Fgf8^(P2A-3×GFP/+):A New Genetic Mouse Model for Specifically Labeling and Sorting Cochlear Inner Hair Cells

The cochlear auditory epithelium contains two types of sound receptors,inner hair cells(IHCs)and outer hair cells(OHCs).Mouse models for labelling juvenile and adult IHCs or OHCs exist;however,labelling for embryonic and perinatal IHCs or OHCs are lacking.Here,we generated a new knock-in Fgf8^(P2A-3×GFP/+)(Fgf8^(GFP)/+)strain,in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements.After confirming that GFP expression accurately reflects the expression of Fgf8,we successfully obtained both embryonic and neonatal IHCs with high purity,highlighting the power of Fgf8^(GFP)/+.Furthermore,our fate-mapping analysis revealed,unexpectedly,that IHCs are also derived from inner ear progenitors expressing Insm1,which is currently regarded as an OHC marker.Thus,besides serving as a highly favorable tool for sorting early IHCs,Fgf8^(GFP)/+will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells:Implications for Tinnitus Generation

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Quantitative analysis of the ribbon synapse number of cochlear inner hair cells in C57BL/6J mice using the three-dimensional modeling method

In mammals,the ribbon synapses of cochlear inner hair cells are a synaptic structure of the first sensory nerve in the pathway of acoustical signal transmission to the acoustic center,and it is directly involved in voice coding and neurotransmitter release. It is difficult to quantitatively analyze the ribbon synaptic number only using an electron microscope,because the ribbon synaptic number is relatively limited and their location is deep. In this study,the specific presynaptic structure-RIBEYE,and non-specific postsynaptic structure-GluR 2 & 3 in C57BL/6J mouse basilar membrane samples were treated by immunofluorescent labeling. Serial section was performed on the samples using a laser scanning confocal microscope,and then the serial sections were used to build three-dimensional models using 3DS MAX software. Each fluorescein color pair indicates one synapse,so the number of ribbon synapses of inner hair cells is obtained. The spatial distribution and the number of ribbon synapses of cochlear inner hair cells were clearly shown in this experiment,and the mean number of ribbon synapses per inner hair cell was 16.10±1.03. Our results have demonstrated the number of ribbon synapses is accurately calculated by double immunofluorescent labeling to presynaptic and postsynaptic structures,serial sections obtained using a laser scanning confocal microscope,and three-dimensional modeling obtained using 3DS MAX software. The method above is feasible and has important significance for further exploring the mechanism of sensorineural deafness.

Forward masking in distinguishing inner and outer hair cell damage

Forward Masking Temporal audiotory resolution is the ability of the auditory system to resolve auditory signals in the time domain. Forward masking is a means of studying temporal resolution where one tone, the probe, is masked by a preceding tone, the masker. Forward masking is

The progenitors of inner ear hair cells and their regulating genes

Hair cells in the mammalian inner ear are very fragile and are often injured as a result of acoustic trauma or exposure to ototoxic drugs (cisplatin, aminoglycosides, etc) [1]. In amphibians and birds, spontaneous

Lack of <i>Rbl</i>1/p107 Effects on Cell Proliferation and Maturation in the Inner Ear

