新霉素对大鼠前庭的损伤及复制缺陷型腺病毒前庭导入的研究

目的 研究新霉素对大鼠前庭毛细胞的毒性作用并探索前庭基因导入的方法和途径,为药物性前庭功能损伤动物模型的制备和基因治疗的相关研究提供参考.方法 将25只成年Wistar大鼠分为正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、腺病毒(缺失E1、E3基因且构建有绿色荧光蛋白报告基因的复制缺陷型腺病毒,Ad-EGFP)鼓阶导入组、腺病毒(Ad-EGFP)前庭阶导入组,每组各5只动物.新霉素组大鼠在右耳通过耳蜗底回鼓阶(鼓阶组)或前庭阶打孔(前庭组)的方法在耳蜗内注入0.1%新霉素5 μl,Ad-EGFP组则以同法导入物理滴度为2.1×1011vp/ml的Ad-EGFP 5 μl,正常对照组大鼠不做任何处理.处理3天后对动物进行颈髓硬膜外短声诱发电位(click- evoked potentials on the surface of the cervical dura mater,CDM-CEP)检查和游泳试验,评价前庭功能,然后将动物处死,进行组织学和形态学观察.结果 3天后新霉素组大鼠的前庭毛细胞即出现严重破坏,并出现严重的前庭功能损伤.正常对照组、新霉素鼓阶给药组、新霉素前庭阶给药组、Ad-EGFP鼓阶导入组、Ad-EGFP前庭阶导入组的游泳时间分别为4.0±0.71、10.2±1.64、9.8±1.48、4.8±0.84、5.0±0.71 s;正常对照组CDM-CEP的阈值为85±3.54 dB SPL,潜伏期为6.59±0.41 s;新霉素组120 dB SPL未引出CDM-CEP;Ad-EGFP鼓阶导入组CDM-CEP阈值为91±5.48 dB SPL,潜伏期为6.76±0.26 s,Ad-EGFP新霉素前庭阶导入组CDM-CEP的阈值为89±6.52 dB SPL,潜伏期为6.78±0.26 s.3天后,Ad-EGFP前庭阶导入组的前庭终末器官均出现Ad-EGFP的转染;而Ad-EGFP鼓阶导入组未见前庭终末器官的表达.结论 新霉素对前庭毛细胞具有极强的破坏作用,新霉素耳蜗内注射的方法可以制备理想的前庭功能障碍动物模型,耳蜗底回前庭阶打孔可以作为基因导入前庭的有效途径.     

碱性成纤维细胞生长因子对前庭毛细胞的保护作用

目的 观察碱性成纤维细胞生长因子对新霉素所致离体椭圆囊毛细胞损害的保护作用。方法 采用离体的豚鼠椭圆囊培养法,对照组为普通前庭培养液培养,实验组按培养液中含和不含ｂＦＧＦ分组,用新霉素造成各实验组２的椭圆囊毛细胞损伤,各组椭圆均行扫描电镜检查并行毛细胞计数。结果 含ｂＦＧＦ的实验组毛细胞的存活数目显著高于不含ｂＦＧＦ的实验组,对照组无缺失或损伤。结论新霉不对离体的椭圆囊毛细胞的有罗强的破坏作用,ｂ     

