年龄相关性听力损失小鼠耳蜗毛细胞的凋亡机制

目的观察年龄相关性听力损失小鼠耳蜗毛细胞的死亡方式,探讨老年性聋的分子机制。方法随机选用5～7只28、30和60天龄的NMF308nmf/nmf小鼠,应用ABR和DPOAE检测听功能,用免疫荧光染色组织化学技术TUNEL、Caspase-3和碘化丙啶（PI）染色标记并观察耳蜗毛细胞。结果NMF308nmf/nmf小鼠从1月龄开始发生听功能减退和毛细胞改变,到2月龄时出现明显的TUNEL阳性标记,是毛细胞凋亡的最早表现;Caspase-3阳性表达的毛细胞凋亡现象稍晚出现;PI标记可见2～3月龄开始出现毛细胞细胞核固缩和碎片;到3月龄时听功能基本丧失,耳蜗毛细胞严重缺失。结论老年性聋的早期首先出现耳蜗毛细胞出现DNA单链断裂,随后Caspase-3信号途径激活,导致耳蜗毛细胞凋亡。     

NMF308nmf/nmf小鼠耳蜗毛细胞凋亡方式及z-VAD-FMK对毛细胞的保护作用

目的 了解年龄相关性听力损失小鼠耳蜗毛细胞的凋亡方式,探讨半胱氨酸蛋白酶(caspase)抑制剂z-VAD-FMK[z-Val-Ala-Asp(Ome)-fluoromethylketone]防治老年性聋的效果。方法选用新生NMF308nmf/nmf小鼠14只和成年同窝NMF308nmf/nmf小鼠32只,分为新生小鼠腹腔注射组(14只)、成年小鼠腹腔注射组(14只)和成年小鼠圆窗膜注射组(18只),各组又分为治疗组(注射z-VAD-FMK)和对照组[注射二甲基亚枫(dimethylsulfoxide,DMSO)],采用圆窗膜局部和腹腔注射两种方法给药,给药前后行ABR检测,用免疫荧光染色化学技术TUNEL、caspase-3和PI(碘化丙啶)染色标记耳蜗毛细胞,观察各组耳蜗毛细胞的凋亡和存活状况。结果NMF308nmf/nmf小鼠从1月龄开始发生听力减退和毛细胞功能改变,到2月龄时,caspase-3激活表达的毛细胞凋亡现象是毛细胞凋亡出现的最早表现;TUNEL阳性标记特征稍晚出现;PI标记可见毛细胞细胞核固缩和碎片出现的时间从2月龄开始;到3月龄时该种小鼠听力基本丧失,耳蜗毛细胞严重缺失;而应用z-VAD-FMK治疗后,各治疗组小鼠ABR反应阈明显好于对照组;耳蜗毛细胞凋亡数目较对照组减少,存活数明显较对照组多,尤以圆窗膜注射组明显。结论 NMF308nmf/nmf小鼠耳蜗毛细胞的死亡方式以caspase-3凋亡途径为主,应用caspase-3抑制剂zVAD-FMK定向内耳给药,可以阻断caspase-3信号途径激活所导致的毛细胞凋亡,从而有效防治老年性聋。     

外源性谷氨酸对豚鼠耳蜗内、外毛细胞易损性的比较

目的观察外源性谷氨酸对豚鼠耳蜗电位及耳蜗内、外毛细胞的影响。方法将健康豚鼠30只随机分为3组,应用豚鼠全耳蜗灌流技术,分别经耳蜗灌流人工外淋巴液和不同浓度的谷氨酸2小时,记录灌流前和灌流后的耳蜗电位(CM、CAP);同时应用透射电镜技术观察灌流前后耳蜗形态学的变化。结果灌流人工外淋巴液后豚鼠耳蜗电位及形态学无改变;灌流10 mmol/L谷氨酸后CM幅度虽有下降,其非线性特点无改变,CAP阈值平均升高了35 dB;灌流20 mmol/L谷氨酸后CM幅度明显下降但仍保持其非线性特点,CAP阈值平均升高了48 dB,灌流谷氨酸后耳蜗内毛细胞及传入神经纤维出现空化。结论谷氨酸是耳蜗主要的兴奋性传入神经递质,应用外源性谷氨酸可以引起耳蜗内毛细胞及传入神经的损伤,但对耳蜗外毛细胞无影响。     

