离体培养时Wnt3a对椭圆囊毛细胞再生的影响

目的 在小鼠椭圆囊体外培养模型上,通过Wnt3a激活经典WNT信号,与DMSO共同作用,研究其对椭圆囊毛细胞再生的影响。方法 40只小鼠随机分成8组。实验第一部分：小鼠椭圆囊经4mmol/L新霉素处理后,在不同浓度(0、25、100、200ng/mL)Wnt3a的培养液中继续培养1d,培养结束后对β-catenin,毛细胞纤毛及细胞核进行免疫荧光染色。实验第二部分：小鼠椭圆囊经4mmol/L新霉素处理后,在含25ng/mL Wnt3a的培养液中培养6d,分别在空白对照组,50μmol/L DAPT、0.1%DMSO、50μmol/L DAPT+25ng/mL Wnt3a培养液中继续培养7d,共培养14d。在所有培养过程中均加入10μmol/L Brdu。培养结束后Brdu,Myocin7a及细胞核进行免疫荧光染色。结果 实验第一部分：在25ng/mL Wnt3a组中可见β-catenin在支持细胞胞浆中有阳性表达,β-catenin阳性细胞数与其他各组比较差异具有统计学意义(P<0.01)。实验第二部分：DMSO组中可见Myosin7a染色阳性细胞,对照组、Wnt3a+DAPT组及DATP组中可见大量Brdu阳性细胞而未见Myosin7a染色阳性细胞,DMSO组中Myosin7a阳性细胞数与其他3组相比较,组间差异具有显著性(P<0.01)。结论 顺序联合应用Wnt3a和DMSO可通过激活经典WNT信号途径,促使椭圆囊内细胞向毛细胞样细胞转分化。     

毛细胞再生研究进展[综述]

感音神经性聋是耳科临床常见疾病,对患者的生活质量和经济负担均有较大影响,目前尚无有效的治疗方法。临床上仅能通过助听器、人工耳蜗植入等人工听觉技术帮助患者恢复部分听觉功能。研究表明哺乳动物可以在某些条件下实现耳蜗毛细胞一定程度的再生,本文就内耳毛细胞再生的研究进展做一综述。     

miRNAs与内耳发育和听觉毛细胞凋亡与再生的研究进展

MicroRNAs(miRNAs)是一种小的、非编码蛋白的RNA,它通过转录后机制的调控参与细胞的分化、增殖、凋亡和代谢等多种生物过程。近年来,许多研究发现miRNAs在脊椎动物内耳的发育、改善听觉毛细胞的凋亡以及促进毛细胞的再生中也发挥了重要作用,miRNAs的出现为听力障碍的机制研究与治疗提供了新的方法。     

顺铂诱导斑马鱼侧线毛细胞损伤及再生模型的建立

目的建立顺铂诱导斑马鱼侧线毛细胞损伤及再生模型。方法采用顺铂溶液直接孵育斑马鱼方法,通过免疫组化、荧光特异性标记侧线细胞的转基因鱼活体成像、原位杂交等方法统计分析顺铂处理前后斑马鱼侧线毛细胞剩余情况以及药物撤除后毛细胞再生情况。结果顺铂引起斑马鱼侧线毛细胞丢失具有剂量依赖效应,相同的孵育时间较高浓度的顺铂孵育后几乎可杀死全部侧线毛细胞;毛细胞再生与顺铂的毒性积累程度有关,较低浓度孵育较短时间毛细胞再生速度较快。毛细胞再生数目随着时间的延长不断增加,72小时后可再生原有数目的90%以上。结论斑马鱼侧线毛细胞经不同浓度顺铂孵育不同时间后,其毛细胞丢失与再生具有剂量和时间依赖效应。     

碱性成纤维细胞生长因子对豚鼠椭圆囊毛细胞再生的影响

目的 利用豚鼠椭圆囊培养的方法观察碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对毛细胞再生的影响。方法 应用新霉素破坏豚鼠离体椭圆囊毛细胞,实验组培养液含有bFGF,对照组培养液则不含有bFGF。培养18d后两组均行扫描电镜检查,并行新生毛细胞计数,比较两组新生毛细胞数目的差异,观察bFGF对椭圆囊毛细胞再生的影响。结果 实验组新生毛细胞数目明显多于对照组(P<0.05)。结论bFGF对椭圆囊毛细胞的再生有促进作用。     

Ganglion cell loss continues during hair cell regeneration

Hair cells and ganglion cells were counted in young adult quail (Coturnix coturnix) after acoustic trauma at 10, 30, 60 and 90 day survival times. Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 mu intervals from basal to apical tip. Hair cells and ganglion cells were counted from 3 mu thick sections at each interval. Hair cells were designated as tall or short within the area 30-70% of length from basal tip of the papilla. Both tall and short hair cells were significantly reduced in number 10 days following trauma. Tall hair cells recovered to within 96% of normal after 60-90 days. Short hair cells recovered but to a lesser extent. Ganglion cell loss did not begin until 30 days after trauma and continued without recovery 90 days after trauma. A good correlation was found for position of both types of hair cell loss and position of ganglion cell loss. These results suggest that the initial loss of hair cells, both tall and short, results in retrograde degeneration of neural fibers and ganglion cells.     