硫酸新霉素对新生大鼠离体培养耳蜗毛细胞的毒性作用研究

目的观察体外培养的新生大鼠耳蜗Corti器经不同浓度硫酸新霉素处理后,各回毛细胞损伤情况。方法取新生大鼠完整耳蜗基底膜,体外培养12小时,存活贴壁后加药处理。对照组10个样本;3个实验组各10个样本,分别用终浓度为1mmol/L、2mmol/L、4mmol/L硫酸新霉素处理24小时。进行MyosinⅦa免疫荧光组织化学染色,在共聚焦显微镜下观察各回毛细胞缺失情况。分别选取10个图片进行毛细胞计数（每张图片截取100μm计数毛细胞个数）,并利用CHISS软件进行统计分析。对照组除不加入硫酸新霉素外,其他培养条件均与实验组相同。结果硫酸新霉素对离体培养的新生大鼠耳蜗毛细胞具有损伤效应,且外毛细胞对硫酸新霉素的毒性作用比内毛细胞敏感。加入硫酸新霉素后,耳蜗毛细胞损失从底回外毛细胞开始,随着药物浓度增加,逐渐累及到中回,最后顶回受累,且损失程度随着浓度的递增而加重,以外毛细胞为甚。单纯体外培养的耳蜗毛细胞没有缺失。经完全随机设计方差分析可得：顶回正常组内毛细胞数量与新霉素1mmol/L组、2mmol/L组无统计学差异,与新霉素4mmol/L组有统计学差异;中、底回正常组内毛细胞数量与新霉素1mmol/L组、新霉素2mmol/L组、新霉素4mmol/L组均有统计学差异;顶回正常组外毛细胞数量与新霉素2mmol/L与4mmol/L组有统计学差异;中、底回正常组外毛细胞数量与新霉素1mmol/L组、新霉素2mmol/L组、新霉素4mmol/L组均有统计学差异。结论新生大鼠体外培养的耳蜗毛细胞随着硫酸新霉素药物浓度的增加损失越严重,且从底回向顶回发展,此与活体动物实验的损害规律基本相同,因此可作为耳毒性药物损伤后毛细胞再生研究的体外模型。     

前庭毛细胞分离法探讨

取豚鼠球囊、椭圆囊及壶腹嵴，置于含胶原酶的Ｈａｎｋ＇ｓ液中消化，再辅以机械分离方法获得单个离体Ｉ型和Ⅱ型前庭毛细胞。Ｉ型细胞为烧瓶状，有明显颈部、皮板及纤毛。Ⅱ型细胞较Ｉ型细胞为短，无显著颈部，但有皮板及纤毛。Ｉ型细胞与Ⅱ细胞数量之比为１２：１。离体５小时后仍有３５％的前庭毛细胞具有活性。观察结果表明，应用胶原酶能获得较多的具有活性的单个离体前庭毛细胞，为前庭毛细胞，为前庭的重、病理及药理实验研究     

新霉素致发育中大鼠毛细胞损伤的电镜观察

目的应用扫描电镜研究氨基甙类抗生素导致出生后发育过程中大鼠耳蜗毛细胞的损伤病理变化。方法７天龄ＳＤ大鼠肌肉注射８０ｍｇｋｇ－１ｄ－１新霉素连续８天,停药后７天处死动物制备样本进行扫描电镜观察。结果新霉素可造成出生后发育过程中的耳蜗毛细胞严重损伤,表现为底回和钩回三排外毛细胞全部损伤缺失,损伤病变累及顶回的外毛细胞,底回和钩回的内毛细胞出现纤毛脱落和细胞损伤丢失;外毛细胞损伤缺失的部位由顶部具有微绒毛的多边形细胞取代,该细胞形态与胚胎发育早期的毛细胞相似;毛细胞损伤区域未发现再生的毛细胞。结论出生后发育过程中大鼠耳蜗毛细胞对氨基甙类抗生素的敏感性较高,表面有微绒毛的多边形细胞在毛细胞损伤区域出现提示听觉感觉上皮细胞有再生的倾向。     

硫酸链霉素对体外培养大鼠前庭毛细胞的损害作用

目的 探讨硫酸链霉素对离体培养条件下大鼠前庭毛细胞的损害特征.方法 取出生后3～4 d的大鼠球囊斑和椭圆囊斑进行离体前庭器官培养,经培养过夜后再用不同浓度的硫酸链霉素培养液继续培养24 h.应用荧光标记的鬼笔环肽对前庭毛细胞的静纤毛和表皮板进行染色,同时应用Topro-3 DNA探针显示细胞核的形态,在激光共聚焦显微镜下计数和摄像.结果 在离体培养条件下,对照组前庭毛细胞生长良好,随着培养液中硫酸链霉素浓度的增加,毛细胞的密度逐渐降低.培养24 h后,0.25 mmol/L硫酸链霉素可造成大约10%的前庭毛细胞缺损,1 mmol/L硫酸链霉素损害的毛细胞数量在50%左右,3 mmol/L硫酸链霉素破坏的毛细胞数量超过75%.细胞核染色显示硫酸链霉索引起的前庭毛细胞损害伴随着细胞核浓缩或破碎现象.存活毛细胞的数量随着硫酸链霉素浓度的增加而减少,但前庭的支持细胞未发生明显病变.结论 在体外培养条件下,硫酸链霉素可引起剂量依赖性的大鼠前庭毛细胞缺失,其损伤机制可能与凋亡有关.     