豚鼠耳蜗外毛细胞的简化分离

目的:为获得能够满足电生理需要的外毛细胞,探讨简单可靠的分离外毛细胞的方法。方法:暴露听泡后去除耳蜗骨壳,将螺旋韧带连同基底膜和蜗轴一起放入酶液消化,减少显微镜下分离基底膜的操作程序,同时避免去除螺旋韧带时部分基底膜片段的丢失。结果:获得较多的保持良好活性的单离外毛细胞,耳大约80%的细胞能保持活性平均约6h。结论:去除骨壳后的残余耳蜗整体消化并适当提高消化液浓度的分离法,操作简单,可以获得能够满足电生理实验需要的较多的单离的耳蜗外毛细胞,有利于内耳研究工作的开展。     

C57BL/6J小鼠听力及耳蜗毛细胞活性的年龄相关性研究

目的 建立年龄相关性听力损失(age-related hearing loss,AHL)的小鼠动物模型,探讨C57BL/6J小鼠发生AHL与毛细胞活性变化的关系,并初步对C57BL/6J小鼠AHL模型进行AHL的病理分类.方法 按3、8、9、10、17、18月龄段分6组培育C57BL/6J小鼠,各组分别进行听性脑干反应(ABR)测试,对耳蜗毛细胞行琥珀酸脱氢酶染色并作基底膜硬铺片,观察各年龄段小鼠内外毛细胞线粒体琥珀酸脱氢酶的活性.结果 C57BL/6J小鼠随年龄增大,ABR阈值明显增高,在3月龄到9月龄期间ABR平均反应阈值增大比较缓慢,差异无统计学意义;在10月龄时,出现明显的听力下降,平均阈值比3月龄时约高18～23 dB,差异有统计学意义(click:t=5.78,P＜0.01;6 kHz:t =3.93,P＜0.01;8 kHz:t=3.01,P＜0.05).10月龄后小鼠听力继续下降,21月龄时平均阈值比3月龄时增高约60～68 dB,差异有显著统计学意义(click:t=31.23,P＜0.01;6 kHz:t=30.44,P＜0.01;8 kHz:t=33.83,P＜0.01).琥珀酸脱氰酶染色显示,随年龄增大,毛细胞线粒体活性丧失逐渐加重:先是底回外毛细胞活性下降,接着发生活性消失,并逐渐向顶回发展,最后累及内毛细胞.结论 C57BL/6J小鼠具有典型的年龄相关性听力损失特点,其听力下降的原因早期可能主要足外毛细胞及内毛细胞活性的丧失,晚期可能是由于基底膜结构混乱,导致电生理屏障消失,致耳蜗内电位(EP)不能维持而引起.C57BL/6J小鼠可作为感音型老年性听力损失动物模型.     

豚鼠耳蜗外毛细胞的共聚焦图像

建立了用激光扫描共聚焦显微镜豚鼠耳蜗外毛细胞生理变化及细胞形态的方法，以荧光染料Ｆｌｕｏ－３作胞内Ｃａ＾２＋指示剂，用共聚焦显微观察乙酰胆碱（Ａｃｈ），ＡＴＰ、高钾引起的外毛细胞（ＯＨＣ）胞内Ｃａ＾２＋浓度的变化，三种物质均引起Ｃａ＾２＋升高，但升高的幅度，进相及所在细胞部位不同，高钾还引起ＯＨＣ短缩，偏折，利用共聚焦显微镜“光学切片”的特性，对Ｆｌｕｏｒｅｓｃｅｉｎ荧光素染色的活的ＯＨＣ形态及磺     

