冲击波对豚鼠耳蜗毛细胞碳酸酐酶的影响

用组织化学、细胞化学方法结合图像分析和听觉电生理技术,研究了冲击波对豚鼠耳蜗毛细胞碳酸酐酶（CA）的影响及其与听阈阈移的关系。结果表明冲击波暴露后6、12、24、48、72小时组,毛细胞中CA阳性产物的灰度值较正常组明显降低（P＜0．01）,其灰度值的变化与听觉脑干反应（ABR）阈移呈负相关（r=-0．9369,P＜0．01）。提示毛细胞中CA活性减低是冲击波致听力损失的因素之一。文中讨论了CA在耳蜗毛细胞的作用及其与听阈的关系。     

冲击波对豚鼠耳蜗毛细胞心钠素免疫反应（ANP—IR）影响...

目的：为探讨冲击波对豚鼠耳蜗毛细胞心钠素免疫反应的影响及与听阈阈移的相关性，对冲击波的致聋机理进行探讨。方法：对冲击波暴露后不同时间组采用免疫细胞化学技术（ＡＢＣ）结合图像分析和听性脑干反应测试技术进行观测。结果：冲击波暴露后２４ｈ内，仙毛细胞（ＩＨＣ）中ＡＮＰ－ＩＲ阳性产物的光密度值较对照组明显增高（Ｐ〈０．０５），且与听阈阈移有明显的正相关性（ｒ＝０．９１８５，Ｐ〈０．０５）；而外毛细胞（ＯＨ     

冲击波对豚鼠耳蜗毛细胞心钠素免疫反应(ANP-IR)影响及与听阈阈移相关性研究

目的：为探讨冲击波对豚鼠耳蜗毛细胞心钠素免疫反应的影响及与听阈阈移的相关性，对冲击波的致聋机理进行探讨。方法：对冲击波暴露后不同时间组采用免疫细胞化学技术（ＡＢＣ）结合图像分析和听性脑干反应测试技术进行观测。结果：冲击波暴露后２４ｈ内，内毛细胞（ＩＨＣ）中ＡＮＰ－ＩＲ阳性产物的光密度值较对照组明显增高（Ｐ＜０．０５），且与听阈阈移有明显的正相关性（ｒ＝０．９１８５，Ｐ＜０．０５）；而外毛细胞（ＯＨＣ）中心钠素免疫反应阳性产物变化不明显。结论：ＩＨＣ和ＯＨＣ中ＡＮＰ－ＩＲ的变化可能是冲击波对其损伤机制的不同表现。     

噪声暴露引起耳蜗内毛细胞谷氨酸样免疫反应的动态变化

目的：观察噪声暴露后不同时间豚鼠耳蜗内毛细胞（inner bair cell,IHC)谷氨酸样免疫反应（glutamate-like immunoreactivity,Glu-IR)的变化。方法：将实验动物随机分为正常对照组与噪声暴露后即刻，8小时，1天，3天和7天组，噪声暴露前，先破坏全部动物的右耳鼓膜，并用磷酸锌牙科水泥堵塞右侧外耳道，然后将实验各组在准自由声场中暴露于120dBlp1/3倍频程的4kHz窄带声中4小时，造成左侧（单侧）耳蜗噪声损害的动物模型，用S对耳蜗Corti器半薄切片作谷氨酸样免疫细胞化学染色，用计算机图像分析系统测量耳蜗IHC中Glu-IR的平均光密度（average optical density,AOD）值，结果：正常对照组，豚鼠两侧耳蜗IHC中Glu-IR的AOD值无差异（P＞0．05），在噪声暴露后即刻组，豚鼠左侧耳蜗IHC中Glu-IR的AOD值较右侧高（P＜0．01），在噪声暴露后8小时组较右侧低（P＜0．05），在噪声暴露后1天，3天和7天组无差异（P＞0．05），结论：噪声暴露后耳蜗IHC中Glu-IR经过一个从增强一减弱－恢复动态变化过程。     