Loss of postnatal mammalian auditory hair cells (HCs) is irreversible. Earlier studies have highlighted the importance of the Retinoblastoma family of proteins (pRBs) (i.e., Rb1, Rbl1/p107, and Rbl2/p130) in the auditory cells’ proliferation and emphasized our lack of information on their specific roles in the auditory system. We have previously demonstrated that lack of Rbl2/p130 moderately affects HCs’ and supporting cells’ (SCs) proliferation. Here, we present evidence supporting multiple roles for Rbl1/p107 inthe developing and mature mouse organ of Corti (OC). Like other pRBs, Rbl1/p107 is expressed in the OC, particularly in the Hensen’s and Deiters’ cells. Moreover, Rbl1/p107 impacts maturation and postmitotic quiescence of HCs and SCs, as evidenced by enhanced numbers of these cells and the presence of dividing cells in the postnatal Rbl1/p107-/-OC. These findings were further supported by microarray and bioinformatics analyses, suggesting downregulation of several bHLH molecules, as well as activation of the Notch/Hes/Hey signaling pathway in homozygous Rbl1/p107 mutant mice. Physiological assessments and detection of ectopic HC marker expression in postnatal spiral ganglion neurons (SGNs) provided evidence for incomplete cell maturation and differentiation in Rbl1/p107﹣/﹣OC. Collectively, the present study highlights an important role for Rbl1/p107 inOC cell differentiation and maturation, which is distinct from other pRBs.

Ephrin A2 protein expression in the regeneration and plasticity of cochlear hair cells in chicken following kanamycin ototoxicity

The results from this study showed that the thresholds of brainstem auditory-evoked potentials peak following 10 successive days of intramuscular injection of Roman chickens with kanamycin, starting 3 days after birth. Fluorescence immunohistochemistry analysis revealed few ganglion cells positively labeled for Ephrin A2 in the cochlea of experimental chickens from 2 days before until 7 days after the last kanamycin injection. The number of Ephrin A2-positive ganglion cell bodies was increased at 15 days after the last injection and was similar to that in normal chickens at 30 days following the cessation of kanamycin treatment. These experimental findings indicate that Ephrin A2 protein expression in the acoustic ganglia is synchronized with the connection damage and regeneration of cochlear hair cells after kanamycin exposure. Ephrin A2 may play an important role in the regeneration and plasticity of cochlear hair cells in the chick cochlea following kanamycin ototoxicity.

斑马鱼tectb内耳基因敲除品系的构建及其作用机制

TECTB基因编码的β-盖膜蛋白(β-tectorin)在人类听觉功能中具有重要的调控作用,是内耳器官中覆盖膜的重要组成成分,在声音转导为神经信号的过程中发挥着关键作用。β-tectorin突变会导致人类非综合征性耳聋的发生,而目前关于听觉障碍发生的分子机制仍然有待研究。斑马鱼(Danio rerio)是一种常用的试验动物模型,能够对内耳发育和相关疾病的研究提供重要的价值。为了研究tectb基因在斑马鱼内耳发育中的作用,本研究利用CRISPR/Cas9基因编辑技术,成功构建tectb内耳基因缺失型斑马鱼模型,并对tectb基因缺失的斑马鱼表型进行初步分析。首先,在tectb基因的第三号外显子上设计靶位点以及基因型检测引物,经体外扩增得到向导DNA(sgDNA),再对其进行体外转录,得到向导RNA(sgRNA);然后,将sgRNA与Cas9酶通过显微注射的方式注射到野生型斑马鱼胚胎中,经基因型鉴定筛选出携带突变的嵌合体F0代鱼,培养至性成熟后与野生型斑马鱼杂交得到F1代突变体杂合子;在遗传稳定性鉴定及基因序列测序之后,保留突变类型为移码突变的F1代鱼,培养至成年后,经过自交得到F2代tectb突变体纯合子;最后,通过体式显微镜初步观察及毛细胞染色试验,发现突变体斑马鱼内耳组织结构正常,耳石形态和大小没有明显差异,毛细胞的发育也没有明显缺陷,但是体内器官与组织的发育是否发生变化,需要进行深入研究。这项研究为深入了解内耳发育的分子机制和耳聋等人类疾病提供了有价值的研究模型,借助tectb基因敲除斑马鱼模型,我们可以探究该基因在疾病发生中的具体作用,更好地研究内耳发育中的关键途径,有利于进一步深入研究听力的调控机制。