小鼠耳蜗感觉上皮细胞的自然培养诱导毛细胞的产生

目的 培养小鼠耳蜗上皮细胞,寻找听觉毛细胞的前体细胞,从而研究听觉毛细胞的再生。方法 改良细胞培养基和培养技术,建立小鼠耳蜗听觉上皮细胞的培养;用免疫细胞化学方法和BrdU标记法检测培养细胞的性质和分裂状态。结果 培养的听觉上皮细胞表现为大而扁平的上皮细胞形态,并且表达上皮细胞的标志F-actin和cytokeratin,部分新生的细胞可被早期毛细胞的特异标志calretinin着染,表明有听觉毛细胞样的细胞产生,这种现象经3次传代培养后仍然存在。结论 自然细胞培养方法可能诱导小鼠听觉毛细胞的产生,在小鼠的耳蜗内可能存在听觉毛细胞的前体细胞,而这些前体细胞是否是组织特异性干细胞还需要更进一步的研究。     

豚鼠耳蜗外毛细胞的变性机制

目的:探讨外毛细胞维持形态及变性过程发生的可能机制。方法:倒置相差显微镜下,对于急性酶分离所得的外毛细胞进行6 h的连续性观察。结果:半数以上活性良好的单离外毛细胞可保持活性平均约6 h,变性过程中均出现自表皮板至基底部的纵行褶线。含钙和不含钙的细胞维持活性时间相同。静纤毛缺失可以同样维持较长时间的活性。结论:钙离子浓度和静纤毛损伤不是外毛细胞变性的决定性因素,而环细胞表面的某种弹性机制可能是维持形态保持活性的根本原因。     

豚鼠耳蜗单离外毛细胞钙波的观察

目的：观察豚鼠耳蜗单离外毛细胞（outer hair cell,OHC)内游离钙浓度（[Ca^2]i)是否存在波动，即有无钙波。方法：健康豚鼠10只断头处死，在无Ca^2的dHanks液和含Ca^2的Hanks液中酶－机械法分离获得单离活性OHC，钙荧光抗体Fluo-3孵育后，分别加入乙酰胆碱或空白对照，用MRC－1024型激光扫描共聚焦显微镜（Bio-Rad，英国）观察OHC约50min内[Ca^2]i的变化。结果：dHanks液中加ACh组的5个OHC，[Ca^2]i呈明显的规律性的波动，即产生了钙波，钙波的周期约600s;Hanks液中加ACh组的6个OHC的[Ca^2]i均迅速升高，无钙波出现，在dHanks液和Hanks液中不加ACh分别观察了3个OHC，[Ca^2]i呈平坦型，无钙波，结论：ACh能刺激诱发豚鼠耳蜗单离OHC产生钙波。     

不同种属实验动物耳蜗毛细胞年龄相关性缺失的不同模式

目的 展示自然衰老和耳聋相关基因遗传缺陷之间耳蜗毛细胞缺失的不同模式。方法 用不同龄的长尾猴、南美栗鼠、豚鼠、Sprague-Dawley 大鼠、CBA/CaJ 小鼠、C57BL/6J 小鼠、A/J小鼠、DBA/2J 小鼠和侏儒灰色突变纯合子 (dwg/dwg) 小鼠作为受试对象。所有测试动物的耳蜗基底膜都被制作成平坦的耳蜗基底膜铺片。沿着耳蜗基底膜的全长,基底膜上所有的内外毛细胞都被完整计数,毛细胞的计数结果被输入到耳蜗图软件并自动生成每组实验条件的平均耳蜗图。结果 在天然衰老的动物中,耳蜗毛细胞的缺失总是发生在老年阶段。与此不同的是,在耳聋相关基因缺陷的动物中,耳蜗毛细胞的缺失却是发生在青年阶段甚至幼年阶段。发生在天然老化动物的耳蜗毛细胞缺失总是呈均匀分布或从耳蜗的顶回向底回扩展。 但是,发生在具有耳聋相关基因遗传缺陷动物的耳蜗毛细胞缺失却通常表现为从耳蜗的底回向顶回扩展。结论 本实验观察结果表明,发生在天然衰老的不具备耳聋相关基因缺陷动物身上的年龄相关性耳蜗毛细胞缺失反映的是真正由衰老引起的耳蜗退化性病变,而发生在伴有耳聋相关基因遗传缺陷的年幼动物身上的年龄相关性耳蜗毛细胞缺失可能与耳聋相关基因的遗传缺陷有关。     

Incidence of otitis media in CBA/J and CBA/CaJ mice.

