成纤维细胞生长因子耳蜗内灌注防治爆震性聋的实验研究

选用豚鼠１９只，震前于圆窗龛放银球电极测复合动作电位（ＣＡＰ）反应阈，爆震后测ＣＡＰ，于耳蜗底回打孔，分别灌注酸性成纤维细胞生长因子（ａＦＧＦ）和碱性成纤维细胞生长因子（ｂＦＧＦ），４８小时后再测ＣＡＰ，处死动物做耳蜗琥珀酸脱氢酶（ＳＤＨ）组化染色铺片。结果爆震后灌注ｂＦＧＦ、ａＦＧＦ两组动物４８小时ＣＡＰ平均阈值分别为８８．７ｄＢ和９３．２ｄＢ，而单纯打孔组和灌注外淋巴组ＣＡＰ平均阈值分别为１１９．４ｄＢ和１０７．５ｄＢ，差异有显著性（Ｐ＜０．０５和０．０１）。铺片结果示灌流生长因子两组动物耳蜗毛细胞损伤程度比其他两组要轻。结果提示ｂＦＧＦ、ａＦＧＦ耳蜗内灌注对爆震性聋ＣＡＰ反应阈恢复有促进作用，在耳蜗声损伤过程中有保护和修复功用。     

内耳色素对爆震性听损伤保护作用的实验观察

目的　探讨内耳色素与爆震性听损伤的关系。方法　利用耳蜗铺片、石蜡切片、透射电镜及扫描电镜观察爆震前后白化豚鼠和杂色豚鼠耳蜗形态结构的变化 ,并对爆震前后白化豚鼠和杂色豚鼠听性脑干反应(ABR)反应阈进行测定。结果　白化豚鼠的耳蜗形态损伤和听功能损伤均较杂色豚鼠严重。结论　爆震后白化豚鼠耳蜗形态学损伤及ABR反应阈的改变均较杂色豚鼠明显。提示内耳血管纹色素颗粒与爆震性内耳损伤有关 ,机理可能为色素颗粒参与调节爆震后内淋巴液中钙离子浓度的平衡及参与清除爆震后耳蜗产生的氧自由基     

用于毛细胞再生研究的噪声性聋动物模型的建立

目的观察高强度脉冲噪声暴露后豚鼠听功能及耳蜗结构的变化,探讨用于毛细胞再生研究的噪声性聋动物模型的建立方法。方法健康成年白色红目豚鼠50只,雌雄不限,体重250～300g。随机分成2组,正常对照组10只,噪声暴露组40只。给予脉冲噪声（压力峰值为175．0dB SPL,脉宽0．25ms,间隔时间20秒）连续暴露200次。于噪声暴露前及暴露后1周、4周、8周检测听性脑干反应（auditory brainstem respons,ABR）,毛细胞计数及耳蜗铺片免疫组化观察耳蜗结构变化。结果高强度脉冲噪声暴露后1周,40只豚鼠中有21只（52．5％）双耳各频率ABR阈值≥95dBSPL。继续观察至噪声暴露后4周及8剧,ABR阈值没有恢复,1周、4周、8周各频率ABR阈值比较无统计学差异（P〉0．05）。毛细胞计数结果显示,噪声暴露敛极重度聋后1周,内毛细胞平均缺失率为91．4％,外毛细胞平均缺失率为97．2％。免疫组化染色分析结果显示,噪声暴露致聋后1周,内、外毛细胞胞核大部分缺失,内毛细胞内侧及外毛细胞外侧的支持细胞的胞核存存。结论高强度脉冲噪声暴露可造成豚鼠极重度感音神经性聋,耳蜗毛细胞广泛缺失且无法内行恢复,而支持细胞夫部分仔留,是进行毛细胞再生研究的理想动物模型。     