The potential of miR-183 family expression in inner ear for regeneration,treatment,diagnosis and prognosis of hearing loss

miRNA-183 family, in normal biology, is expressed in a harmonious and stable manner in the neurosensory organs and cells. Studies have also shown that miRNA-183 family, in different pathways, affects the neurosensory development, maintenance, survival and function. In addition, it has potential neuroprotective effects in response to neurosensory destructive stimulations. miRNA-96 mutation causes hereditary deafness in humans and mice, and therefore affects the inner ear activity and its maintenance. Certain roles have been identified for mi R-96 in the maintenance and function of the inner ear. The comparison of the target genes of family-183 in transcriptomes of newborn and adult hair cells shows that hundreds of target genes in this family may affect development and maintenance of the ears. Identifying the genes that are regulated by miRNA-183 family provides researchers with important information about the complex development and environmental regulation of the inner ear, and can offer new approaches to the maintenance and regeneration of hair cells and auditory nerve.

PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

Studies have shown that phosphatase and tensin homolog deleted on chromosome ten(PTEN)participates in the regulation of cochlear hair cell survival.Bisperoxovanadium protects against neurodegeneration by inhibiting PTEN expression.However,whether bisperoxovanadium can protect against noise-induced hearing loss and the underlying mechanism remains unclear.In this study,we established a mouse model of noise-induced hearing loss by exposure to 105 dB sound for 2 hours.We found that PTEN expression was increased in the organ of Corti,including outer hair cells,inner hair cells,and lateral wall tissues.Intraperitoneal administration of bisperoxovanadium decreased the auditory threshold and the loss of cochlear hair cells and inner hair cell ribbons.In addition,noise exposure decreased p-PI3K and p-Akt levels.Bisperoxovanadium preconditioning or PTEN knockdown upregulated the activity of PI3K-Akt.Bisperoxovanadium also prevented H_(2)O_(2)-induced hair cell death by reducing mitochondrial reactive oxygen species generation in cochlear explants.These findings suggest that bisperoxovanadium reduces noise-induced hearing injury and reduces cochlear hair cell loss.

基于内耳干细胞的毛细胞再生调控研究进展

听力损失的发病率和致残率很高,给社会和经济带来沉重负担。毛细胞的不可逆性损失是感音神经性耳聋的主要原因。明确损伤修复的机制并开发新的防治策略迫在眉睫。利用多种信号和转录因子触发内耳干细胞的激活并再生功能性毛细胞是有希望的治疗手段。本文对诱导退变毛细胞有效功能再生的信号通路和调控策略进行了回顾和总结,并讨论了未来潜在的治疗方法,如小分子药物和基因治疗用于临床再生功能性毛细胞的可行性。

γ-Synuclein在不同年龄段C57小鼠内毛细胞中的差异表达

目的观察不同年龄段C57BL/6J小鼠内毛细胞中γ-Synuclein的表达差异性,探讨其与老年性聋听力损失的可能关系。方法随机选取出生后1月龄、3月龄、6月龄和9月龄C57BL/6 J小鼠各12只,对其进行ABR检测,取其耳蜗基底膜顶、底回进行免疫荧光染色,观察γ-Synuclein的表达差异情况。结果各年龄段C57小鼠ABR32kHz(高频)听力阈值分别为38.75±7.42、45.00±7.39、66.25±9.80、79.58±8.38dB SPL,从6月龄开始32kHz听阈明显升高(P<0.05),9月龄时相比6月龄进一步升高(P<0.05)。各年龄组免疫荧光染色耳蜗顶回γ-Synuclein染色完整未见缺失,底回γ-Synuclein出现选择性表达缺失,各年龄组选择性表达缺失数分别为1.08±1.00、1.25±1.22、2.75±1.91、4.42±2.11个,从6月龄开始γ-Synuclein在底回选择性表达缺失明显增加(P<0.05),9月龄时相比6月龄进一步增加(P<0.05)。结论γ-Synuclein选择性表达缺失增加的变化趋势与ABR测听高频听阈升高的变化趋势相符,提示γ-Synuclein与老年性聋听力损失存在可能关系。