The inbred CBA/J mouse has become a standard experimental animal for auditory study because of its lifelong good hearing. In a newly established mouse breeding colony that housed CBA/J and CBA/CaJ mice to reared as auditory subjects, otitis media frequently afflicted CBA/J mice, reaching an incidence of 90% in animals greater than 400 days of age. Otitis media was not found in CBA/CaJ mice. Three attempts to establish a colony that was free of otitis were unsuccessful. Although the primary pathogen was not clearly established, Pasteurella pneumotropica was isolated from infected bullae. Partial control of otitis media followed the introduction of tetracycline prophylaxis. The CBA/CaJ mice may be suitable replacements for CBA/J mice in studies that require inbred mice with good hearing, since their auditory thresholds did not differ significantly from those of otitis-free CBA/J mice.     

Divergence of noise vulnerability in cochleae of young CBA/J and CBA/CaJ mice

CBA/CaJ and CBA/J inbred mouse strains appear relatively resistant to age- and noise-related cochlear pathology, and constitute the predominant 'good hearing' control strains in mouse studies of hearing and deafness. These strains have often been treated as nearly equivalent in their hearing characteristics, and have even been mixed in some studies. Nevertheless, we recently showed that their trajectories with regard to age-associated cochlear pathology diverge after one year of age (Ohlemiller et al., 2010a). We also recently reported that they show quite different susceptibility to cochlear noise injury during the 'sensitive period' of heightened vulnerability to noise common to many models, CBA/J being far more vulnerable than CBA/CaJ (Fernandez et?al., 2010 J. Assoc. Res. Otolaryngol. 11:235-244). Here we explore this relation in a side-by-side comparison of the effect of varying noise exposure duration in young (6 week) and older (6 month) CBA/J and CBA/CaJ mice, and in F1 hybrids formed from these. Both the extent of permanent noise-induced threshold shifts (NIPTS) and the probability of a defined NIPTS were determined as exposure to intense broadband noise (4-45?kHz, 110?dB SPL) increased by factors of two from 7?s to 4?h. At 6 months of age the two strains appeared very similar by both measures. At 6 weeks of age, however, both the extent and probability of NIPTS grew much more rapidly with noise duration in CBA/J than in CBA/CaJ. The 'threshold' exposure duration for NIPTS was <1.0?min in CBA/J versus >4.0?min in CBA/CaJ. F1 hybrid mice showed both NIPTS and hair cell loss similar to that in CBA/J. This suggests that dominant-acting alleles at unknown loci distinguish CBA/J from CBA/CaJ. These loci have novel effects on hearing phenotype, as they come into play only during the sensitive period, and may encode factors that demarcate this period. Since the cochlear cells whose fragility defines the early window appear to be hair cells, these loci may principally impact the mechanical or metabolic resiliency of hair cells or the organ of Corti.     

Absence of Voltage-Dependent Compliance in High-Frequency Cochlear Outer Hair Cells

Cochlear outer hair cells are the key element in a mechanical amplification process that enhances auditory sensitivity and tuning in the mammalian inner ear. The electromotility of outer hair cells, that is, their ability to extend or contract at acoustic frequencies, is proposed to be the source of the mechanical amplification. For amplification to take place, some stiffness is required for outer hair cells to communicate force to the organ of Corti, the sensory epithelium of the inner ear. Modulation of this stiffness would be expected to have a significant effect on inner ear function. Outer hair cell compressive stiffness has recently been shown to be dependent on membrane potential, but this has only been demonstrated for cells originating in the apical, low-frequency segment of the cochlea, whereas cochlear amplification is arguably more important in the more basal high-frequency segment. The voltage-dependent compliance (the reciprocal of stiffness) of high-frequency outer hair cells was investigated by two methods in cells isolated from the basal turns of the guinea pig cochlea. In contrast to previous findings, no evidence was found for voltage-dependent changes in compliance. The results call into question the importance of outer hair cell voltage-dependent compliance as a component of cochlear amplification.     

