听力学

88030876 大鼠内耳Corti氏器扫描电镜样品的制备与观察/孙爱华…∥临床耳鼻咽喉科杂志.-1987,1(4).-205～207 本文首次报告非脱钙的大鼠耳蜗Corti氏器扫描电镜样品制备方法,观察和测量了听毛细胞静纤毛及其表面结构。结果表明,经本方法制备的样品,Corti器表面超微结构保存良好,显示清晰。大鼠耳蜗可根据外毛细胞静纤毛特点分为顶、中、底三个区域。本文认为,以Corti氏器表面超微结构特征为标志的耳蜗分区法对于研究和分析听毛细胞的生理功能及其病理改变     

大鼠内耳Corti氏器扫描电镜样品的制备与观察

本文首次报告非脱钙的大鼠耳蜗Corti氏器扫描电镜样品制备方法,观察和测量了听毛细胞静纤毛及其表面结构。结果表明,经本方法制备的样品,Corti器表面超微结构保存良好,显示清晰。大鼠耳蜗可根据外毛细胞静纤毛特点分为顶、中、底三个区域。本文认为,以Corti氏器表面超微结构特征为标志的耳蜗分区法对于研究和分析听毛细胞的生理功能及其病理改变具有重要意义。     

新生小鼠耳蜗基底膜体外培养的实验研究

目的建立可靠的新生小鼠离体耳蜗基底膜的培养方法,为内耳的研究提供新的条件和模型。方法在显微镜下完整分离出新生1～5 d小鼠的耳蜗基底膜组织,采用组织贴壁法在无血清培养基中进行培养,免疫荧光法检测新生小鼠离体耳蜗基底膜培养组织中毛细胞及螺旋神经元的生长状态。结果新生小鼠耳蜗基底膜离体培养24 h后,显微镜下观察可见基底膜贴壁生长良好,外周有新生上皮细胞和成纤维细胞长出;高倍显微镜下可见耳蜗内外毛细胞和支持细胞等结构;免疫荧光法检测可见毛细胞和螺旋神经元生长良好,并能保持较长一段时间。结论组织贴壁法无血清培养新生小鼠离体耳蜗基底膜是一种理想的耳科实验造模方法。     

电离辐射对耳蜗毛细胞超微结构的影响

目的 观察电离辐射对耳蜗毛细胞超微结构的损伤情况.方法 将健康豚鼠15只随机分成二组:单纯放射组(单放组)10只,对照组5只,每组观察10耳.对照组不放射,单放组用直线加速器所产生的6Mev电子线对豚鼠的耳颞部予以分次放射(2 Gy/天),总剂量达到60 Gy,行透射电镜及扫描电镜观察两组豚鼠耳蜗毛细胞的形态学变化.结果 透射电镜观察见对照组耳蜗外毛细胞边界清楚,细胞无肿胀,表皮板完整,线粒体嵴完整,核膜完整;单放组耳蜗外毛细胞边界不清,细胞肿胀变形,线粒体空泡变,线粒体嵴断裂,核膜不完整,异染色质增多.扫描电镜观察见对照组耳蜗外毛细胞的纤毛排列整齐无倒伏、缺失;单放组耳蜗外毛细胞的纤毛明显倒伏、缺失、排列紊乱.结论 分割剂量电离辐射对豚鼠耳颞部放射可造成耳蜗毛细胞超微结构损害.     

荧光下豚鼠听觉离体毛细胞的凋亡改变

目的：探讨离体耳蜗毛细胞的凋亡改变。方法：采用胶原酶制备离体耳蜗毛细胞、0．01％吖啶橙进行荧光染色,荧光显微镜观察凋亡改变。结果：凋亡毛细胞的主要形态特征包括：胞核皱缩;荧光显微镜下凋亡毛细胞呈红色或红黄色荧光,毛细胞形态完整,无胞膜破裂或胞质溢出。结论：离体耳蜗毛细胞的凋亡改变使细胞呈红色或红黄色荧光,胞膜完整。     