铜绿假单胞菌感染小鼠中耳后对耳蜗及毛细胞损伤的观察分析

目的探讨铜绿假单胞菌(PA)感染小鼠中耳后对内耳及毛细胞的损伤。方法选取出生后6~8周的健康CBA小鼠,分为对照组(PBS种植组)和PA左侧中耳种植组,每组小鼠再分为PBS/PA种植后饲养3天组、7天组、14天组;使用耳内镜、小动物体内成像系统、免疫荧光等方法对小鼠中耳、内耳感染情况及耳蜗毛细胞的损伤情况进行观察分析。结果PA感染中耳后3天、7天、14天中耳腔出现不同程度的黏膜充血水肿,鼓膜穿孔及积脓等化脓性中耳炎的症状;圆窗可见颗粒样物质沉积;小动物体内成像检查可检测到部分耳蜗出现PA感染信号;免疫荧光染色及细胞计数发现,PA感染后7天底转毛细胞计数88.33±66.40,感染后14天小鼠底转、中转毛细胞计数分别为85±33.45,166±33.41,均出现不同程度的缺失,与PBS对照组比较差异显著(P值均小于0.05)。结论PA感染小鼠中耳后细菌及其产物可进入内耳并引起耳蜗毛细胞的损伤;引起毛细胞损伤的可能是PA的慢性毒性。

缺氧对体外培养大鼠耳蜗毛细胞的损伤作用

目的:建立耳蜗器官体外培养模型,观察缺氧对体外培养耳蜗内毛细胞(IHC)及外毛细胞(OHC)的影响。方法:分离生后3d Wistar大鼠耳蜗基底膜,平铺在含有DMEM培养液的培养基内(每盘6条基底膜),2盘为1组,随机分为8组,放置在37℃、5%CO2培养箱培养,其中7组作为实验组,按不同时间段进行缺氧(37℃、90%N2、5%CO2、5%O2)培养;1组作为对照组放置在37℃、5%CO2培养箱。采用Phalloidin荧光标记观察耳蜗中IHC及OHC形态变化并计数单位面积(24 mm×36 mm)细胞数即细胞密度。结果:在缺氧早期(0.5 h)IHC形态及密度无明显变化;1 h细胞轻度肿胀,体积增大,单位面积细胞数减少,细胞密度与对照组比较差异无显著性(P>0.05);2 h时IHC散在缺失,细胞密度与对照组比较差异有显著性(P<0.01);6 h时IHC缺失增多,并可见毛细胞坏死后遗留的“空神经杯”现象,细胞密度与对照组比差异有显著性(P<0.01);随缺氧时间延长,IHC缺失逐渐加重。OHC在缺氧早期(0.5 h、1 h)形态学改变不明显,2 h偶见细胞缺失,随缺氧时间延长,6 h后出现不同程度的排列拥挤、紊乱及缺失,单位面积细胞数目减少,细胞密度与对照组比较差异有显著性(P<0.01),以48 h最严重。结论:缺氧造成体外培养耳蜗毛细胞缺失,以IHC为重。

盖膜与内毛细胞静纤毛相互作用的生物力学分析

目的探讨在Corti器振动过程中盖膜与内毛细胞静纤毛的相互作用关系;进一步阐明感音传导过程中,内耳Corti器微观结构可能的存在状态。方法以豚鼠耳蜗底回Corti器的电镜图像为原型,建立盖膜与内毛细胞最长静纤毛接触和不接触的两种二维计算模型各一个,并作力学分析比较。结果模拟Corti器振动过程,在相同剪切流的压力作用下,静纤毛在不接触盖膜时表现偏移度较大;两种模型均表现在静纤毛顶连接及小根基部应力较高、固有频率近似。结论静纤毛在不与盖膜直接接触时更容易受兴奋性刺激。