压力波对豚鼠耳蜗外侧壁微循环及听阈的影响

本研究应用生物微球技术、ＨＲＰ示踪显色法，结合光学显微镜，分别将对照组及爆震后２、６、２４ｈ组豚鼠耳蜗外侧壁血流量、微血管数量进行测量，并测试听性脑子反应。结果：爆震前、后各组ＡＢＲ平均阈值，组间差异显著（Ｐ＝０．０００４），６ｈ组阈移最大，其次为２４ｈ组，２ｈ组最小。爆震后豚鼠耳蜗侧壁血流量，无显著差异（Ｐ＞０．０５），而各回血流量有波动；爆震后耳蜗外侧壁血流量与阈移呈负相关（ｒ＝－０．５９３，Ｐ＜０．０５），外侧壁微血管的数量，因时间而变化，与血流量变化相吻合。讨论了爆震后耳蜗外侧壁血流量变化与听力损失、外侧微血管数量变化之间的关系。     

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。     

内耳色素与爆震性听损伤

目的 探讨内耳色素与爆震性听损伤的关系。方法 选用豚鼠４８只，分为：白化豚鼠爆震组，杂色豚鼠爆震组，正常对照组。爆震前及爆震后６小时、１、２、７、１４、２１天测定Ａ、Ｂ组豚鼠ＡＢＲ阈值。处死豚鼠做耳蜗铺片、耳蜗树脂包埋半薄切片、耳蜗扫描电镜制样，观察耳蜗内色素及耳蜗损伤情况。结果 光镜下杂色豚鼠可见耳蜗内色素颗粒，而白化豚鼠未见。爆震后白化豚鼠听力损伤比杂色豚鼠严重，其听力恢复亦较杂色豚鼠慢。爆震     

成纤维细胞生长因子耳蜗内灌注防治爆震性聋的实验研究

选用豚鼠１９只，震前于圆窗龛放银球电极测复合动作电位（ＣＡＰ）反应阈，爆震后测ＣＡＰ，于耳蜗底回打孔，分别灌注酸性成纤维细胞生长因子（ａＦＧＦ）和碱性成纤维细胞生长因子（ｂＦＧＦ），４８小时后再测ＣＡＰ，处死动物做耳蜗琥珀酸脱氢酶（ＳＤＨ）组化染色铺片。结果爆震后灌注ｂＦＧＦ、ａＦＧＦ两组动物４８小时ＣＡＰ平均阈值分别为８８．７ｄＢ和９３．２ｄＢ，而单纯打孔组和灌注外淋巴组ＣＡＰ平均阈值分别为１１     

降钙素基因相关肽在豚鼠耳蜗中的分布

目的：观察降钙素基因相关肽(CGRP)在成年豚鼠耳蜗中的分布。方法：采用免疫组织化学，光镜观察的方法，对CGRP在豚鼠耳蜗中的分布进行研究。结果：CGRP阳性反应物分布于螺旋神经元．骨螺旋板神经孔，内毛细胞、外毛细胞与外支持细胞交界区，螺旋韧带血管纹。结论：CGRP广泛分布于豚鼠耳蜗，是耳蜗传出神经系统重要的神经递质或神经凋质，CGRP可能通过调节螺旋神经元、毛细胞和支持细胞等影响听功能．也可能影响耳蜗的血流。     

5-磷酸二酯酶抑制剂对噪声性聋影响的实验研究

目的探讨5-磷酸二酯酶(PDE5)抑制剂对豚鼠噪声性聋的影响。方法豚鼠随机数字表法分为对照组、噪声暴露组和西地那非给药组,每组15只。西地那非组及噪声组豚鼠在白噪声暴露1周后分别腹腔注射西地那非10mg/(kg.d)及生理盐水4 ml/(kg.d),连续给药4周。分别测试噪声暴露前1天、噪声暴露后1、2及4周听性脑干反应(ABR)阈值及80dB HL下ABRⅠ波潜伏期,并通过扫描电镜观察噪声暴露后4周豚鼠耳蜗毛细胞的形态变化。结果与噪声暴露前相比,西地那非组ABR阈值及Ⅰ波潜伏期均小于噪声组,差异具有统计学意义(P值均<0.01)。扫描电镜显示,噪声组豚鼠耳蜗内、外毛细胞均出现听毛紊乱、融合及缺失;而西地那非组耳蜗病变较轻,听毛仅有轻微倒伏、融合现象。结论西地那非能够减轻噪声对豚鼠耳蜗毛细胞的损害,降低噪声性听觉损伤引起的ABR阈值升高,缩短其引起的Ⅰ波潜伏期延长。     