实验性冲击波负压暴露后豚鼠耳蜗基底膜AChE和SDH活性的变化

目的 探讨冲击波负压（BUP）暴露对豚鼠耳蜗基底膜乙酰胆碱酯酶（AChE）和琥珀酸脱氢酶（SDH）活性的影响。方法 中等强度BUP重复暴露3次后2h～3d检测豚鼠听性脑干反应（ABR）阈值，然后处死并应用酶组织化学技术观察其耳蜗基底膜两种酶的活性产物的变化。结果 负压峰值为-44．5kPa～-48．8kPa的BUP暴露后2h，耳蜗基底膜外毛细胞下神经末梢区域AChE活性产物明显减少，在负压暴露后24h和3d时有所恢复，但仍低于正常对照组。基底膜SDH活性下降主要发生于外毛细胞，且以第二转第三排最为明显；BUP暴露后2h活性下降幅度最大，至24h和3d时有所恢复。上述两种酶活性产物的变化，与动物的ABR阈移幅度变化是一致的。结论 BUP暴露后豚鼠耳蜗基底膜的AChE和SDH活性有不同程度的下降，这可能是造成BUP暴露后听力损失的一个主要原因。     

CO2激光照射豚鼠耳蜗对内耳结构和功能的影响

目的探讨不同功率CO2激光照射豚鼠耳蜗后其内耳结构和功能的影响.方法 24只红目豚鼠分3组,每组8只,分别以功率为1、2、3 W,功率密度分别为796、1 592、2 388 W/cm2的CO2激光照射左耳耳蜗底周,以右耳为对照,术前及处死前分别行听性脑干反应(ABR)检查,分术后即刻和术后3周两批断头处死,应用光镜和扫描电镜技术观察耳蜗形态学变化.结果激光照射术后即刻3组豚鼠听阈较术前明显升高(1 W组P<0.05;2 W组P<0.05;3 W组P<0.01),照射后3周1 W组听阈基本恢复(P>0.05),2 W、3 W组听阈有所下降但仍高于术前(P<0.05).照射后即刻,光镜下见3组豚鼠耳蜗外毛细胞出现肿胀,硝酸银浓染,血管纹血管扩张;照射后3周,扫描电镜观察1 W组内耳结构轻微改变, 2 W组出现散在的外毛细胞静纤毛的倒伏,排列紊乱,3 W组出现外毛细胞静纤毛严重缺失、破坏和融合呈球状.结论应用CO2激光行耳科手术时,应注意功率及功率密度的选择,以免造成内耳损伤.     

听力学

20030941内毛细胞的分离和形态学特点/杨仕明…//中华耳鼻咽喉科杂志一2002,37(6)一462~464 目的:探讨内毛细胞(l HC)的分离技术和形态学特征。方法:从豚鼠耳蜗制备基底膜活体铺片,微分干涉倒置显微镜下用显微分离结合酶消化的方法单离IHC。高倍显微镜下,用两个钨丝电极沿IHC和第一排外毛细胞(OHC)之间的Corti隧道显微切割,游离的IHC团被吸引至玻璃微吸管内。结果:从每只豚鼠耳蜗可分离到30一50个活性良好的单离IHC,并存活Zh左右。典型的IHC胞体具有特征性,呈梨状或长颈的烧瓶形状,胞体中间可见大的圆形细胞核。胞体内包含较多细胞…     

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.     

Prestin基因敲除小鼠听力和毛细胞改变的相关性研究

目的研究发育和成熟过程中的prestin基因敲除小鼠听力特征,比较其听力损害和外毛细胞(OHC)丧失的相关性,初步探讨OHC丧失的机制.方法利用听性脑干反应(ABR)、抗小鼠耳蜗毛细胞特异性的肌浆球蛋白7a(Mysojn 7a)抗体免疫染色和耳蜗连续切片观察出生后(postnatal,P)14天(P14)～P56的prestin基因敲除纯合子(prestin-/-)、杂合子(prestin /-)和野生型(prestin / )小鼠听阈、耳蜗毛细胞和Corti器的改变.结果P14的prestin-/-小鼠听阈较prestin / 升高25 dB SPL,至P21和P28,听阈分别提高49 dB和52 dB,P35后听阈改变不明显.在P21的prestin-/-小鼠,32 kHz听阈提高46 dB;prestin /-也显示约3.5 dB的听阈升高(p＜0.0001).Prestin-/-小鼠在P28以前无明显的OHC丧失,但是,OHC的长度较prestin / 明显缩短,P28以后,OHC丧失进行性加重.内毛细胞(IHC)丧失明显延迟和轻于OHC.结论Prestin-/-小鼠在毛细胞丧失之前呈现明显的听力损害,OHC的丧失可能与其本身的结构改变和成熟过程中的代谢异常有关.     