外源性谷氨酸对豚鼠耳蜗内、外毛细胞易损性的比较

目的观察外源性谷氨酸对豚鼠耳蜗电位及耳蜗内、外毛细胞的影响。方法将健康豚鼠30只随机分为3组,应用豚鼠全耳蜗灌流技术,分别经耳蜗灌流人工外淋巴液和不同浓度的谷氨酸2小时,记录灌流前和灌流后的耳蜗电位(CM、CAP);同时应用透射电镜技术观察灌流前后耳蜗形态学的变化。结果灌流人工外淋巴液后豚鼠耳蜗电位及形态学无改变;灌流10 mmol/L谷氨酸后CM幅度虽有下降,其非线性特点无改变,CAP阈值平均升高了35 dB;灌流20 mmol/L谷氨酸后CM幅度明显下降但仍保持其非线性特点,CAP阈值平均升高了48 dB,灌流谷氨酸后耳蜗内毛细胞及传入神经纤维出现空化。结论谷氨酸是耳蜗主要的兴奋性传入神经递质,应用外源性谷氨酸可以引起耳蜗内毛细胞及传入神经的损伤,但对耳蜗外毛细胞无影响。

老年性聋耳蜗带状突触数量在时空上的改变

目的研究年龄相关性听力损失过程中听力阈值变化与耳蜗内毛细胞带状突触标记物数量之间的关系。方法应用C57BL/6J小鼠进行本研究。在本研究中,对鼠龄为1、2、4、6和12个月的C57BL/6J小鼠分别进行ABR阈值检测分析(Click&Tone burst)。对被检测小鼠的耳蜗基底膜标本进行突触前特异蛋白RIBEYE/CtBP2进行免疫荧光标记,采用激光共聚焦观察结合三维重建的方法来计数突触标记物(RIBEYE/CtBP2)的数量并进行统计学分析。结果小鼠的ABR阈值从鼠龄4个月时开始出现显著抬高(P<0.05),以后在鼠龄为6和12个月时抬高更加明显。在频率分布上,这种阈值的抬高在高频区域(8KHz&16KHz)表现的最为明显。同时,本研究发现标记物的数量和ABR阈值的变化呈现一致性的改变:突触标记物数量也在4个月时开始出现减少(P<0.05),且随着鼠龄的增加数量减少更加明显。这种数量减少的表现在高频区域(8KHz&16KHz)表现得尤为显著(P<0.01)。在听力减退的早期阶段(鼠龄4-6个月),小鼠毛细胞,尤其是内毛细胞数量并未现明显的减少。结论老年性听力损失和耳蜗带状突触的数量改变密切相关,突触数量的减少可能是导致年龄相关性听力损失的一个重要因素。

模拟载人飞船内微重力和噪声环境下大鼠耳蜗毛细胞的形态学变化

目的:观察模拟载人飞船中微重力和噪声环境下大鼠耳蜗毛细胞的形态学特点,探讨耳蜗三回(底回、中回和顶回)毛细胞的不同变化。方法:选取32只雄性健康SD大鼠,随机分为空白组、失重组、噪声组和失重+噪声组4组,每组8只。失重组以持续尾吊法模拟微重力,噪声组以持续2周的(72±2)d B SPL的稳态噪声及之后的3次高达160 d B SPL的脉冲噪声模拟飞船内复合噪声环境,失重+噪声组同时给予模拟微重力和噪声环境,空白组不做任何处理,常规饲养2周。暴露后检测听性脑干反应(auditory brainstem response,ABR)阈值,处死大鼠即刻取耳蜗基底膜行免疫荧光及扫描电子显微镜(scanning electron microscope,SEM)观察。结果:给予模拟环境暴露后,各组大鼠ABR阈值升高,各实验组大鼠暴露前后ABR阈值差异均有统计学意义(P<0.05)。免疫荧光结果显示:失重组底回以内毛细胞缺失为主,中回可见毛细胞肿胀,顶回毛细胞杂乱不清。噪声组底回以外毛细胞缺失为主,多见于最外层毛细胞,中回毛细胞肿胀,顶回杂乱并有毛细胞的缺失。失重+噪声组底回核缺失较明显,主要存在最外层毛细胞,内毛细胞也存在较多缺失。中回和顶回缺失发生在最内层外毛细胞。电子显微镜观察发现失重组底回纤毛大片倒伏,偶有缺失,顶回和中回毛细胞纤毛无明显异常。噪声组底回纤毛大片倒伏伴缺失,顶回和中回均有缺失,其中顶回最为严重。失重+噪声组底回纤毛大片融合伴缺失,中回纤毛融合、倒伏并缺失,顶回内外毛细胞纤毛大量缺失。4组的损伤程度:失重+噪声组>噪声组>失重组>空白组;三回毛细胞损伤程度:底回>中回>顶回。结论:模拟载人飞船内微重力和噪声环境中暴露2周可致大鼠耳蜗形态学结构发生显著变化,尤以失重+噪声组变化最明显,三回毛细胞中以底回最重,顶回最轻。