豚鼠单个离体前庭毛细胞的光镜观察

分离豚鼠前庭器感觉毛细胞，并使其在体外保持特有的形态和活性，用倒置显微镜进行观察。结果显示，细胞在形态和大小上有较大的差异。说明哺乳动物的前庭毛细胞仅分为Ｉ型和Ⅱ型是不够的。笔者认为，将哺乳动物的前庭毛细胞分成４型，似在细胞形态学上更具代表性。     

庆大霉素致豚鼠前庭毛细胞破坏后再生及功能恢复

为探讨哺乳动物前庭毛细胞破坏后能否再生及前庭功能能否恢复，以豚鼠为研究对象。实验组动物每日给予庆大霉素皮下注射，连续１０天，对照组动物给予等量生理盐水。于停药后的第２、４、１２和２４周分别处死动物，扫描电镜观察发现，停药２周椭圆囊和壶腹嵴毛细胞破坏最明显的区域可见到不成熟的毛细胞纤毛丛，至停药２４周后继续存在。与对照组相比，停药１２周毛细胞密度下降明显（Ｐ〈０．０１），停药后２４周时毛细胞密度较１     

实验动物前庭感觉毛细胞的定量观察

目的 测量常用实验动物内耳前庭感觉区的实际面积和量化分析前庭各个感觉区的毛细胞总数或密度。方法 ①制作CBA/CaJ小鼠、裸鼠、SD大鼠、豚鼠、南美栗鼠、新西兰白兔和非洲黑长尾猴的球囊斑铺片和椭圆囊斑铺片及壶腹嵴铺片,所有铺片样品来自每种受试动物的6个颞骨,在放大100倍的光学显微镜下拍摄2个囊斑铺片的整体照片;②应用Image J软件的图像测量程序,测量了上述7种常用实验动物球囊斑和椭圆囊斑的实际面积;③用网格将球囊斑铺片和椭圆囊斑铺片照片上的前庭感觉区划分为一个个方块区域。在放大400倍的光学显微镜下准确计数每个方格内的毛细胞数量,然后将每个方格的毛细胞计数结果相加以获得每种受试动物球囊斑和椭圆囊斑上的毛细胞总数;④应用前庭小视野定量观察技术计算出前庭各个感觉区小视野范围内的毛细胞密度。结果 ①从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑面积依次为(0.193±0.009)、(0.216±0.008)、(0.323±0.010)、(0.528±0.035)、(0.687±0.065)、(1.237±0.075)、(1.371±0.032)mm²;椭圆囊斑的面积依次为(0.193±0.020)、(0.208±0.013)、(0.321±0.011)、(0.526±0.034)、(0.795±0.017)、(1.224±0.082)、(1.388±0.048)mm²;②从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑毛细胞的总数依次为(2476.3±64.4)、(2389.8±47.8)、(3135.3±191.6)、(4882.2±208.7)、(6128.5±242.9)、(10572.2±464.4)、(10992.7±397.4)个;椭圆囊斑毛细胞的总数依次为(2491.4±54.8)、(2368.0±46.1)、(3218.8±82.9)、(4925.3±271.1)、(7794.0±386.1)、(11347.4±435.7)、(11114.5±410.6)个;③从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的球囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.007mm²)依次为101.0±5.79(微纹区)/120.8±4.15(周边区),95.5±3.91(微纹区)/109.2±5.26(周边区),78.4±6.54(微纹区)/94.8±4.38(周边区),60.0±4.74(微纹区)/84.6±2.61(周边区),57.2±3.83(微纹区)/80.0±3.54(周边区),53.8±4.21(微纹区)/68.0±4.18(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的椭圆囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.007mm²)依次为103.8±5.02(微纹区)/119.2±3.70(周边区),91.2±2.49(微纹区)/106.4±4.16(周边区),74.1±3.54(微纹区)/90.8±3.56(周边区),60.4±4.98(微纹区)/81.6±2.07(周边区),57.8±1.92(微纹区)/77.8±3.70(周边区),54.0±2.74(微纹区)/66.4±2.51(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的壶腹嵴毛细胞密度(毛细胞数量/0.007mm²)依次为112.4±6.38,105.5±3.51,95.2±3.42,84.0±7.16,78.2±2.86,70.8±2.39。可见由于体型较小动物毛细胞的细胞体比体型较大动物毛细胞的细胞体小,因而体型较小动物的前庭毛细胞密度高于体型较大动物的前庭毛细胞密度。另外,每种实验动物球囊斑和椭圆囊斑微纹区的毛细胞密度相似,周边区的毛细胞密度也大致相同,但是同种实验动物囊斑微纹区的毛细胞密度却低于周边区的毛细胞密度。此外,壶腹嵴毛细胞的密度与球囊斑和椭圆囊斑周边区的毛细胞密度几乎相同。鉴于某些损害因素往往具有选择性破坏囊斑微纹区毛细胞的表现,因此囊斑微纹区的毛细胞密度应该与囊斑周边区的毛细胞密度区分开来进行统计,必要时甚至需要把Ⅰ型毛细胞和Ⅱ型毛细胞也区分开来分别予以病理学改变的定量评估。结论 本研究采用的前庭测量方法和获得的前庭各个感觉区的测量数据和毛细胞总数及毛细胞密度,为前庭病理学研究的定量分析提供了有益的参考经验和必要的参考数据。     