噪声暴露引起耳蜗内毛细胞谷氨酸样免疫反应的动态变化

目的 :观察噪声暴露后不同时间豚鼠耳蜗内毛细胞 (inner hair cell,IHC)谷氨酸样免疫反应 (glutamate- likeimmunoreactivity,Glu- IR)的变化。方法 :将实验动物随机分为正常对照组与噪声暴露后即刻、8小时、1天、3天和 7天组。噪声暴露前 ,先破坏全部动物的右耳鼓膜 ,并用磷酸锌牙科水泥堵塞右侧外耳道 ,然后将实验各组在准自由声场中暴露于 12 0 d BL p1/ 3倍频程的 4 k Hz窄带声中 4小时 ,造成左侧 (单侧 )耳蜗噪声损害的动物模型。用 SP法对耳蜗 Corti器半薄切片作谷氨酸样免疫细胞化学染色 ,用计算机图像分析系统测量耳蜗 IHC中 Glu- IR的平均光密度 (average opticaldensity,AOD)值。结果 :正常对照组 ,豚鼠两侧耳蜗 IHC中 Glu- IR的 AOD值无差异 (P >0 .0 5 ) ;在噪声暴露后即刻组 ,豚鼠左侧耳蜗 IHC中 Glu- IR的 AOD值较右侧高 (P <0 .0 1) ,在噪声暴露后 8小时组较右侧低 (P <0 .0 5 ) ,在噪声暴露后 1天、3天和 7天组无差异 (P >0 .0 5 )。结论 :噪声暴露后耳蜗 IHC中 Glu- IR经过一个从增强 -减弱 -恢复动态变化过程     

Effects of a single high dose of cisplatin on the melanocytes of the stria vascularis in the guinea pig

The antineoplastic drug cisplatin is known to cause a reduction in endocochlear potential. The hypothesis to be tested was whether a single high dose of cisplatin affects the melanocytes by altering the expression of melanin. Pigmented guinea pigs received a bolus injection of cisplatin (8 mg/kg as a 15-second intravenous infusion). Auditory brainstem response (ABR) thresholds and morphological analysis of the hair cells and the stria vascularis were made 96 h after injection. ABR thresholds were elevated (15-40 dB) at 12-30 kHz and a significant loss of outer hair cells in the more basal regions was found. Cisplatin caused a significantly lower density of melanin in the intermediate cells in the basal region without any signs of apoptosis. Changes in melanin content were not noted in the middle or apical cochlear regions. Significant correlations were found between melanin density, ABR threshold shifts and outer hair cell loss in the region corresponding to 30 kHz. The findings reported here further support the multiple cytotoxic effect of cisplatin on the inner ear.     

Ultrastructural pathology in the stria vascularis of the MRL-Fasl(lpr) mouse.

The MRL-Fas(lpr) mouse, a model of multisystemic, organ nonspecific autoimmune disease, has been proposed as a model of immune-mediated inner ear disease. A preliminary study employing light microscopy indicated that it develops cochlear pathology that appeared most striking in the stria vascularis, where cells underwent edema and degeneration. However, other structures, including the inner and outer hair cells and the supporting cells, also appeared to display pathology. The current study analyzed cochlear ultrastructure using transmission electron microscopy to better delineate the cochlear lesions found in these animals. MRL-Fas(lpr) animals were allowed to develop systemic disease (20 weeks old) and then had auditory brainstem response (ABR) thresholds determined. Animals were then killed and their cochleas prepared for electron microscopy. Age-matched MRL-+/+ and BALB/c mice served as controls. Results indicated that MRL-Fas(lpr) mice demonstrated elevated ABR thresholds. In contrast to a preliminary report, the cochlear pathology was observed exclusively in the stria vascularis, where cells demonstrated hydropic degeneration. Strial capillary structure was normal as were the rest of the cellular cochlear constituents. No inflammatory infiltrate was noted. These studies confirm that the MRL-Fas(lpr) mouse develops cochlear abnormalities focused in the stria vascularis. Whether the mechanism of the cellular degeneration involves autoimmune, genetic, or uremic processes has yet to be determined.     

