环损伤不引起ＥＰ的明显变化，说明耳蜗局部代偿能力能够在一定程度上维持内耳功能。     

细胞增殖及凋亡与耳蜗感觉上皮的分化

目的观察耳蜗发育中细胞增殖、凋亡的变化规律，探讨它们与耳蜗感觉上皮分化的关系。方法利用透射电镜观察从胚胎第8天到刚出生的C57BL/6小鼠耳蜗感觉上皮的增殖、凋亡及分化的变化。结果胚胎第10天，耳蜗才开始发育；到胚胎第11天耳蜗感觉上皮出现核分裂像并逐渐增多，胚胎第14天时已开始减少；细胞凋亡现象出现在胚胎第13天，从胚胎第14天到胚胎第15天细胞凋亡最明显，后逐渐减少；同时在胚胎第14天毛细胞开始分化形成，到胚胎第16天，Corti器原基形成时，毛细胞和支持细胞形态趋向成熟，但到出生时，Corti器尚未发育成熟。结论细胞增殖和凋亡是耳蜗发育中的必然现象，细胞增殖、凋亡及毛细胞的分化成熟相继发生但又互相重叠；细胞增殖与凋亡的动态平衡在耳蜗感觉上皮分化成熟中起着重要作用。     

大鼠耳蜗器官培养及其组织学检查技术

目的探讨大鼠耳蜗器官体外培养的方法和观察毛细胞、神经纤维及螺旋神经节细胞的组织学检查技术。方法将出生3天的SASCO Sprague Dawley大鼠耳蜗取出平铺培养。应用神经丝免疫组织化学方法对螺旋神经节细胞及神经纤维染色,同时应用鬼笔环肽染色特异性显示耳蜗毛细胞的静纤毛和表皮板。在共聚焦显微镜下应用不同的激发荧光分别显示耳蜗毛细胞、螺旋神经节和神经纤维。结果耳蜗器官经1～3天离体培养,内、外毛细胞生长良好,无衰亡和缺损;神经纤维排列有序;螺旋神经节细胞形态正常。结论本文介绍的耳蜗器官体外培养方法和组织学检查指标,将在常规评估耳蜗毛细胞、螺旋神经节细胞和神经纤维的离体培养损害实验模型中,有一定应用价值。     

Cochlear ultrastructure in two-phase endolymphatic hydrops in the guinea-pig

Endolymphatic hydrops continues to be considered as a pathological factor in the etiology of Menière’s disease. We have developed the two-phase endolymphatic hydrops model, which seems to represent a functional model combining multiple etiologies, and which may resemble the fluctuating characteristics of Menière’s disease. A transmission electron microscopic study was performed on the endolymphatic sacs of four groups of guinea-pig cochleas: (1) controls, (2) non-operated, aldosterone-treated cochleas, (3) operated (dissection of the endolymphatic sac) cochleas, (4) operated and aldosterone-treated cochleas. Light and electron microscopy showed a normal morphology in the controls. Aldosterone as a single treatment resulted in an increased activity of the marginal cells in the stria vascularis. Dissection induced a gradient of degenerative effects and cell loss in the intracellular and extracellular structures of the sensory cells, the stria vascularis and Reissner’s membrane, which may be reversible. Subsequent administration of aldosterone induced severe damage and increased cell loss, which may be irreversible. Our findings demonstrate changes that may be reversible due to the compromising effect of a single treatment, and irreversible changes due to interaction of both compromising factors. These findings support our two-phase concept. Received: 30 October 2000 / Accepted: 14 June 2001     

