大鼠椭圆囊再生毛细胞前体细胞来源的初步研究

目的 探讨哺乳动物椭圆囊再生毛细胞前体细胞的可能来源.方法 取出生1天的大鼠的椭圆囊,经嗜热菌蛋白酶(thermolysin)处理,消化法进行原代培养.在倒置显微镜下观察椭圆囊感觉上皮细胞(utricular sensory epichelial cell,USEC)的形态、生长特征;透射电镜观察、免疫细胞化学法检测上皮细胞角蛋白18、波形蛋白等鉴定USEC上皮细胞来源.免疫细胞化学法、RT-PCR技术检测支持细胞标记物p27k1plmRNA及毛细胞的特征性标记物Brn3a、Calretinin及AchRa9、Myosin Ⅶ a mRNA的表达.结果 原代培养的USEC呈扁平、多角形、核大而圆的上皮细胞形态,细胞之间连接紧密,形成单层时呈"铺路石样"外观.可见由数百个USEC包绕液体而成的dome(穹窿样)结构,表达细胞角蛋白,不表达波形蛋白,微绒毛丰富,细胞间连接紧密,提示其上皮来源.原代培养的USEC表达支持细胞标记物p27k1pl mRNA及毛细胞的特征性标志物Brn3a、Calretinin及AchRa9、MyosinⅦa mRNA,表明培养的USEC可能来源于支持细胞并具有毛细胞的特性.结论 原代培养的USEC能产生毛细胞样细胞且表达支持细胞标记物,表明其来源为支持细胞.椭圆囊感觉上皮的支持细胞可能为毛细胞再生的前体细胞之一.     

前庭诱发肌源性电位临床研究现状与展望

 前庭诱发肌源性电位与前庭功能的关系逐步被发现并应用于临床检查,并在一部分前庭功能障碍疾病,如梅尼埃病、良性阵发性位置性眩晕、前庭神经炎等病中发挥了协助诊断及评估病情的作用。本文主要概述前庭诱发肌源性电位的历史、原理、方法及其临床应用。     

Acoustic Trauma Increases Ribbon Number and Size in Outer Hair Cells of the Mouse Cochlea

Outer hair cells (OHCs) in the mouse cochlea are contacted by up to three type II afferent boutons. On average, only half of these are postsynaptic to presynaptic ribbons. Mice of both sexes were subjected to acoustic trauma that produced a threshold shift of 44.2 ± 9.1 dB 7 days after exposure. Ribbon synapses of OHCs were quantified in post-trauma and littermate controls using immunolabeling of CtBP2. Visualization with virtual reality was used to determine 3-D cytoplasmic localization of CtBP2 puncta to the synaptic pole of OHCs. Acoustic trauma was associated with a statistically significant increase in the number of synaptic ribbons per OHC. Serial section TEM was carried out on similarly treated mice. This also showed a significant increase in the number of ribbons in post-trauma OHCs, as well as a significant increase in ribbon volume compared to ribbons in control OHCs. An increase in OHC ribbon synapses after acoustic trauma is a novel observation that has implications for OHC:type II afferent signaling. A mathematical model showed that the observed increase in OHC ribbons considered alone could produce a significant increase in action potentials among type II afferent neurons during strong acoustic stimulation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10162-020-00777-w.     

Age-Related Blood Flow and Capillary Changes in the Rat Utricular Macula: A Quantitative Stereological and Microsphere Study

Vascular change may contribute to age-related vestibular dysfunction. Previously, we reported a significant age-related decrease in blood flow (BF) and mean capillary diameter (D(cap)) in the rat posterior canal crista. The purpose of this study was to examine an otolith organ, the utricle, for similar changes. Old male Fischer 344 rats (O; 28-31 mos) were anesthetized, and the left cardiac ventricle was transcutaneously injected with radioactive microspheres to determine BF. The temporal bones were removed, fixed, and decalcified. The utricles were dissected free and placed into a gamma counter with the reference samples. The specimens were then plastic embedded and serially sectioned at 1 microm according to the vertical section technique. Microsphere surface counts were made and neuroepithelial BF calculated. A systematic random set of sections was sampled and analyzed using stereological techniques for estimates of D(cap), capillary surface area/unit volume (S(v,cap)), capillary length/ unit volume (L(v,cap)), and volume of utricular neuroepithelium (V(ut)). Using these data, total capillary surface (S(cap)) and total length (L(cap)) were calculated. Statistical comparisons were made with data from our previous study of young animals (Y; 3-6 mos). Results indicate a significant age-related decrease in BF (Y = 0.125 microL/min, O = 0.062 microL/min; P = 0.003), D(cap) (Y = 5.95 micro, O = 4.57 microm; P = 0.0002), S(vcap) (Y = 12.33 mm2/mm3, = 9.87 mm2/mm3, P = 0.016), S(cap) (Y = 0.178 mm2, O = 0.129 mm2; p = 0.01), and V(ut) (Y = 0.014 mm3, O = 0.013 mm3; P = 0.04) with no significant change in L(v,cap) (Y = 655 mm/mm3, O = 686 mm/mm3, P = 0.41) or L(cap) (Y = 9.47 mm, O = 8.96 mm; P = 0.49). These age-related vascular changes are likely to have a significant impact on utricular physiological and thus, dysequilibrium.     