心钠素免疫反应物质在豚鼠耳蜗Corti器中的分布

目的 为了解豚鼠耳蜗Ｃｏｒｔｉ器组织中心钠素免疫反应（Ａｔｉａｌ ｎａｔｒｉｕｒｅｔｉｃ ｐｅｐｔｉｄｅｓ ｉｍｍｕｎｏｒｅａｃｔｉｏｎ，ＡＮＰ－ＩＲ）物质的分布及形态学特征。方法 对耳蜗切片组织采用免疫组织化学ＡＢＣ法进行研究。结果 Ｃｏｒｔｉ器中内侧第一、二排外毛细胞，Ｂｏｅｔｔｃｈｅｒ细胞和靠近螺旋韧带嵴的基底膜ＡＮＰ－ＩＲ为阴性，其余的组织细胞均为阳性反应。结论 ＡＮＰ在Ｃｏｒｔｉ器功能方     

An experimental study of auditory dysfunction associated with hyperlipoproteinemia

We sought to clarify whether or not hyperlipoproteinemia induces auditory dysfunction. In so doing, we studied the general states and cochlear pathologies of guinea pigs after the administration of a hyperlipid diet for 3 months. Serum biochemistries indicated marked elevations of cholesterol, high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL) levels. An increased auditory threshold varying from 10 to 20 dB was observed in 40% of the guinea pigs using auditory brainstem responses. Histochemical study of the inner ear revealed variations in lipid metabolism and partial disorders of the outer hair cells. Electron microscopic observations showed vacuolar and parenchymal protrusions on the surfaces of the stria vascularis and Corti's organ, and vacuolar degeneration was seen around the capillary vessels of the vascular stria. Our data has shown that the auditory dysfunction present in the inner ear was less marked than were the morphological changes seen. Our findings suggest that other factors besides hyperlipoproteinemia are involved in the development of severe auditory damage.     

An experimental study of auditory dysfunction associated with hyperlipoproteinemia

Summary We sought to clarify whether or not hyperlipoproteinemia induces auditory dysfunction. In so doing, we studied the general states and cochlear pathologies of guinea pigs after the administration of a hyperlipid diet for 3 months. Serum biochemistries indicated marked elevations of cholesterol, high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL) levels. An increased auditory threshold varying from 10 to 20 dB was observed in 40% of the guinea pigs using auditory brainstem responses. Histochemical study of the inner ear revealed variations in lipid metabolism and partial disorders of the outer hair cells. Electron microscopic observations showed vacuolar and parenchymal protrusions on the surfaces of the stria vascularis and Corti's organ, and vacuolar degeneration was seen around the capillary vessels of the vascular stria. Our data has shown that the auditory dysfunction present in the inner ear was less marked than were the morphological changes seen. Our findings suggest that other factors besides hyperlipoproteinemia are involved in the development of severe auditory damage.     

Age-related auditory pathology in the CBA/J mouse

Commercially obtained aged male CBA/J mice presented a complex pattern of hearing loss and morphological changes. A significant threshold shift in auditory brainstem responses (ABR) occurred at 3 months of age at 4kHz without apparent loss of hair cells, rising slowly at later ages accompanied by loss of apical hair cells. A delayed high-frequency deficit started at 24kHz around the age of 12 months. At 20-26 months, threshold shifts at 12 and 24kHz and the accompanying hair cell loss at the base of the cochlea were highly variable with some animals appearing almost normal and others showing large deficits. Spiral ganglion cells degenerated by 18 months in all regions of the cochlea, with cell density reduced by approximately 25%. There was no degeneration of the stria vascularis and the endocochlear potential remained stable from 3 to 25 months of age regardless of whether the animals had normal or highly elevated ABR thresholds. The slow high-frequency hearing loss combined with a modest reduction of ganglion cell density and an unchanged endocochlear potential suggest sensorineural presbycusis. The superimposed early hearing loss at low frequencies, which is not seen in animals bred in-house, may complicate the use of these animals as a presbycusis model.     

Alterations of inner ear morphology in experimental hypercholesterolemia.