神经营养因子对噪声引起豚鼠耳蜗外毛细胞损伤的防护作用

目的 定量观察胶质细胞源性神经营养因子(glial cell ine-derived neurotrophic factor,GDNF)和神经营养-3(neurotrophin-3,NT-3)对噪声引起豚鼠耳蜗外毛细胞损伤的防护作用。方法 将微渗透压泵埋置于豚鼠背部,经固定于耳蜗底回鼓阶内的改良微导管将GDNF(100ng/ml)和NT-3(2.5μg/ml)的混合液缓慢注入12只豚鼠左侧内耳,以左侧内耳灌注人工外淋巴液的9只豚鼠为对照,检测噪声暴露后豚鼠听功能和耳蜗外毛细胞形态、数量的变化。结果 噪声暴露10天后,实验组手术耳和非手术耳的脑干诱发电反应阈值低于对照组(P<0.05,p<0.01)。毛细胞表皮板和纤毛肌动蛋白荧光染色计数发现,实验组手术耳和非手术耳的外毛细胞缺失率低于对照组(p<0.001,p<0.01)。毛细胞核荧光染色计数发现,实验组手术耳和非手术耳的外毛细胞核肿胀率低于对照组(p<0.01,p<0.01)。结论 GDNF和NT-3对噪声引起豚鼠耳蜗外毛细胞损伤具有较好的防护作用。     

Identification of factors that maintain mammalian outer hair cells in adult organ of Corti explants

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for 1 month in a minimal essential medium. In contrast, under the same culture conditions, only IHCs survive in explants from adult guinea pig organ of Corti while many of the OHCs are lost within the first 48 h. Hair cell counts show OHCs loss to be greater in the lower portion (i.e. middle turn) of the cochlea than at the apex. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) indicates that there is DNA damage in adult OHCs within 8 h of explantation. Treatment of the adult organ of Corti explants with either actinomycin D (10⁻⁷ M) or cycloheximide (10⁻⁶ M) prevents most OHC losses. According to these results apoptosis may be the mechanism of OHC loss in adult organ of Corti explants. Stable membrane potentials recorded from the OHCs in both uncultured and actinomycin D-treated organ of Corti explants cultured for 72 h demonstrate the functional integrity of these hair cells. OHC losses in the adult guinea pig organ of Corti cultures can also be prevented by treatment with several of the growth factors tested, i.e. acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), and glial cell-derived neurotrophic factor (GDNF). The results of this study suggest that growth factor therapy may be applicable to the treatment of some hearing disorders.     

手机微波辐射对豚鼠耳蜗外毛细胞膜电位的影响

目的探讨手机微波辐射对豚鼠耳蜗外毛细胞膜电位的影响。方法取成年豚鼠20只随机分为对照组和辐射组,每组各10只。对照组不做任何处理;辐射组:距豚鼠右侧耳廓2cm处对辐射组豚鼠进行手机微波辐射,辐射时间分别为1、6、12、24和48h,然后分别于各时间点随机活杀两组中的豚鼠各2只,取出听泡,分离耳蜗外毛细胞,用2 000Hz纯音进行不同强度的声刺激,观察离体单个外毛细胞(OHC)膜电位的变化特征。结果对照组离体单个OHC予2 000Hz、10dB HL纯音刺激时,于第40s细胞内荧光值开始升高,并于第50s达到峰值(1.112u),随后细胞内荧光值开始回落,于第60s至最低值(1.000u)。辐射组微波辐射1小时后,OHC细胞膜的去极化和复极化时间明显增加,并且不能完全去极化和复极化;辐射6和12h后,随着间断的声刺激可见OHC内荧光值缓慢上升,辐射6小时组之毛细胞于第70s荧光值达峰值(1.100u),而辐射12小时组之毛细胞于第90s荧光值达峰值(1.050u),随后整条曲线呈微上升趋势;辐射24和48h后,经过声刺激,曲线呈微上升、微下降波动,无明显峰值及回落。结论长时间的手机微波辐射能导致离体豚鼠耳蜗OHC膜离子通透性的改变,影响OHC膜电位的形成及细胞去极化、复极化,从而导致离体OHC膜电位对不同强度纯音刺激的敏感性逐渐降低。     