PD-L1及CD8^+TIL在前庭神经鞘膜瘤中的表达及意义

目的探讨程序性死亡蛋白配体1(PD-L1)及CD8+肿瘤浸润性淋巴细胞(Tumor infiltrating lymphocyte,TIL)在散发性前庭神经鞘膜瘤(vestibular schwannoma,VS)中的表达,为前庭神经鞘膜瘤免疫靶向治疗提供理论和实验基础。方法回顾性收集2018年11月至2019年4月于上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科手术治疗的15例散发性VS患者的临床资料及病理蜡块,采用免疫组化法检测15例散发性前庭神经鞘膜瘤手术切除组织中PD-L1及CD8^+淋巴细胞表达情况,分析它们之间相关性及与临床病理特征的关系。结果显微镜下观察15例散发性VS组织中有14例(93.3%)PD-L1表达阳性,其中7例(46.7%)为高表达。在15例(100%)样本中均观察到CD8^+淋巴细胞的阳性表达,其表达量与PD-L1表达量呈正相关(P=0.02,r=0.82)。结论在散发性VS中存在PD-L1广泛表达,PD-L1表达与CD8^+TIL表达呈正相关,该肿瘤可能对免疫检查点抑制剂的免疫治疗有反应,在未来临床试验中可进一步探索。     

DAPT对离体培养大鼠基底膜毛细胞发育的影响

目的研究将γ-分泌酶抑制剂(DAPT)与新生大鼠基底膜共培养,观察其对毛细胞发育和分化的影响。方法采用新生P0大鼠基底膜中圈及顶圈贴壁培养,随机分为正常培养组、DAPT组,按培养时间又分为4天、7天和9天组;其中DAPT为γ-分泌酶抑制剂,溶解于终浓度低于0.1%的DMSO,每日换液均加入DAPT。采用扫描电镜观察基底膜毛细胞数量变化。结果正常组顶圈呈1排内毛细胞,2-3排外毛细胞;中圈呈1排内毛细胞,3排外毛细胞,内外毛细胞排列规则,随着培养天数的增加数量无明显变化。DAPT组顶圈呈1排内毛细胞,4-7排外毛细胞;中圈呈1排内毛细胞,4-5排外毛细胞。结论 DAPT能够诱导基底膜顶圈产生大量额外的外毛细胞     

Antioxidant and Anti-Apoptotic Effect of Melatonin on the Vestibular Hair Cells of Rat Utricles

Objectives

Aminoglycosides are commonly used antibiotic agents, and they are known to generate free oxygen radicals within the inner ear and to cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Melatonin, a pineal secretory product, has the properties of being a powerful direct and indirect antioxidant. The aim of the present study was to prove the antioxidant effect of melatonin against gentamicin-induced ototoxicty.

Methods

The utricular maculae of Sprague-Dawley rats were prepared from postnatal day 2-4, and these maculae were were divided into 6 groups as follows: 1) control, 2) melatonin only, 3) gentamicin only, and 4), 5), and 6) gentamicin plus melatonin (10, 50, and 100 µM, respectively). To count the number of hair cells, 5 utricles from each group were stained with phalloidin-FITC on the 1st, 4th, and 7th days after drug administration. Reactive oxygen species (ROS) was assessed by using the fluorescent probe hydrofluorescent diacetate acetyl ester. The caspase-3 activity was also examined with using the fluorescent caspase-3 substrate and performing Western blotting.

Results

The result of this study showed that gentamicin induced the loss of utricular hair cells, and this loss of hair cells was significantly attenuated by co-administration of melatonin. Melatonin reduced ROS production and caspase-3 activation in the gentamicin treated utricular hair cells.