The minimum peptides of IGF-1 and substance P protect vestibular hair cells against neomycin ototoxicity

Conclusions: Our data indicate that SSSR and SSSR + FGLM-NH2 protect sensory hair cells against neomycin-induced death in the vestibular epithelium. In addition, the results show that SSSR and FGLM-NH2 can be used as protective molecules against aminoglycoside ototoxicity. Objectives: This study investigated the role of the peptides SSSR and SSSR + FGLM-NH2 in mammalian vestibular hair cell death induced by aminoglycoside. Methods: Cultured utricles from mature CBA/N mice were used in this study. The cultured utricles were assigned to five groups (control group, neomycin group, neomycin + SSSR group, neomycin + FGLM-NH2 group, and neomycin + SSSR + FGLM-NH2 group). Aat 24 h after exposure to neomycin, the hair cells were labeled immunohistochemically, and the rate of survival of vestibular hair cells was evaluated using a fluorescence microscope. Results: The rate of survival of vestibular hair cells was significantly higher in the neomycin + SSSR and neomycin + SSSR + FGLM-NH2 groups than in the neomycin group. The results suggest that SSSR could protect hair cells against aminoglycoside ototoxicity.     

Type I hair cell regeneration induced by Math1 gene transfer following neomycin ototoxicity in rat vestibular sensory epithelium

Conclusions. Antibiotic treatment does not absolutely prevent the development of otogenic intracranial complications (ICC); however, their incidence is relatively low (0.36%).Various pathogens can be isolated in cultures of patients with these complications, but combinations of third- or fourth-generation cephalosporins with chloramphenicol, vancomycin, metronidazole or aminoglycosides can provide good results. Underlying cholesteatoma is common and is usually associated with intracranial abscess or sinus thrombosis. High morbidity rates warrant long-term follow-up. Objective. To evaluate the cause and nature of otogenic ICC in patients treated at 1 medical center over an 18-year period. Material and methods. This was a retrospective chart review of 28 patients admitted to Sheba Medical Center, Israel with otogenic ICC between 1984 and 2002. Results. Meningitis was the commonest complication (46.4%), followed by brain abscess, epidural abscess, sigmoid sinus thrombosis, subdural empyema, perisinus abscess and transverse and cavernous sinus thrombosis. Twelve patients (42.9%) had received antibiotic treatment prior to admission. Chronic otitis media, cholesteatoma and brain abscess were diagnosed mainly in adults, while acute otitis media and epidural abscess were more frequent in children. Twenty-one patients underwent mastoidectomy to eradicate the source of infection. The commonest finding at surgery was granulations (81%). Cholesteatoma was seen in 38.1% of cases. Cholesteatoma and brain abscess were usually associated with Gram-negative bacterial infection. Meningitis, however, was caused by Streptococcus pneumoniae in 40% of cases. CT showed a sensitivity of 92.75% for diagnosing otogenic ICC. There was no mortality. The morbidity rate was high (71.4%) and included hearing impairment, hemiparesis, hydrocephalus, mental retardation, polyneuropathy and epilepsy.     