Central nervous system plasticity during hair cell loss and regeneration

Following cochlear ablation, auditory neurons in the central nervous system (CNS) undergo alterations in morphology and function, including neuronal cell death. The trigger for these CNS changes is the abrupt cessation of afferent input via eighth nerve fiber activity. Gentamicin can cause ototoxic damage to cochlear hair cells responsible for high frequency hearing, which seems likely to cause a frequency-specific loss of input into the CNS. In birds, these hair cells can regenerate, presumably restoring input into the CNS. This review summarizes current knowledge of how CNS auditory neurons respond to this transient, frequency-specific loss of cochlear function. A single systemic injection of a high dose of gentamicin results in the complete loss of high frequency hair cells by 5 days, followed by the regeneration of new hair cells. Both hair cell-specific functional measures and estimates of CNS afferent activity suggest that newly regenerated hair cells restore afferent input to brainstem auditory neurons. Frequency-specific neuronal cell death and shrinkage occur following gentamicin damage to hair cells, with an unexpected recovery of neuronal cell number at longer survival times. A newly-developed method for topical, unilateral gentamicin application will allow future studies to compare neuronal changes within a given animal.     

Apical hair cells and hearing

This study assessed the contribution of the apical hair cells to hearing. Guinea pigs, chinchillas and monkeys were behaviorally trained using positive reinforcement to respond to pure-tone stimuli. When a stable audiogram had been determined, each subject received one of three experimental treatments: ototoxic drug administration, low-frequency noise exposure, or the application of a cryoprobe to the bony wall of the cochlear apex. After post-treatment audiograms stabilized, subjects were euthanized and the percentage of hair cells remaining was assessed by light microscopy. Results indicate that a redundancy of encoding mechanisms exist in the mammalian cochlea for low-frequency stimuli. They also suggest that a very small percentage of apical hair cells are sufficient for some low-frequency hearing. Finally, data from this and other studies suggest that the low-frequency threshold shift caused by the loss of a certain percentage of apical hair cells is less pronounced than the high-frequency threshold shift caused by the loss of a comparable percentage of basal hair cells. These data agree with anatomical and electrophysiological evidence that functional as well as anatomical differences may exist between the apex and base of the cochlea.     

Salicylate-induced morphological changes of isolated inner hair cells and outer hair cells from guinea-pig cochlea

Objectives

The aim of this study is to investigate the salicylate-induced morphological changes of cochlear inner hair cells (IHCs) and outer hair cells (OHCs).

Methods

IHCs and OHCs were acutely isolated from the guinea-pig cochlea. Cells were observed under the inverted microscope and 10 mmol/L sodium salicylate solutions or 0.01 mmol/L dexamethasone-plus-salicylate solutions were applied. The cell length or the ratio between the length and width was the indices of the morphological changes in cells.

Results

Isolated IHCs did not demonstrate any significant changes in sodium salicylate solutions in 20 min and in 40 min, whereas OHCs were shortened by the 10 mmol/L sodium salicylate to 83% in 20 min and 75% in 40 min. There were no significant differences between in the dexamethasone-plus-salicylate solutions and in the control solutions after 20 min and 40 min both in IHCs and OHCs.

Conclusions

Although salicylate affected the isolated OHCs from guinea-pig cochlea, IHCs were not changed morphologically by sodium salicylate applications. Dexamethasone inhibited the salicylate-induced morphological changes of OHCs.     

Triggers of hair cell regeneration in the avian inner ear

We performed an in vitro study in order to determine possible triggers of hair cell regeneration in the chick basilar papilla following degeneration. We compared the response of sensory epithelium damaged by collagenase treatment with that damaged by acoustic trauma. The former exhibited no proliferative activity, but the latter did. The basilar papillae damaged by acoustic trauma could have proliferating activity in medium containing fetal bovine serum (FBS) or epidermal growth factor (EGF) but not in the medium without FBS or EGF. These findings indicate that regeneration of basilar papillae depends on the manner of cell death and that FBS or EGF is required for regeneration.     

