Inner ear barriers to nanomedicine-augmented drug delivery and imaging

There are several challenges to inner ear drug delivery and imaging due to the existence of tight biological barriers to the target structure and the dense bone surrounding it. Advances in imaging and nanomedicine may provide knowledge for overcoming the existing limitations to both the diagnosis and treatment of inner ear diseases. Novel techniques have improved the efficacy of drug delivery and targeting to the inner ear, as well as the quality and accuracy of imaging this structure. In this review, we will describe the pathways and biological barriers of the inner ear regarding drug delivery, the beneficial applications and limitations of the imaging techniques available for inner ear research, the behavior of engineered nanomaterials in inner ear applications, and future perspectives for nanomedicine-based inner ear imaging.

Expression ofα2-Na/K-ATPase in C57BL/6J Mice Inner Ear and Its Relationship with Age-related Hearing Loss

K^(+)cycling in the cochlea is critical to maintain hearing.Many sodium-potassium pumps are proved to participate in K^(+)cycling,such as Na/K-ATPase.Theα2-Na/K-ATPase is an important isoform of Na/K-ATPase.The expression ofα2-Na/K-ATPase in the cochlea is not clear.In this study,we used C57BL/6 mice as a model of presbycusis and implemented immunohistochemistry staining and quantitative real time-PCR,and theα2-Na/K-ATPase expression pattern was confirmed in the inner ear.It was foundα2-Na/K-ATPase was expressed widely in cochlea and its mRNA and protein expression was gradually reduced with aging(4-,14-,26-and 48-weeks old mice).We suspected that,the down-regulation ofα2-Na/K-ATPase expression might be associated with the remodeling of K^(+)cycling,degeneration of morphological structure and decrease of hearing function in aging C57 mice.In conclusion,we speculated that the reduction ofα2-Na/K-ATPase might play an important role in the pathogenesis of age-related hearing loss.

Glutamate receptor antagonist and neurotrophin can protect inner ear against damage

In this study,I focused on finding a mean of protecting against hearing loss.By infusing the cochlea with the neurotrophin factor,NT-3 alone or combined treatment with MK 801,a NMDA receptor antagonist I found hearing loss was attenuated and spiral ganglion neuron loss was nearly totally protected indicating that the importance of the combined treatment of NT-3 and NMDA receptor antagonists in the treatment of hearing disorders.

Reaction-Diffusion Algorithm for Quantitative Analysis of Periodic V-Shaped Bundles of Hair Cells in the Inner Ear

Columnar outer hair cells of the inner ear form V-shaped bundles, which are arranged in three rows in the cochlea. If the formation of these V-shaped bundles is disturbed, sensorineural hearing loss occurs. To quantify the distribution of V-shaped bundles, the use of a Turing-type reaction-diffusion (RD) system, including anisotropic diffusion, should be considered. We found that a periodic triangle pattern appears based on the anisotropy in the RD system. Then, using the proposed RD system, the image of the V-shaped bundle was examined. The results showed that the correlation between the RD pattern and V-shape bundles not only classifies the experimental result but also significantly differs in the number of directional changes in a set of V-shape bundle. Therefore, this process can help quantify the images of V-shaped bundles.

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

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.

A child with severe inner ear malformations with favorable hearing utilization and balance functions after wearing hearing aids

Infants with congenital deafness caused by severe bilateral inner ear malformations frequently suffer from severe hearing loss and poor balance. Unfortunately, the use of hearing aids is usually ineffective in recovering hearing, necessitating cochlear implants. We report a case of a 6-year-old boy with congenital deafness and bilateral inner ear malformations(right side, incomplete partition type I [IP-I]; left side, common cavity deformity). Hearing aids had a remarkable effect in this patient, enabling sufficient and favorable hearing recovery such as to allow the patient to engage in daily conversations. Per-rotatory nystagmus was recorded on an electronystagmogram for both right and left rotations in a damped rotational chair test. It is rare for deaf children with severe bilateral inner ear malformation to demonstrate favorable development in hearing and good equilibrium function. Our findings suggest that auditoryevestibular hair cells in this patient may have been partially preserved despite IP-I in the right ear and common cavity deformity of the left ear.