乙酰胆碱对豚鼠外毛细胞的电压依赖性外向整流钾电流的影响

目的 观察乙酰胆碱（ＡＣｈ）对不同长度豚鼠耳蜗外毛细胞（ＯＨＣ）电压依赖性外向整流钾电流的影响，分析 ＡＣｈ对钾电流激活动力学的影响。方法 全细胞膜片钳技术。结果１００μｍｏｌ／Ｌ的ＡＣｈ对短ＯＨＣ电压依赖性外向整流钾电流的影响较大，刺激电压为５０ｍＶ时，最大外向电流的幅度增加了３４．８％。ＡＣｈ对峰电流的影响大于稳态电流，改变了外向整流钾电流的动力学特征。ＡＣｈ将ＯＨＣ的零电流电位向超极化方向移位约５ｍＶ。1００μｍｏｌ／Ｌ的ＡＣｈ使ＯＨＣ电压依赖性外向整流钾电流的激活动力学发生改变，Ｖ（１／２）＝（－５２．３８±３．９８）ｍＶ，较作用前明显超极化，激活的电压敏感性也提高，Ｓ＝（４０±４．１４）ｍＶ（ｎ＝５）。结论ＡＣｈ增加了ＯＨＣ电压依赖性外向整流钾通道的电导，使通道的激活电压向超极化方向移位。ＡＣｈ的作用是使ＯＨＣ超极化。     

Kinetics of Gentamicin in Cochlear Hair Cells After Chronic Treatment

Presence of gentamicin (GM) in cochlear hair cells was detected by immunohistochemistry in guinea pigs (GPs) cochlea 1, 9 and 41 days after a 6-day treatment with GM at 60 mg/kg/day (s.c.). The number of GPs in each group was respectively 7, 12 and 6. Twelve other non-treated GPs served as controls. Cochlear function was measured, just before sacrifice, by VIIIth nerve compound action potential (CAP) audiograms. Functional and immunohistological evaluations were performed by two independent naive observers respectively. Functional changes were minimal: only one out of the 25 treated GPs, from the 41-day group, showed significant threshold elevations on high frequencies. Meanwhile GM labelling was observed in most outer hair cells (OHCs) from the three rows of all the treated GPs, with radial and longitudinal gradients, and found similar in the 3 groups. These results 1) confirm that GM is significantly present in OHCs before the development of ototoxicity and 2) indicate that GM accumulates and is maintained inside the OHCs for very long periods of time, i.e. that its clearance from the hair cells, if any, would be very slow.     

Auditory brainstem responses in neonatally sound deprived CBA/J mice

Auditory brainstem responses (ABR) from ten normal (3 males, 7 females) and ten environmentally sound deprived CBA/J mice (6 males, 4 females) were elicited by 30 dB HL click stimuli delivered via specially constructed, matched insert earphones. The mice were tested at 44–49 days of age under chloral hydrate sedation using an electrode montage of vertex/linked bilateral bullae/presacrum. The deprived mice were killed immediately after testing and serial transverse sections were prepared from their brains. Examination of globular cells in the ventromedial ventral cochlear nuclei revealed significantly smaller cross-sectional areas of these cells than in previously studied normal mice.

Although monaural thresholds did not differ between the two groups, significantly shorter mean latencies were observed in the ABRs of the sound deprived group in wave I as well as in the interpeak latencies of the later ABR components (I–IV. I–V. and III–V). These electrophysiologic alterations in brainstem conduction presumably reflect the anatomical changes demonstrated in neonatally sound deprived mice.     

豚鼠耳蜗外毛细胞的共聚焦图像

建立了用激光扫描共聚焦显微镜观察豚鼠耳蜗外毛细胞生理变化及细胞形态的方法。以荧光染料Ｆｌｕｏ－３作胞内Ｃａ￣（２＋）指示剂，用共聚焦显微镜观察了乙酰胆碱（ＡＣｈ）、ＡＴＰ、高钾引起的外毛细胞（ＯＨＣ）胞内Ｃａ￣（２＋）浓度的变化。三种物质均引起Ｃａ￣（２＋）升高，但升高的幅度、时相及所在细胞部位不同。高钾还引起ＯＨＣ短缩、偏折。利用共聚焦显微镜“光学切片”的特性，对Ｆｌｕｏｒｅｓｃｅｉｎ荧光素染色的活的ＯＨＣ形态及碘化吡啶（ＰＩ）染色的细胞核形态进行了三维重建。讨论了共聚焦显微镜在ＯＨＣ生理及形态学研究中的应用前景。     

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。