Streptomycin ototoxicity and hair cell regeneration in the adult pigeon utricle

OBJECTIVE: The purpose of this study was to develop a technique to investigate the regeneration of utricular hair cells in the adult pigeon (Columba livia) following complete hair cell loss through administration of streptomycin. STUDY DESIGN: Experimental animal study. METHODS: Animals were divided into four groups. Group 1 received 10 to 15 days of systemic streptomycin injections. Animals in Groups 2 and 3 received a single direct placement of a 1-, 2-, 4-, or 8-mg streptomycin dose into the perilymphatic space. Animals in Groups 1 and 2 were analyzed within 1 week from injection to investigate hair cell destruction, whereas Group 3 was investigated at later dates to study hair cell recovery. Group 4 animals received a control injection of saline into the perilymphatic space. Damage and recovery were quantified by counting hair cells in isolated utricles using scanning electron microscopy. RESULTS: Although systemic injections failed to reliably achieve complete utricular hair cell destruction, a single direct placement of a 2-, 4-, or 8-mg streptomycin dose caused complete destruction within the first week. Incomplete hair cell loss was observed with the 1-mg dose. Over the long term, regeneration of the hair cells was seen with the 2-mg dose but not the 8-mg dose. Control injections of saline into the perilymphatic space caused no measurable hair cell loss. CONCLUSIONS: Direct placement of streptomycin into the perilymph is an effective, reliable method for complete destruction of utricular hair cells while preserving the regenerative potential of the neuroepithelium.     

骨形态发生蛋白4在内耳发育及在毛细胞与螺旋神经节细胞再生中的研究进展

听力损失是人类最常见的感觉缺陷,主要涉及到毛细胞(HCs)和螺旋神经节细胞(SGCs)的损失等。在内耳发育过程中,骨形态发生蛋白4(BMP4)的表达具有时序性和特异性,并且通过调控分泌蛋白Tsukushin(TSK)、性别决定相关基因簇2蛋白(SOX2)等细胞因子,与WNT和SHH等信号通路之间相互作用,参与耳泡的诱导、前庭和耳蜗等器官的形成、HCs和SGCs等细胞的分化过程。此外,近些年在哺乳动物与非哺乳动物的耳蜗外植体中,发现BMP4在HCs和SGCs的再生中起重要作用。综述BMP4调控内耳发育、诱导HCs和SGCs再生的作用,以及相关的研究进展,以期为HCs和SGCs再生相关机制的阐明奠定基础,为听力损失的治疗带来新的思路与策略。     

Harvesting human hair cells

The sensory hair cells of the inner ear are responsible for converting balance and hearing stimuli into electrical signals. Until recently, all previous studies of hair cell physiology had been performed on tissue obtained from non-mammals and rodents. In primates, hair cells are difficult to access, because they rest within the densest structure of the body, the otic capsule of the temporal bone. In this report, we describe a technique that we have used in physiological studies to harvest living human hair cells. We collected vestibular and cochlear tissue specimens from adult humans undergoing translabyrinthine and transotic surgical approaches for resection of lateral skull base tumors. Viable hair cells were identified and visualized with light microscopy. The ability to study normal hair cells from humans may further the study of normal and pathological human sensation, hair cell regeneration, and genetic causes of balance and hearing disorders.     

Cytodifferentiation of cochlear hair cells

Cytodifferentiation of a limited number of cochlear hair cells in the mouse starts on the 14th gestational day. One day later, the number of identified hair cells increases considerably. Cytodifferentiation apparently occurs in a gradient from the hair cell surface to the base. First, the irregular microvilli covering the future hair cell surfaces begin to show a regular pattern but are of the same thickness and length as microvilli on supporting cells. Second, a polarization of sensory hairs occurs with a stepwise increase in stereocilia length in the different rows toward the kinocilium. Finally, the cuticle is formed, giving an anchorage for sensory hair rootlets in the hair cell. At birth some hair cells can be found with immature surface morphologic features, e.g., stereocilia of the same length on the entire hair cell surface and lack of a cuticular plate. The onset of hair cell differentiation takes place without morphologic contact with ingrowing nerve fibers.     

Distribution and time course of hair cell regeneration in the pigeon utricle.

Vestibular and cochlear regeneration following ototoxic insult from aminoglycoside antibiotics has been well documented, particularly in birds. In the present study, intraotic application of a 2 mg streptomycin paste was used to achieve complete vestibular hair cell destruction in pigeons (Columba livia) while preserving regenerative ability. Scanning electron microscopy was used to quantify hair cell density longitudinally during regeneration in three different utricular macula locations, including the striola, central and peripheral regions. The utricular epithelium was void of stereocilia (indicating hair cell loss) at 4 days after intraotic treatment with streptomycin. At 2 weeks the stereocilia began to appear randomly and mostly in an immature form. However, when present most kinocilia were polarized toward the developing striola. Initially, regeneration occurred more rapidly in the central and peripheral regions of the utricle as compared to the striola. As regeneration proceeded from 2 to 12 weeks, hair cell density in the striola region equaled the density noted in the central and peripheral regions. At 24 weeks, hair cell density of the central and peripheral regions was equal to normal values, however the striola region had a slightly greater hair cell density than that observed for normal animals.