Sirtuin 1激活在氯化钴诱导的离体耳蜗缺氧损害中的作用研究

目的观察氯化钴（CoCl2）对耳蜗毛细胞和神经节细胞的损伤模式并探讨Sirtuin 1（SIRT1）在耳蜗缺氧过程中的作用。方法采用CoCl2作用于离体培养的新生大鼠耳蜗组织,通过细胞化学染色和免疫荧光染色确定CoCl2对耳蜗毛细胞存活的影响,采用实时定量PCR方法检测CoCl2作用耳蜗组织后Sirtuin 1mRNA表达的变化。同时使用Sirtuin1激活剂白藜芦醇和Sirtuin 1抑制剂Sirtinol,观察SIRT1在缺氧介导的耳蜗损伤过程中的作用。结果300μmol／L以上浓度CoCl2作用于耳蜗24h可引起内外毛细胞缺失。细胞肿胀明显,神经元胞体变得不规则,数目明显减少,有些神经纤维甚至断裂。CoCl2处理后可引起早期耳蜗组织Sirtuin1基因表达增强,6h达到峰值。白藜芦醇预处理可以显著提高外毛细胞（P〈0．01）和内毛细胞的存活率（P〈0．05）。同时给予Sirtuin1抑制剂Sirtinol,白藜芦醇的保护效应被抵消。结论CoCl2导致耳蜗组织缺氧损害。耳蜗在缺氧过程中Sirtuin1早期上调,可保护耳蜗组织对抗缺氧诱导的细胞损伤。     

强噪声暴露后不同时期豚鼠耳蜗外毛细胞死亡方式的研究

目的观察强噪声暴露后不同时期豚鼠耳蜗外毛细胞的死亡方式。方法选用健康雄性白色豚鼠48只,随机分为健康对照组及噪声暴露后3h、1d、4d、14d、30d组(实验组),每组8只,实验组动物暴露于120dBSPL、4kHz窄带噪声环境4h,各组豚鼠于实验前及噪声暴露后相应时间点处死前测试听性脑干反应(auditorybrainstem response,ABR),然后完成耳蜗基底膜铺片、Hoechst33342荧光染色,共聚焦荧光显微镜观察毛细胞核的变化并计数。对照组不作任何处理。结果实验组噪声暴露后各组ABR反应阈明显高于对照组和噪声暴露前各组(P<0.01),实验组豚鼠耳蜗可见外毛细胞出现凋亡、坏死和缺失三种变化,噪声暴露后3h、1d、4d组凋亡的细胞数明显多于坏死(P<0.05),噪声暴露后14d组凋亡与坏死的细胞数没有明显差别(P>0.05),噪声暴露后30d组坏死细胞数多于凋亡(P<0.05)。结论强噪声暴露后早期豚鼠耳蜗外毛细胞损害以凋亡为主,且凋亡过程迅速,耳蜗基底膜外毛细胞核的缺失主要源于凋亡细胞,强噪声暴露后中晚期外毛细胞损害以坏死为主。     

豚鼠耳蜗微血管SP受体阳性结构的观察

目的探讨ＳＰ（ｓｕｂｓｔａｎｃｅＰ）调节耳蜗血流的作用机理。方法本研究采用免疫组织化学ＡＢＣ技术结合葡萄糖氧化酶－ＤＡＢ－镍染色法,观察豚鼠耳蜗微血管ＳＰ受体的分布状态。结果豚鼠耳蜗基底膜各圈均可见ＳＰ受体阳性的微血管,阳性结构主要位于血管内膜,呈密度增高的线状分布。结论作用于耳蜗微血管内膜ＳＰ受体的ＳＰ,主要来源于血液及内皮细胞自身。     

光化学法建立豚鼠耳蜗微循环障碍模型的初步报告

为建立较好的耳蝇微循环障碍模型，采用豚鼠股静脉内注射四碘四氯荧光素二钠，以波长为５５０±２０ｎｍ绿色光束照射耳蜗的方法，诱导豚鼠耳蜗血管内发生光化学反应，损伤血管内皮细胞，血小板被受损的内皮细胞激活，粘附、聚集于内皮细胞表面或暴露的基膜上形成微血栓，导致耳蜗微循环障碍，耳蜗血流量（ＣＢＦ）骤降，复合动作电位（ＣＡＰ）振幅下降乃至消失。随着时间的延长，耳蜗血管纹、Ｃｏｒｔｉ器、螺旋神经节细胞等结构先后出现不同程度的缺血性病变，与某些病理条件下的耳蜗微循环障碍有很大的相似性。这种方法，可以为研究内耳微循环障碍或缺血性损伤机制，以及筛选治疗药物建立较为理想的动物模型。     