Canalicular reticulum in vestibular hair cells

A membrane limited system referred to as canalicular reticulum (CR) has been demonstrated in the apical cytosol of the cochlea's inner and outer hair cells. Similarities between cochlear and vestibular hair cells prompted investigation of the presence of CR in hair cells of the gerbil vestibular labyrinth. A method of fixation with glutaraldehyde followed by an osmium-ferrocyanide mixture demonstrated abundant CR in the apex of both type I and type II hair cells. The CR was closely associated with numerous Golgi zones in the apex of the vestibular hair cells, indicating its genesis from Golgi cisternae. Also preserved in upper cytosol were discrete complexes of mitochondria with granular reticulum. These complexes offered a possible site for generating the membrane in Golgi zones and CR. Single and parallel cisternae of granular reticulum were observed in the basal half of the hair cells together with numerous synaptic-like vesicles. These cisternae with their terminal blebbing and accompanying canaliculi were interpreted as novel structures mediating synaptic vesicle genesis in vestibular hair cells in a manner comparable to that postulated for cochlear inner hair cells.     

Quantitative analysis of stereociliary arrays on vestibular hair cells

We have developed a method for quantifying the number, spacing, and distribution of stereocilia on the apical surface of hair cells using spatial autocorrelation analysis and statistics for directional data. Here, we illustrate the method using idealized hair bundles, and we apply it to scanning micrographs of turtle hair cells from the utricle and posterior canal, and to freeze-fracture preparations of bullfrog saccule. The analysis suggests three common features of stereociliary bundles. First, bundle geometries form a continuum from ‘loose’ to ‘tight’ (Acta Otolaryngol. 106 (1988) 393) rather than two distinct groups. Second, interciliary spacing along the three hexagon axes is not equal; spacing is usually widest along the hexagon axis closest to the bundle’s axis of bilateral symmetry (the presumptive excitatory axis). Third, spacing between stereocilia changes with distance from the kinocilium. All three features will influence predictions of the tip link tensions that accompany bundle deflection.     

鼠尾胶原在毛细胞膜片钳实验中的应用

目的　探讨应用鼠尾胶原在豚鼠前庭毛细胞膜片钳实验中的促进毛细胞贴壁效果。方法　制作鼠尾胶原 ,观察全细胞膜片钳实验中 ,自制的鼠尾胶原对前庭毛细胞的贴壁黏附作用。结果　无鼠尾胶原时 ,前庭毛细胞悬浮于外液 ,不宜封接 ;有鼠尾胶原时 ,前庭毛细胞贴附于培养皿底壁 ,易于封接和长时间 (约 8h)的观察和记录。鼠尾胶原对前庭毛细胞具有良好的贴壁黏附促进作用。结论　鼠尾胶原能促进毛细胞贴壁 ,涂布鼠尾胶原有利于膜片钳实验的长时程记录 ;并且制作简便 ,成本低廉     

新霉素损伤后小鼠耳蜗毛细胞的改变[论著]

目的　观察新霉素对小鼠听功能及耳蜗毛细胞形态学变化的影响。方法　选取C57BL/6小鼠16只在出生后第8天开始连续10 d皮下注射硫酸新霉素,对照组（10只）皮下注射等量生理盐水。停药后1、4、8、12 w进行听性脑干反应（ABR）检测。每次ABR检测后,新霉素组随机取3只小鼠,对照组随机取2只小鼠,处死后,取耳蜗进行冰冻切片,用免疫荧光方法观察耳蜗Corti器及毛细形态学变化。结果　新霉素可造成小鼠耳蜗毛细胞严重损伤,且小鼠ARB阈值显著增高,不可恢复;新霉素对耳蜗Corti器毛细胞的损伤顶圈最轻,中圈次之,底圈最重,且随着时间推移Corti器形态结构破坏逐渐加重且不可自我修复。结论　硫酸新霉素造成小鼠Corit器毛细胞的损伤是不可逆的。     

Fodrin immunocytochemical localization in the striated organelles of the rat vestibular hair cells.