豚鼠冲击波负压暴露后耳蜗毛细胞损害定量观察

目的 探讨冲击波负压（blast underpressure,BUP）暴露后豚鼠耳蜗毛细胞损害特点.方法将豚鼠暴露实验性BUP 14天后处死,硝酸银染色硬铺片法计数观察耳蜗基底膜毛细胞损伤情况.结果压力峰值介于-22.4kPa和-63.3kPa之间的实验性BUP暴露后,豚鼠耳蜗外毛细胞出现了明显的病理性改变,损伤的程度以第二转最重,第二排和第三排的病变比第一排更为严重.BUP强度越高,毛细胞损害越重.各实验组动物的外毛细胞总缺失率明显高于正常对照组（P＜0.01）;重复暴露3次的动物外毛细胞缺失率明显高于暴露1次的动物（P＜0.01）.结论BUP暴露可引起明显的豚鼠耳蜗外毛细胞缺失等损害,其损害程度与负压峰值及暴露次数密切相关;毛细胞损害越重,ABR阈移也就越明显.     

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.     

碱性成纤维细胞生长因子真核表达载体的构建及其在内耳基因治疗中的实验研究

目的　观察碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)双顺反子真核表达载体在豚鼠耳蜗中的表达 ,及噪声损伤后对毛细胞的保护作用。方法　利用脑炎心肌炎病毒的内部核糖体进入位点 (internalribosomeentrysite,IRES) ,构建人bFGF与增强型绿色荧光蛋白 (enhancegreenfluorescenceprotein ,EGFP)基因真核表达载体pIRES bFGF EGFP。采用硬脂胺 (stearylamine ,SA)脂质体介导bFGF基因转染豚鼠内耳 ,噪声暴露即刻通过圆窗向豚鼠耳蜗注入含有bFGF基因作为治疗基因的真核表达载体 ;或在噪声暴露前 7d ,作为保护因子转导转染豚鼠耳蜗。结果　构建的pIRES bFGF EGFP在转染 2 4h后开始表达 ,48h达到最高 ,表达时间可持续 1个月。治疗组在噪声后听性脑干反应平均阈值均低于噪声组 ,在保护组 :pIRES bFGF EGFP对耳蜗毛细胞有明显的保护作用。结论本研究成功地构建了pIRES bFGF EGFP真核共表达载体 ,具有bFGF、EGFP的双重活性 ,IRES可引导外源基因bFGF在内耳毛细胞表达     

Assessment criteria for rotated stereociliary bundles in the guinea pig cochlea.