Conclusion

Our findings conclusively reveal that melatonin has protective effects against gentamicin-induced hair cell loss in the utricles of rat by inhibiting both ROS production and caspase-3 activity.     

Changes in Conformation of Stereocilia in Hair Cells of the Rat Spiral Organ of Corti after 6-Hydroxydopamine as Revealed by Scanning Electron Microscopy

Newborn rats were chemically sympathectomized by injection of 6-hydroxydopamine every day from day 1 to day 7, then every 5th day from day 10 until they were sacrificed. Litter mats left untreated or injected with the vehicle only (asorbic acid or physiological saline) were used as controls. Animals were sacrificed on days 15, 22, 30 and 34 by intracardiac perfusion with glutaraldehyde-paraformaldehyde in Millonig buffer, and their cochlease were dissected and prepared for scanning electron microscopy

Stereocilia of outer and of inner hair cells were generally upright and not clumped in the 15-day-old treated rats. At 22 days, some of the outer hair cells were clumped and the tips of the hairs showed a tendency to stick to shreds of the tectorial membrane. Hairs of the inner hair cells were elongated and often in disarray. At 30 days, the changes in conformation of the hairs of both inner and outer hair cells had progresses, especially apically. Hairs of the outer hair cells were clumped, sometimes missing, and frequently bent over as though lacking stiffness. Tips of the hair were often firmly attached to shreds of the tectorial membrane. Hairs of the inner hair cells were thread-like and in disarray

The only known effect of 6-hydroxydopamine under the experimental condition used here is the destruction of the peripheral adrenergic nervous system. Thus, present findings are interpreted to indicate that loss of sympathetic fibers to the internal ear results in changes in fluid and/or ion balance, perhaps due to an interference with protein metabolism, secondarily causing abnormalities in stereocilia. It is of interest that present results mimic in some respects the early effects of ototoxic drugs     

Fgf22在神经系统中的研究进展及在耳蜗带状突触中的作用

成纤维细胞生长因子22(Fibroblast growth factor 22, Fgf22)是成纤维细胞生长因子家族(Fibroblast growth factor family, Fgfs)的成员之一。Fgf22与哺乳动物胚胎期大脑发育、中枢神经系统兴奋性突触的形成、维持与调控等都有着密切联系,最近研究发现Fgf22与听觉系统耳蜗带状突触密切相关。因此,充分认知Fgf22在神经系统中的研究进展及其在听觉系统耳蜗带状突触中的意义,将为进一步深入探讨相关遗传性疾病的分子机制研究提供重要的理论依据,也将为发现潜在耳聋新基因提供新思路。     

渗透浓度变化和电刺激诱发离体耳蜗外毛细胞运动的初步观察

观察细胞外环境改变对外毛细胞功能的影响，对揭示神经性耳聋的机理很有意义。用显微机械法分离豚鼠耳蜗外毛细胞，观察到细胞外环境渗透浓度降低（２８０Ｏｓｍｍｏｌ／Ｌ）１ｍｉｎ即可诱发离体外毛细胞短缩３．１４±０．７０％（ｔ检验，以下同，Ｐ＜０．０１），伴直径增加。外环境渗透浓度升高（３２０Ｏｓｍｍｏｌ／Ｌ）５ｍｉｎ可使离体外毛细胞伸长３．３４±１．０９％（Ｐ＜０．０５），伴直径缩小；交变电场（刺激电流Ｉ＜０．１ｍＡ，刺激频率ｆ＝１～１０Ｈｚ）的变化可使外毛细胞产生伸缩运动，在Ｋ￣＋１００ｍｍｏｌ／Ｌ，渗透浓度３２０Ｏｓｍｍｏｌ／Ｌ环境下外毛细胞随交变电场变化而产生的运动幅度，负相时减少４３．１４％（Ｐ＜０．０５），正相时减少４６．１７％（Ｐ＜０．０５）。外环境恢复正常后外毛细胞形状亦可恢复。讨论了外毛细胞的运动机制和生物学基础。     

Phosphorylation of MYL12 by Myosin Light Chain Kinase Regulates Cellular Shape Changes in Cochlear Hair Cells

Journal of the Association for Research in Otolaryngology - The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape...     