The immunocytochemical distribution of a spectrin-related protein, fodrin was studied at the electron microscopic level in the rat vestibular hair cells. As previously demonstrated [Scarfone et al., Neurosci. Lett. 93, 13-18, 1988], an intense immunoreactivity was found in the cuticular plates. We demonstrate furthermore, here, for the first time the association of fodrin immunoreactivity with the striated infracuticular structures called striated organelles (SO). Fodrin was found in striated structures clearly identified as SO in both Type I and Type II hair cells. SO were labelled regardless of their location, subcuticular or associated with the plasma membrane of the cells. We suggest that fodrin, as in the cuticular plate, could participate to the Ca2+ dependent cross-linking of the actin filaments of the striated organelles and could play a role in their interaction with the submembraneous cytoskeleton.     

Application of rat tail collagen in patch clamp experiment with vestibular hair cells]

OBJECTIVE: To study the feasibility of application of rat-tail collagen in patch clamp research in vestibular hair cells. METHODS: The effect of self-made rat-tail collagen on promoting adhesion of vestibular hair cells in whole cell patch clamp experiment was observed. RESULTS: A seal was hard to be formed when the vestibular hair cells suspended among the external solution without rat-tail collagen. However, when the vestibular hair cells were firmly adhesive to the bottom of the recording chamber with rat-tail collagen, a seal can be formed easily, which fitted to the long-term observation and recording. The effect of rat-tail collagen on adhesive to vestibular hair cells is obvious. CONCLUSION: Rat-tail collagen could facilitate the vestibular hair cells to adhesive with the bottom of the recording chamber, which is helpful for long-term patch clamp research, and the collagen is considered as an optimal adhesive reagent to vestibular hair cells for patch clamp research.     

Ocsyn and mitochondrial-canalicular complexes in vestibular hair cells

Ocsyn, a syntaxin-interacting protein characterized by Safieddine et al. [Safieddine, S., Ly, C.D., Wang, Y.-X., Kachar, B., Petralia, R.S., Wenthold, R.J., 2002. Ocsyn, a novel syntaxin-interacting protein enriched in the subapical region of inner hair cells. Mol. Cell. Neurosci., 20, 343-353] in the guinea pig organ of Corti was primarily identified in organelles located at the subapical region of inner hair cells. They proposed that in cochlear inner hair cells, ocsyn was involved in protein trafficking associated to recycling endosomes. Ocsyn happens to be highly homologous to syntabulin with an almost identical syntaxin-binding domain. Syntabulin is believed to attach syntaxin-containing vesicles to kinesin for their axonal transport along microtubules. The present study shows the distribution of ocsyn in guinea pig and rat vestibular hair cells using immunocytochemistry and confocal microscopy. Ocsyn was characterized by intense immunolabeled spots distributed exclusively in type I and II vestibular hair cells. The subcuticular region under the cuticular plate exhibited particularly densely packed spots. In the neck region of the sensory cells, where microtubules are abundant, there was no colocalization of ocsyn and alpha-tubulin. Ocsyn labeled spots were also present in the medial and basal hair cell regions, particularly in the supranuclear and infranuclear regions. Mitochondria are particularly numerous in these three regions (subcuticular, supranuclear and infranuclear). Double labeling of ocsyn and cytochrome c showed that ocsyn was present in the same zones that mitochondria. This, together with the great similarity of ocsyn and syntabulin, suggest that, akin to syntabulin, ocsyn is involved in addressing organelles. We propose that ocsyn is involved in the formation of the canalicular-mitochondrial complexes depicted by Spicer et al. [Spicer, S.S., Thomopoulos, G.N., Schulte, B.A., 1999. Novel membranous structures in apical and basal compartments of inner hear cells. J. Comp. Neurol., 409, 424-437].