OBJECTIVE: Our study examined the relationship between variant stereociliary bundles of cochlear outer hair cells (OHCs) and auditory function to analyze assessment criteria for rotated stereociliary bundles in the guinea pig cochlea. METHODS: Auditory brainstem response and distortion product otoacoustic emission (DPOAE) were recorded on 100 guinea pigs. Variant hair cells were identified and counted by scanning electron and light microscopy. RESULTS: The most common variation observed was rotation of stereociliary bundles in the first-row OHCs (OHC1), with most 13.3% variant OHC1 rotated 90 degrees and a few 2.5% rotated 180 degrees. Occasionally, the length and angle of the 2 arms of an OHC deviated from the norm. The auditory brainstem response threshold of affected animals increased only slightly, 20- to 30-dB sound pressure level. More importantly, amplitude of DPOAE increased significantly (40.5 dB sound pressure level). CONCLUSION: Our study suggests that rotation of stereociliary bundles in the cochlear OHC was found to be prevalent in 28% of the animals. We established the assessment criteria for rotated stereociliary bundles that were more than 10% OHC1 rotated. This hair bundle seemed to be rotated by 90 degrees from the normal orientation and was accompanied with changes of auditory function. Increased amplitude of DPOAE is associated with the variation of rotated OHC that might result in hearing loss.     

Some aspects of temporal coding by single cochlear fibres from regions of cochlear hair cell degeneration in the guinea pig.

Some temporal coding properties of cochlear nerve fibers are investigated in kanamycin-treated guinea pigs (GPs) with various degrees of outer hair cell (OHC) degeneration. In particular, the phase locking ability of fibres from pathological cochleas, and also their adaptation properties are compared with the properties of normal cochlear fibres. No systematic effects of OHC loss on these properties have so far been found. These preliminary results therefore suggest (in so far as these animals can be regarded as models of sensorineural hearing loss of cochlear origin in man) that little deterioration should be expected in functions purely dependent upon faithful temporal coding of the stimulus waveform.     

锌,铁预防豚鼠钻井井场噪声性听力损失的实验观察

为观察给予饲料中添加锌,铁喂养豚鼠,采用听性脑干反应(ABR)和耳蜗电图(EcochG)及扫描和透射电镜技术观察,在钻井井场柴油机旁噪声[102 dB(A)]暴露下豚鼠听功能和耳蜗毛细胞的变化.结果表明,当脱离噪声后听力逐渐恢复,于第7d组可见明显恢复.耳蜗毛细胞改变明显减轻,并逐渐恢复正常.而喂普通饲料组未见恢复.证明锌、铁能有效地防治钻井井场噪声所致的听力损伤.     

表皮生长因子及其受体在中耳慢性鼓膜穿孔病变中的作用

OBJECTIVE: To evaluate the possible roles of epidermal growth factor(EGF) and its receptor (EGFR) on the chronic tympanic membrane perforations. METHODS: A phosphate buffer saline of EGFR was administered to a Gelfoam pledget placed over chronic tympanic membrane perforations in guinea pigs. The EGFR of 10 specimens from the acquired middle ear cholesteatoma of the adjacent skin around perforation was examined by immunohistochemical SP method and computer image analysis. Results Complete closure of the tympanic membrane perforations was observed in 82.6% of EGF-treated ears, but only 33.3% in the controls(P < 0. 01). No case was led to middle ear cholesteatoma in the experiment group (0/23). The positive expression in the adjacent skin around perforation was (39.3 -/+ 7.4)%; and the normal external ear skin was (25.4 +/- 3.7)%; There were distinctly significant differences between the adjacent skin around perforation and the normal external ear skin (P < 0. 01). CONCLUSIONS: EGF is effective on closing chronic tympanic membrane perforations in the guinea pigs. Present data suggests that EGF-treated may induce the occurrence of middle ear cholesteatoma.     

Some aspects of temporal coding by single cochlear fibres from regions of cochlear hair cell degeneration in the guinea pig

Summary Some temporal coding properties of cochlear nerve fibres are investigated in kanamycin-treated guinea pigs (GPs) with various degrees of outer hair cell (OHC) degeneration. In particular, the phase locking ability of fibres from pathological cochleas, and also their adaptation properties are compared with the properties of normal cochlear fibres. No systematic effects of OHC loss on these properties have so far been found.These preliminary results therefore suggest (in so far as these animals can be regarded as models of sensorineural hearing loss of cochlear origin in man) that little deterioration should be expected in functions purely dependent upon faithful temporal coding of the stimulus waveform.