SD大鼠损伤Corti器中毛细胞样细胞的演变

目的：研究氨基糖类抗生素引起哺乳类动物Corti器损伤及损伤后的修复情况。方法：用新霉素皮下注射致新生SD大鼠Corti器损伤，然后用扫描电镜观察停药后不同时期的Corti器毛细胞的损伤及修复表现。结果：在外毛细胞损伤区有细胞表面长有微绒毛簇的毛细胞样细胞出现，且其在损伤Croti器中有发生-发展-退化的变化过程。结论：毛细胞样细胞的出现是受损伤Corti器的一种修复形式。     

毒蕈碱受体在大鼠前庭神经核中表达的免疫组织化学与原位杂交研究

目的探讨毒蕈碱受体(muscarinic receptor ,M受体)在大鼠前庭神经核中(vestibular nucleus )的表达.方法以免疫组织化学及原位杂交组织化学的方法对M受体M1、M3、M4亚型在大鼠前庭神经核中的表达进行研究.结果免疫组织化学研究表明M1、M3、M4在前庭神经核中均有表达;原位杂交组织化学也证实了M3 mRNA在前庭神经核中的表达.结论本实验首次表明在大鼠前庭神经核中存在着多种M受体亚型,但它们在前庭神经核中如何发挥功能性作用还需要作进一步研究.     

c-Maf基因在小鼠耳蜗内外毛细胞中的表达及意义

目的研究c-Maf基因在小鼠耳蜗内、外毛细胞中的表达、分布特点及意义。方法 1)根据形态上的差异分离收集成年小鼠内、外毛细胞,利用Microarray基因芯片技术分别检测内、外毛细胞c-Maf基因RNA水平的表达;2)全耳蜗基底膜铺片,免疫组织化学染色后激光共聚焦显微镜观察c-Maf蛋白在不同发育阶段小鼠耳蜗内、外毛细胞中的分布特点及表达,并使用软件Image Pro Plus进行c-Maf蛋白定量分析。结果 1)耳蜗内、外毛细胞c-Maf基因RNA水平相近;2)c-Maf蛋白主要表达于成年小鼠的耳蜗内外毛细胞胞质,在内毛细胞的核下区表达强于核上区,且在内、外毛细胞的表达强度相近;3)c-Maf蛋白在小鼠出生后0天、7天、12天及成年小鼠耳蜗内、外毛细胞稳定表达。结论 c-Maf蛋白从出生开始在小鼠耳蜗内、外毛细胞上稳定表达,且在成年小鼠内毛细胞的核下区表达较强,表明其可能在毛细胞生长发育以及功能维持中发挥重要的作用。     

Neurod1与内耳感觉神经及毛细胞发育的相关研究

本文主要介绍了Neurod1在内耳感觉神经元及毛细胞发育中的相关研究。Neurod1是b HLH转录因子家族中的重要一员,它能促进感觉神经元前体细胞分化为成熟感觉神经元细胞。在内耳中,Neurod1主要表达于感觉神经元细胞,部分感觉上皮也有表达。Neurod1基因条件性敲除小鼠可出现听力及平衡障碍,其内耳感觉神经元细胞大量缺失,同时内耳中的传入及传出神经也存在缺陷。Neurod1基因敲除小鼠螺旋神经节中还可出现异位毛细胞的生长。在内耳发育过程中,Neurod1基因与多种基因相互调控,协同促进内耳发育成熟。如Neurog1、Atoh1、Sox2、Nhlh1等。     

Preliminary Characterization of Voltage-Activated Whole-Cell Currents in Developing Human Vestibular Hair Cells and Calyx Afferent Terminals

We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11–14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15–18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G _K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a “collapsing” tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15–18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.     

中等强度带通噪声对C57小鼠内毛细胞形态与功能的影响

目的探讨C57小鼠在8-16kHz,98dB SPL,2h的噪声暴露前后听力以及耳蜗内的病理改变。方法 1.以98dB SPL,8-16kHz的带通噪声对C57小鼠噪声暴露2小时。2.分别在噪声暴露前、噪声暴露后第一天及第十五天进行听觉脑干反应测试(Auditory Brainstem Response, ABR)以观测噪声造成的损伤及恢复。3.将非噪声小鼠设为对照组,噪声暴露后第15天的小鼠设为实验组,取对照组与实验组小鼠耳蜗基底膜进行免疫荧光染色,对内外毛细胞以及内毛细胞突触前结构Ribbon进行计数。4.取对照组与实验组小鼠耳蜗基底膜,对8-11kHz频率区域的内毛细胞进行全细胞膜片钳记录,比较两组内毛细胞钙电流大小以及突触囊泡的释放能力。结果噪声暴露后第十五天C57小鼠中高频阈值没有完全恢复。在22kHz,实验组小鼠ABR I波波幅显著下降,潜伏期显著延长。免疫荧光染色发现噪声后外毛细胞在高频区域有部分丢失,突触前Ribbon在中圈存在部分丢失,在底圈显著丢失。膜片钳记录结果显示实验组小鼠内毛细胞钙电流幅值无明显改变。长时程去极化刺激时,实验组小鼠内毛细胞ΔCm显著低于对照组。结论 8-16kHz,98dB SPL噪声暴露使C57小鼠产生轻度永久性阈移,噪声后基底膜受损区域主要集中在中高频区。相对于外毛细胞来说,噪声后内毛细胞突触结构与功能损伤的频率范围更加广泛。     