豚鼠耳蜗内毛细胞、外毛细胞及外指细胞钙成像

用ｆｌｕｏ－３／ｆｕｒａ－ｒｅｄ荧光比值作细胞内游离钙指标，用激光扫描共聚焦显微镜测定豚鼠耳蜗分离的内、外毛细胞及外指细胞的游离钙，并利用钙成像技术进行细胞形态计量。单个外毛细胞呈试管状。内毛细胞呈烧瓶状，有明显的颈部。外指细胞分为体部及指状突部。毛细胞的胞内游离钙浓度明显高于外指细胞。毛细胞的胞质、胞核及表皮板的游离钙浓度不同。外指细胞的细胞体与指状突的游离钙浓度也不同。本研究认为，有无颈部为区别分离的内、外毛细胞的重要标准，外指细胞可凭借其特异的指状突结构以资识别。内耳毛细胞及外指细胞胞内游离钙分布的不均一性可能与各部位生理功能不同有关。

内毛细胞缺损对噪声引起外毛细胞损害的潜在影响

目的 观察噪声对正常灰鼠和内毛细胞缺损灰鼠的外毛细胞是否具有不同的破坏效应。方法 6只正常灰鼠和 12只卡铂致聋灰鼠作为受试对象。提前 30天给 12只灰鼠按照 10 0mg/kg的剂量腹腔注射卡铂 ,以达到预先选择性破坏其内毛细胞的目的。将 6只正常灰鼠和 6只经卡铂处理的灰鼠移入噪声室 ,在强度为 12 0dB、中心频率在 4kHz的脉冲噪声环境暴露 6小时 ;另外 6只经卡铂处理的灰鼠不经噪声暴露作为对照组。在噪声暴露后 30天取出耳蜗 ,常规制备耳蜗铺片 ,然后进行全耳蜗毛细胞计数 ,并将各组动物的毛细胞计数结果制备成平均受损情况分布图 ,观察和比较噪声引起的外毛细胞损害水平在具有正常内毛细胞的灰鼠和丧失内毛细胞的灰鼠是否存在差别。结果 单纯注射卡铂的灰鼠内毛细胞平均损失在 90 %左右 ,但外毛细胞无损害。经噪声暴露的正常灰鼠外毛细胞在耳蜗底回中部约相当于 4kHz共振频率区域平均损失 6 0 % ,但是在该区域两侧靠近底回和顶回的外毛细胞损失少于 2 0 %。预先经卡铂破坏 95 %内毛细胞的灰鼠经噪声暴露后 ,外毛细胞在耳蜗底回中部平均损失达到 90 % ,在该区域两侧的外毛细胞损失百分比大于 4 0 %。结论 内毛细胞缺损可以造成更多的外毛细胞在噪声刺激中被破坏 ,提示正常内毛细胞的存在可能有?