中药健耳Ⅱ号胶囊对抗C57BL/6J小鼠老年性耳蜗损害的实验研究

目的 观察中药健耳Ⅱ号胶囊是否对C57小鼠老年性耳蜗损害具有保护作用。方法 选择刚出生的C57BL／6J小鼠40只，随机平均分为对照组、中药组各20只，其中对照组小鼠为常规饲料喂养，分别在出生后2．4和7个月终止实验；中药组小鼠常规饲料喂养至出生后2个月时开始饮用健耳Ⅱ号胶囊溶液以代替日常饮水，至7月龄时终止实验。取出小鼠耳蜗分别制成耳蜗铺片和耳蜗切片，在光学显微镜下定量观察并比较两组小鼠耳蜗毛细胞的密度和疆孔内的神经纤维数量以及螺旋神经节密度。结果 对照组C57BL／6J小鼠随着月龄的递增出现耳蜗毛细胞损伤加重的趋势，且这种随着年龄增长而发生的耳蜗损害遵循着从底回逐渐向顶回发展的规律，外毛细胞的损害比同区域的内毛细胞严重。7月龄对照组动物的耳蜗毛细胞损害范围比较广泛，底回螺旋神经节细胞数量明显减少，存活神经元细胞亦呈现异常的形态学改变，同时疆孔内的神经纤维数量也有所减少。但在中药组小鼠，无论是耳蜗毛细胞的数量还是疆孔内的神经纤维数量，以及蜗轴螺旋管内的螺旋神经节细胞密度均比对照组动物的损害轻。经统计学分析，两组动物耳蜗底回的螺旋神经节细胞密度和底回疆孔内的神经纤维数量差异均有统计学意义（P〈0．001，P〈0．05）。结论 ①C57BL／6J小鼠在出生后4个月即开始发生耳蜗进行性损害，可作为研究老年性聋的理想动物模型；②鉴于在老年性耳蜗损害的早期可见毛细胞和螺旋神经节均有损害，提示老年性耳蜗损害可能是同时发生在听觉神经元；③中药健耳Ⅱ号胶囊不但可以延缓C57BL／6J小鼠随着年龄增长而发生的耳蜗毛细胞损坏，而且对于听觉神经元和听神经纤维也具有一定的保护作用。     

光化学法诱导豚鼠耳蜗微循环障碍模型

目的 通过光化学法建立豚鼠耳蜗微循环障碍模型以及观察血管纹、耳蜗毛细胞形态学变化.方法 ①将豚鼠随机分成5个组.实验组分成3个组,颈外静脉注入四氯四碘荧光素二钠(rose bengal,RB),用波长为(540±40)nm、光强为(500～600)mW/cm2绿光照射打开的听泡,各组选用不同光照时间诱导耳蜗微循环变化.对照组分2个组,对照Ⅰ组仅颈外静脉注入RB而不行光照;对照Ⅱ组不注入RB而仅行光照耳蜗.②分别对5个组动物行听性脑干反应及形态学检测.结果 ①每个实验组两次ABRⅢ波反应阈差异均有统计学意义(P＜0.05);②形态学观察实验组均出现耳蜗微循环障碍的变化,螺旋器以外毛细胞破坏为主.结论 采用光化学法可致耳蜗微循环障碍及毛细胞破坏,方法简便,易于实施,重复率高.     

Cochlear origin of early hearing loss in vestibular schwannoma

OBJECTIVE: To test whether early hearing loss (HL) is cochlear in origin in patients with vestibular schwannoma (VS). STUDY DESIGN: Retrospective case review in an academic tertiary referral center. METHODS: A group of 19 VS patients with normal/symmetrical hearing and a group of 20 VS patients with mild HL (threshold at any tested frequency better than 45 dB HL) on the tumor ear side. Differences of the amplitudes of the distortion products of otoacoustic emissions (DPOAEs) between the tumor ear and the nontumor ear were studied at frequencies of 1, 1.4, 2, 2.8, and 4 kHz. The Wilcoxon test was used to compare the ears for both groups and to test for possible differences in tumor size between groups. RESULTS: DPOAE amplitudes do not differ strongly between the ears in VS patients with normal/symmetrical hearing (two-sided P values: .050 at 1 kHz, .182 at 1.4 kHz, .378 at 2 kHz, .293 at 2.8 kHz, and .238 at 4 kHz) but are decreased compared with the nontumor ear at frequencies 1, 1.4, 2, and 2.8 kHz in VS patients with even mild HL (two-sided P values: .013 at 1 kHz, .007 at 1.4 kHz, .033 at 2 kHz, .010 at 2.8 kHz, and .156 at 4 kHz). Tumor size did not differ significantly between the two groups (P = .436). CONCLUSION: Amplitudes of DPOAEs begin to decrease even at the early stages of HL in VS patients, which suggests a cochlear origin of early HL in these patients. DPOAEs may be used in a clinical setting to monitor progression of cochlear damage at the early stages of hearing impairment in VS patients.     