Changes in Cytochemistry of Sensory and Nonsensory Cells in Gentamicin-Treated Cochleas

Effects of a single local dose of gentamicin upon sensory and nonsensory cells throughout the cochlea were assessed by changes in immunostaining patterns for a broad array of functionally important proteins. Cytochemical changes in hair cells, spiral ganglion cells, and cells of the stria vascularis, spiral ligament, and spiral limbus were found beginning 4 days post administration. The extent of changes in immunostaining varied with survival time and with cell type and was not always commensurate with the degree to which individual cell types accumulated gentamicin. Outer hair cells, types I and II fibrocytes of the spiral ligament, and fibrocytes in the spiral limbus showed marked decreases in immunostaining for a number of constituents. In contrast, inner hair cells, type III fibrocytes and root cells of the spiral ligament, cells of the stria vascularis, and interdental cells in the spiral limbus showed less dramatic decreases, and in some cases they showed increases in immunostaining. Results indicate that, in addition to damaging sensory cells, local application of gentamicin results in widespread and disparate disruptions of a variety of cochlear cell types. Only in the case of ganglion cells was it apparent that the changes in nonsensory cells were secondary to loss or damage of hair cells. These results indicate that malfunction of the ear following gentamicin treatment is widespread and far more complex than simple loss of sensory elements. The results have implications for efforts directed toward detecting, preventing, and treating toxic effects of aminoglycosides upon the inner ear.     

高强度低频噪声对豚鼠听力及耳蜗毛细胞的影响

目的观察高强度低频噪声对听力及耳蜗的影响。方法听力正常豚鼠,体重在250~300g之间,经强度为130dBSPL中心频率在100Hz的窄带噪声持续爆震4小时,分别于即刻、震后1天,作脑干诱发电位检测,以及耳蜗形态学观察。结果经130dBSPL强噪声暴露后,豚鼠听力有20dB左右的暂时性阈移产生。1天后,听力有所恢复,但听阈仍然高于正常。耳蜗形态学观察到,强噪声暴露后,耳蜗毛细胞虽未有损伤的表现,但凋亡前期蛋白Caspase3已经出现。结论高强度的低频噪声能产生暂时性阈移和永久性阈移,但可部分恢复。噪声对耳蜗毛细胞短期虽未观察有明确的损伤,但却开启了凋亡的程序。     

谷氨酸／天冬氨酸转运体抗体对豚鼠听性脑干反应及毛细胞形态的影响

目的 研究谷氨酸/天冬氨酸转运体(glutamate--aspartate transporter,GLAST)抗体对豚鼠耳蜗听性脑干反应(ABR)和耳蜗毛细胞形态的影响.方法 健康豚鼠20只随机分为实验组和对照组,每组10只.实验组耳蜗鼓阶内灌注GLAST抗体,对照组灌注人工外淋巴液,观察两组术后3、6、9天ABR反应阈、耳蜗基底膜铺片和透射电镜的形态学改变.结果 实验组术后第3天ABR波形消失,术后第9天无恢复;对照组术后第3天8只动物ABR波形消失,术后第6天和第9天全部动物引出ABR波形,平均阈值分别为62.50±5.25、47.50±6.18dB SPL,差异有统计学意义(P<0.05).随着GLAST抗体灌注后时间延长,实验组内、外毛细胞及纤毛出现不同程度缺失,透射电镜显示内、外毛细胞及神经末梢胞浆、线粒体空化,细胞核染色质边集等凋亡早期征象.对照组的损伤较轻,与ABR阈值改变相一致.结论 耳蜗内GLAST抗体灌注后出现耳蜗毛细胞、神经末梢的损伤及ABR波形消失,提示GLAST抗体阻断耳蜗Corti器中的GLAST,导致谷氨酸的神经毒性表达.