不同基因转染方法对小鼠内耳毛细胞转染效率的比较

目的对比不同的基因转染方法对小鼠内耳毛细胞的转染效率,以探寻简便、高效转染耳蜗毛细胞的方法。方法分离111只新生昆明小鼠(P2)耳蜗螺旋韧带及感染上皮,分别采用电穿孔法介导质粒载体(pGPHI/GFP/Neo)、腺病毒载体(Ad5/CMV/GFP)和慢病毒载体(LV/CMV/GFP)3种方法转染小鼠内耳毛细胞,于转染48小时后在荧光显微镜下观察并比较三种方法基因转染后小鼠内耳毛细胞绿色荧光蛋白(green fluorescence protein,GFP)的表达。结果电穿孔法介导质粒载体转染组和慢病毒载体转染组GFP阳性细胞极少;腺病毒载体转染组效率最高,耳蜗中回外毛细胞GFP阳性率为90.0%±4.1%,内毛细胞GFP阳性率为5%±0.4%。结论腺病毒载体能更有效地将外源基因导入耳蜗毛细胞内表达,是基因转染耳蜗毛细胞的理想载体。     

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。     

Smad5 基因敲除小鼠耳蜗扫描电镜观察

目的观察Smad5基因敲除杂合（3月组2只小鼠4只耳蜗）子小鼠内耳形态学超微结构变化。方法野生型动物组：1个月组4只小鼠5只耳蜗，2个月组及3个月组各有2只小鼠4只耳蜗：杂合子动物组1个月组3只小鼠3只耳蜗，2个月组2只小鼠4只耳蜗，3个月组3只小鼠6只耳蜗。按常规方法制成标本作扫描电镜观察。结果Smad5基因敲除杂合子小鼠扫描电镜观察：1月组耳蜗中均观察到耳蜗第一和第二圈外毛细胞有不同程度的静纤毛融合，未见外毛细胞缺失，内毛细胞未见明显地病理变化。2月组耳蜗中观察到第一和第二圈均可见内外毛细胞静纤毛不同程度缺失，有的部位以内毛细胞静纤毛缺失为主，而有的部位以外毛细胞静纤毛缺失为主，有的以片状缺失为主，有的则以散在性缺失为主。3个月组3只小鼠6只耳蜗中均观察到不同程度第一和第二圈内外毛细胞静纤毛广泛性缺失，有外毛细胞缺失的部位多，以第一排外毛细胞的静纤毛缺失为主．第二和第三排外毛细胞静纤毛多以散在性缺失为主，有内毛细胞缺失的部位多伴有外毛细胞静纤毛的大片状缺失。野生型动物组扫描电镜观察：1个月组4只小鼠5只耳蜗，2个月组2只小鼠4只耳蜗均未观察到内外毛细胞缺失和静纤毛的变化，3月组有少量的外毛细胞缺失。结论野生型小鼠2个月以内耳蜗毛细胞无明显变化．杂合子小鼠随着月龄的增加内外毛细胞缺失的程度逐渐加重。     

单次不同放射线剂量和时间对Balb/c小鼠耳蜗显微和超微结构的影响

目的：观察单次不同放射剂量及时间对Balb／c小鼠耳蜗显微和超微结构的影响,探讨放射线所致感音神经性聋机制的形态学依据。方法：将16只健康Balb／c小鼠随机分成4组（对照组和3个实验组,每组4只）,实验组分别给予8、12、16Gy剂量的放射线照射,在照射后第3、7天处死小鼠,取耳蜗标本后行石蜡包埋、组织切片及苏木精-伊红染色,每组取耳蜗标本行扫描电镜观察。结果：扫描电镜观察发现,对照组内毛细胞及外毛细胞排列整齐,无倒伏、紊乱及缺失;各放射组内毛细胞出现轻微倒伏及排列紊乱,外毛细胞偶有缺失。苏木精-伊红染色结果示各放射组耳蜗内毛细胞、外毛细胞、血管纹细胞及支持细胞在照射后第3、7天与对照组比较未出现明显病理形态学异常。结论：在一次给予≤16Gy放射剂量照射后早期小鼠耳蜗形态仅有轻微改变。     