Cochlear findings in the white spotting (Ws) rat

White spotting (Ws) rats possess a c-kit gene mutation at the W locus, resulting in a variety of characteristics including a lack of intermediate cells of the stria vascularis. The present study employs a light microscope (LM), scanning (SEM) and transmission electron microscopes (TEM), diaminobenzidine (DAB) staining techniques and auditory brainstem response (ABR) to investigate the structure and function of the cochlea in 26 homozygous Ws/Ws rats aged 1–6 months. A slight thinning of the stria vascularis and moderate elevation of ABR threshold were about the only defects noted in 1 month animals, while older animals displayed various defects that tended to worsen with age. At 3 months LM revealed pigment granules in the basal turn of most animals, with a loss of pigmentation in the upper turns. The stria vascularis and organ of Corti tended to be well preserved in the lower, pigmented portion, while the upper, unpigmented portion showed severe strial degeneration and some outer hair cell loss. DAB staining revealed a well developed strial capillary net throughout the pigmented portion of the cochlea, with severe degradation in the unpigmented apical portion. ABR thresholds were slightly elevated over 1 month values. At 6 months great differences in degeneration were noted between right and left ears of the same animal.     

卡那霉素和速尿联合用药后的毛细胞死亡*

目的观察卡那霉素和速尿联合用药后豚鼠耳蜗毛细胞的死亡时程和方式.方法选用健康成年白色红目豚鼠,雌雄不限,随机分成健康对照组和药物致聋后6 h、9 h组(实验组),每组5只.实验组在选取的时间点完成听性脑干反应(ABR)检测后行耳蜗基底膜铺片、PI荧光染色,共聚焦显微镜下观察毛细胞.对照组不做处理.结果实验组半数豚鼠致聋(ABR阈值>95 dB SPL),致聋豚鼠耳蜗可见外毛细胞核固缩和核肿胀,与给药6 h组相比,给药9 h组外毛细胞核肿胀数目增多.检测给药6 h组和9 h组豚鼠末端脱氧核苷酸转移酶介导的dUTP缺口末端标记物(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL),没有观察到阳性着色细胞.正常对照组所有豚鼠ABR正常,其耳蜗各回毛细胞没有出现毛细胞核固缩或者核肿胀.结论卡那霉素和速尿联合用药后数小时即可导致豚鼠耳蜗毛细胞死亡,毛细胞损害为两种类型,即坏死和凋亡.     

Cochlear implantation in infants less than 12 months of age

OBJECTIVE: Infants are diagnosed with severe to profound hearing loss at an earlier age due to the advent of universal newborn hearing screening. This offers the opportunity to provide intervention in the form of cochlear implantation at an earlier age than was previously possible. The purpose of this investigation is to evaluate the risk of cochlear implant surgery in children less than 12 months of age. DESIGN: Retrospective review of children who underwent cochlear implantation before 12 months of age. SETTING: Patients were identified from a database of pediatric cochlear implant patients at a tertiary care center. All patients were diagnosed with severe to profound hearing loss by otoacoustic emission and auditory brainstem response. Follow-up ranged from 2 months to 5 years. RESULTS: Fourteen of 15 patients had full insertions of the electrode hardware. Less than full insertion and post-operative CSF otorrhea occurred in one patient with severe cochlear abnormalities. There were no other perioperative surgical complications. The average speech detection threshold was 27.6 dB (20-45 dB) at approximately 1-3 months post-stimulation and 25 dB (15-30 dB) at approximately 5-7 months. CONCLUSION: In our experience, we feel cochlear implantation is safe for infants as young as 6 months of age. The current standard at our institution is to implant by 7 months of age for prelingual deafness as opposed to waiting additional time until 12 months of age before the brain is presented with speech.