Previous clinical and experimental studies have indicated that auditory function may be compromised by hypercholesterolemia. In this investigation, inner ear tissue from chinchillas maintained on a cholesterol-supplemented diet for 3 months was examined for morphological alterations which might underlie the physiological changes observed with this condition in earlier studies. Ultrastructural analysis of cochleas from 16 hypercholesterolemic chinchillas revealed alterations in both the stria vascularis and outer hair cells. Strial marginal cells throughout the cochlea and outer hair cells of the apical turn, contained electron-lucent patches of an amorphous material. These patches had the morphological characteristics and histochemical properties of glycogen. Mild extracellular edema and increased numbers of lysosomes were also noted in the stria vascularis of experimental animals. These alterations suggest that chronic hypercholesterolemia metabolically stresses inner ear tissue. It is hypothesized that such changes could increase susceptibility of the cochlea to ototraumatic agents.     

Morphological changes of cochlea in a strain of new-mutant mice

The hearing ability and histological characteristics of the cochlea of a strain of new-mutant mice were analyzed. This new mutant arose as a spontaneous mutation in the C3H/He stock. The genetic mode is autosomal recessive and the animals show abnormal behavior such as circling, head-tossing and hyperactivity. The audiological findings exhibited no recordable auditory brain stem response (ABR) in any homozygotes at ages ranging from 11 days to 117 days. For morphological examination, we used 36 homozygote with ages ranging from 10 days to 18 months. The primary morphological abnormalities were observed in the organ of Corti. The stereocilia of the outer hair cells showed disarray throughout the whole cochlea, although outer hair cell cytoplasm became fully developed, including the nerve terminals. Age-dependent degeneration of the outer hair cells subsequently occurred from the basal to the apical part of the cochlea. The earliest change demonstrated in the outer hair cells was cuticular degeneration. Although the abnormalities of the inner hair cells occurred late, a complete loss of inner and outer hair cells was demonstrated. The stria vascularis was well preserved at a later age as were spiral ganglion cells. These histological findings confirm that this mouse is classified as a neuroepithelial-type mutant. As this animal was expected to have a single gene abnormality, molecular genetic studies on this animal can provide important information on the nature of histological changes of the hair cell from a mode of gene action.     

卡那霉素联合呋塞米快速诱导小鼠耳蜗损伤

目的 探讨卡那霉索和呋塞米联合应用对小鼠耳蜗的毒性作用,建立一种可靠的小鼠感音神经性聋模型.方法 选用3～4周龄的CBA/J小鼠为实验对象,按1 g/kg的剂量皮下注射卡那霉素,30～45 min后按0.4 g/kg的剂量腹腔注射呋塞米.在注射前、注射后12 h、24 h、48 h、7 d、2周、4周及12周分别应用听性脑干反应(ABR)检测小鼠听觉功能的改变;应用异硫氰酸荧光素标记的鬼笔环肽及碘化丙啶染色、半薄切片甲苯胺蓝染色、脱氧核苷酸末端转移酶介导的dUTP缺口末端标记技术、扫描电镜等观察小鼠毛细胞死亡的模式和程度;同时通过透射电镜观察记录耳蜗血管纹形态及厚度的变化.结果 序贯应用卡那霉素及呋塞米后12 h小鼠ABR阈值开始上升,随后的36 h内持续进行性上升,继而稳定在90 dB左右.应用激光共聚焦显微镜在药物注射后12 h观察到耳蜗底回外毛细胞开始出现死亡,24 h时底回外毛细胞基本全部消失,同时顶回外毛细胞开始出现死亡,至48 h时整个耳蜗外毛细胞绝大部分死亡;而内毛细胞的损伤至给药后7 d时才开始出现,随时间推移仍有部分内毛细胞完好无损.死亡的毛细胞均具有典型的凋亡细胞特征.扫描电镜观察发现在药物注射后受损毛细胞出现纤毛消失、表皮板塌陷,随后支持细胞增生并在该处形成瘢痕.血管纹表面边缘细胞在给药后出现部分胞体融合并且有坏死物自胞内排出,随后边缘细胞表面大部分微绒毛消失,胞体呈"石块"样改变.透射电镜结果 显示血管纹厚度在给药后进行性下降,主要为边缘细胞萎缩造成.结论 单次序贯应用卡那霉素及呋塞米能快速诱导小鼠耳蜗毛细胞大量死亡,适合应用于建立小鼠感音神经性聋模型.