重组腺病毒Ad-GFP转染新生小鼠离体耳蜗基底膜的实验分析

目的观察重组腺病毒Ad-GFP转染新生小鼠离体耳蜗基底膜培养组织的情况。方法取新生1～5 d小鼠的耳蜗基底膜,离体培养1 d后,加入重组腺病毒Ad-GFP继续培养1 d,在荧光显微镜及Confocal显微镜下观察病毒对离体耳蜗基底膜的转染情况。结果耳蜗基底膜离体培养1 d后,在显微镜下观察,可见基底膜贴壁生长良好,外周有新生上皮细胞和成纤维细胞长出;高倍显微镜下可见耳蜗内外毛细胞和支持细胞等结构。离体耳蜗基底膜培养组织加入重组腺病毒Ad-GFP继续培养1 d后,可见重组腺病毒Ad-GFP能高效转染新生小鼠离体耳蜗基底膜及其外周长出的新生上皮细胞和成纤维细胞;在新生小鼠离体耳蜗基底膜上,不仅大上皮嵴细胞区域、小上皮嵴细胞区域的细胞能被高效转染,毛细胞也能被高效转染。结论重组腺病毒Ad-GFP能高效转染新生小鼠离体耳蜗基底膜培养组织。     

Hush puppy: a new mouse mutant with pinna, ossicle, and inner ear defects

OBJECTIVES/HYPOTHESIS: Deafness can be associated with abnormalities of the pinna, ossicles, and cochlea. The authors studied a newly generated mouse mutant with pinna defects and asked whether these defects are associated with peripheral auditory or facial skeletal abnormalities, or both. Furthermore, the authors investigated where the mutation responsible for these defects was located in the mouse genome. METHODS: The hearing of hush puppy mutants was assessed by Preyer reflex and electrophysiological measurement. The morphological features of their middle and inner ears were investigated by microdissection, paint-filling of the labyrinth, and scanning electron microscopy. Skeletal staining of skulls was performed to assess the craniofacial dimensions. Genome scanning was performed using microsatellite markers to localize the mutation to a chromosomal region. RESULTS: Some hush puppy mutants showed early onset of hearing impairment. They had small, bat-like pinnae and normal malleus but abnormal incus and stapes. Some mutants had asymmetrical defects and showed reduced penetrance of the ear abnormalities. Paint-filling of newborns' inner ears revealed no morphological abnormality, although half of the mice studied were expected to carry the mutation. Reduced numbers of outer hair cells were demonstrated in mutants' cochlea on scanning electron microscopy. Skeletal staining showed that the mutants have significantly shorter snouts and mandibles. Genome scan revealed that the mutation lies on chromosome 8 between markers D8Mit58 and D8Mit289. CONCLUSION: The study results indicate developmental problems of the first and second branchial arches and otocyst as a result of a single gene mutation. Similar defects are found in humans, and hush puppy provides a mouse model for investigation of such defects.     

Distribution of serotonin immunoreactivity in the spiral ganglion neurons of mouse cochlea

OBJECTIVE: Serotonin (5-hydroxytryptamine, 5-HT) is a neuromodulator/neurotransmitter with multiple biological functions. Spiral ganglion cells in the cochlea are the primary neurons of the afferent system in the auditory transmission. In this study, we used the immunohistochemical technique to investigate the distribution of serotonin in the spiral ganglion of mouse cochlea. MATERIALS AND METHODS: The cochlea tissue of four adult mice was dissected and fixed. The immunohistochemical staining was applied by using goat anti-serotonin polyclonal antibody as primary antibody. Tissue sections were treated with biotin-labeled rabbit anti-goat immunoglobulin G, followed by adding streptavidin-biotin-peroxidase complex. Finally, the sections were stained with 3,3-diaminobenzidine (DAB) solution. RESULTS: The spiral ganglion exhibited pronounced immunoreactivity for serotonin. Specifically, serotonin immunoreactivity was detected in the cytoplasma of spiral ganglion neurons located in Rosenthal's canal of the bony modiolus of mouse cochlea. CONCLUSIONS: Since spiral ganglion neurons are the afferent neurons to the auditory sense organ, our result strongly suggests that serotonin molecule may function as a neuromodulator/neurotransmitter in the peripheral auditory processing.     

强噪声暴露后大鼠听觉电生理及形态学改变

目的:观察强噪声暴露后大鼠听觉电生理及形态学的改变,为探讨噪声性聋的发病机制提供实验依据.方法:大鼠随机分为对照组和实验组,实验组暴露于中心频率为4 kHz的窄带噪声中,给声强度为120 dBSPL,暴露时间为4 h.观察2组听觉脑干诱发电位(ABR)及耳蜗形态学的变化.结果:实验组出现ABR反应阈上升,与对照组比较差异有统计学意义(P<0.01);暴露后第1天,基底膜铺片经DNA荧光染料碘化丙锭染色后示;实验组3排外耳毛细胞(OHC)中可出现不同的毛细胞细胞核形态变化(正常、凋亡、坏死和缺失),而第21天未见明显的细胞凋亡;OHC的缺失2组差异有统计学意义(P<0.01),螺旋神经节细胞计数2组差异无统计学意义(P>0.05).扫描电镜示;实验组OHC纤毛异常(散乱、倒伏)及OHC的缺失,以第3排OHC最严重.结论;所应用的强噪声能引起大鼠听觉系统的损害,产生永久性阈移(PTS).该噪声条件下,耳蜗毛细胞的死亡模式在暴露后早期包括凋亡和坏死,而晚期则主要是坏死;PTS的产生可能和OHC纤毛异常及OHC的缺失有关.     

Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea

Objective: This study aimed in fully investigating the toxicities of ouabain to mouse cochlea and the related cellular environment, and providing an optimal animal model system for cell transplantation in the treatment of auditory neuropathy (AN) and sensorineural hearing loss (SNHL).

Methods: Different dosages of ouabain were applied to mouse round window. The auditory brainstem responses and distortion product otoacoustic emissions were used to evaluate the cochlear function. The immunohistochemical staining and cochlea surface preparation were performed to detect the spiral ganglion neurons (SGNs), Schwann cells and hair cells.

Results: Ouabain at the dosages of 0.5?mM, 1?mM and 3?mM selectively and permanently destroyed SGNs and their functions, while leaving the hair cells relatively intact. Ouabain at 3?mM resulted in the most severe SGNs loss and induced significant loss of Schwann cells started as early as 7 days and with further damages at 14 and 30 days after ouabain exposure.

Conclusions: The application of ouabain to mouse round window induces damages of SGNs and Schwann cells in a dose- and time-dependent manner, this study established a reliable and accurate animal model system of AN and SNHL.     

快速老化痴呆小鼠听功能及耳蜗螺旋神经节细胞MeCP2增龄性变化

目的探讨快速老化痴呆小鼠听觉功能、耳蜗螺旋神经节细胞（spiral ganglion cell,SGC）甲基化CpG结合蛋白2（methyl-CpG-binding protein 2,MeCP2）表达的增龄性变化。方法检测5、7月龄快速老化小鼠亚系8（senescence accelerated dementia mouse/prone,SAMP8）和同龄抗快速老化小鼠亚系1（senescenceaccelerated mouse/resistance 1,SAMR 1）8 kHz短纯音听觉脑干反应阈值以及SGC的MeCP2免疫组化染色等方面的增龄性变化。结果①第5、7月龄SAMP8小鼠双耳听觉脑干反应阈值较同龄SAMR1小鼠明显提高（5月龄左侧t=5.84,P〈0.05,右侧t=3.31,P〈0.05;7月龄左侧t=5.11,P〈0.05,右侧t=5.11,P〈0.05）;②MeCP2蛋白在不同月龄快速老化小鼠耳蜗组织中均有表达,第5、7月龄SAMP8小鼠SGC中的MeCP2蛋白较同龄SAMR1小鼠明显降低（5月龄t=2.80,P〈0.05;7月龄t=4.64,P〈0.05）。结论 SGC中MeCP2蛋白的表达水平随着快速老化小鼠听觉功能增龄性减退而降低,说明MeCP2蛋白可能与听觉形成有关。     

Morphological changes in the tectorial and basilar membranes of aged rats

Using both light and transmission electron microscopy presbycusic degeneration of the cochlea was observed in particular in the tectorial and basilar membranes, in naturally aged rats. These animals showed a descending auditory pattern as determined by auditory brainstem response. Ultrastructurally, the number of collagen fibers in the tectorial membrane was reduced and straight type A fibers were increased relative to branched, coiled type B fibers. The basilar membrane in the basal turn was also thickened by an increased homogeneous ground substance. These findings indicate that the specificity of vibration of the tectorial and basal membranes is very different in aged and young rats.     

Scanning electron microscopy of cochlear blood vessels in the mouse

Summary Resin replicas of the microvasculature of the cochlear lateral wall of the mice were studied by scanning electron microscopy. The vascular system of the mouse cochlea showed a similar architecture to that of the guinea pig, cat, and rat. Several thin connecting branches were found between the vessels of the osseous spiral lamina and the cochlear lateral wall at the apical end. The apical turn and the basal part of the lower basal turn were innervated by several radiating arterioles originating from a single stem arteriole. The hook portion and the apical end had a particular vascular meshwork different from other parts of the cochlea.     

Venous drainage through the internal auditory meatus of the guinea pig cochlea

Summary The angioarchitecture of the guinea pig cochlea has been investigated closely using light microscopy and resin injections. However, detailed information concerning the vasculature of the modiolus is still unavailable, and even the existence of venous drainage through the internal auditory meatus is not agreed upon. In the present investigation, vascular casts of guinea pig temporal bones were studied using scanning electron microscopy. A vessel, formed by the confluence of the vascular network on the modiolar wall and having a spiral course into the internal auditory meatus was found in the modiolus of the basal turn. The vessel had a venous pattern on its cast surface and, after exiting from the internal auditory meatus, drained finally into the dural sinus. These scanning electron microscopic findings were confirmable by serial sections of the dural veins in the internal auditory meatus and the modiolus. The vessel found may correspond to the so-called internal auditory vein, but it would be more appropriate to call it the vein of the internal auditory meatus, since it appears to be an independent route of venous drainage from the modiolus.     

Cochlear distribution of Na,K-ATPase and corticosteroid receptors in two mouse strains with congenital hearing disorders.

As corticosteroid hormones, via their receptors, and Na,K-ATPase are thought to be involved in the regulation of endolymph production, two mouse models were used to investigate whether degeneration of the stria vascularis (SV) and disturbed endolymph composition are correlated with changes in the amounts and distribution of corticosteroid receptors and Na,K-ATPase in the cochlea. Both the shaker-2 mouse and the newly discovered mix mouse are deaf at birth and show vestibular dysfunction. In both mouse strains, the SV is degenerated and endolymph production is severely disturbed. In the shaker-2 mouse, using the C57Bl mouse as a normal control, immunohistochemical staining of mineralo- and glucocorticoid receptors (MR and GR) and the Na,K-ATPase subunits alpha1, alpha3 and beta1 showed a weaker reaction in all structures of the cochlea. The inner ear morphology of the mix mouse is described and compared to that of asymptomatic littermates. Immunostaining of MR, GR and the different Na,K-ATPase subunits in this mouse was considerably weaker in the SV, while staining intensities were normal in the remaining cochlea. The reduced corticosteroid receptor levels may lead to a reduction in Na,K-ATPase expression in the same tissues, although this conclusion should be treated with caution. The conclusion that reduced Na,K-ATPase levels in both mouse strains may be an important mechanism of the disturbed endolymph production is less controversial.