New prospects in the diagnosis and treatment of immune-mediated inner ear disease

Autoimmune inner ear disease(AIED) represents a very fertile research field and the advancements in the understanding of this disease have a direct application not only in patients affected with this condition but also in other inner ear disorders that share the same injury mechanism, damage to the inner ear hair cells. AIED also presents many challenges that have still to be overcome. Firstly, access to the inner ear is limited, as many interventions such as biopsies can result in great irreversible damage. Secondly, there are no completely specific markers for AIED. Lack of a definitive diagnosis can result in the treatment of patients not affected with the disease and, therefore, no response. Finally, some patients become refractory to glucocorticoids and new therapies are needed. This review offers an overview of the animal models that have contributed to the understanding of AIED pathophysiology, the value of currently available diagnostic tests, and therapeutic options, with a special focus on new therapies for non responders or patients refractory to glucocorticoids. Among these new options for therapy, biological agents have been tested recently, whereas gene and stem celltherapy may have a role in the future. The intratympanic route of administration avoids the systemic side effects associated with currently used drugs, and may become a more frequent approach in the future.

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.

Genetically modified pigs:Emerging animal models for hereditary hearing loss

Hereditary hearing loss(HHL),a genetic disorder that impairs auditory function,significantly affects quality of life and incurs substantial economic losses for society.To investigate the underlying causes of HHL and evaluate therapeutic outcomes,appropriate animal models are necessary.Pigs have been extensively used as valuable large animal models in biomedical research.In this review,we highlight the advantages of pig models in terms of ear anatomy,inner ear morphology,and electrophysiological characteristics,as well as recent advancements in the development of distinct genetically modified porcine models of hearing loss.Additionally,we discuss the prospects,challenges,and recommendations regarding the use pig models in HHL research.Overall,this review provides insights and perspectives for future studies on HHL using porcine models.

Current strategies for drug delivery to the inner ear

For many years,drug delivery to the inner ear has been a challenge to physicians in the treatment of inner ear disorders.In the past decade,the field of inner ear drug delivery has emerged with the development of new biomaterials and drug delivery technologies to improve the effectiveness of inner ear drug therapy.This paper reviews a number of inner ear drug delivery strategies including systemic,intratympanic,and intracochlear delivery.A focus of this review is the recent advances in intratympanic delivery of medications;approaches utilizing novel biomaterials as well as other recent developments are also discussed.Biotechnology-based approaches,such as gene and stem cell therapy methods are also reviewed.Among the various strategies,local drug delivery approaches including intratympanic and intracochlear drug delivery methods that limit systemic exposure are particularly promising.These inner ear drug delivery systems provide a new opportunity to improve the treatment of inner ear disorders.

Unidirectional and stage-dependent roles of Notch1 in Wnt-responsive Lgr5+ cells during mouse inner ear development

Wnt and Notch signaling play crucial roles in the determination of the prosensory domain and in the differentiation of hair cells(HCs)and supporting cells during mouse inner ear development;however,the relationship between the two signaling pathways in the mouse cochlea remains largely unknown.Here,we investigated the interactions between Notch and Wnt signaling on the basis of the bidirectional regulation of Notch1 specifically in Wnt-responsive Lgr5+progenitors during different cochlear development stages.We found that the downregulation of Notch1 in Lgr5+cells from embryonic day(E)14.5 to E18.5 can drive the quiescent Lgr5+cells to re-enter the cell cycle and differentiate into extra HCs,whereas the upregulation of Notch1 expression did not affect the proliferation or differentiation of otic progenitor cells.No effect was observed on the upregulation or downregulation of Notch1 in Lgr5+cells from E10.5 to E14.5.We concluded that the roles of Notch1 in Wnt-responsive Lgr5+cells are unidirectional and stage dependent and Notch1 serves as a negative regulator for Lgr5+progenitor activation during cochlear differentiation.Our findings improved the understanding of the interactions between Notch and Wnt signaling in cochlear development.

Phosphatidylinositol 4-kinase β mutations cause nonsyndromic sensorineural deafness and inner ear malformation

Congenital hearing loss is a common disorder worldwide.Heterogeneous gene variation accounts for approximately 20-25%of such patients.We investigated a five-generation Chinese family with autosomaldominant nonsyndromic sensorineural hearing loss(SNHL).No wave was detected in the pure-tone audiometry,and the auditory brainstem response was absent in all patients.Computed tomography of the patients,as well as of two sporadic SNHL cases,showed bilateral inner ear anomaly,cochlear maldevelopment,absence of the osseous spiral lamina,and an enlarged vestibular aqueduct.Such findings were absent in nonaffected persons.We used linkage analysis and exome sequencing and uncovered a heterozygous missense mutation in the PI4 KB gene(p.Gln121 Arg)encoding phosphatidylinositol 4-kinaseβ(PI4 KB)from the patients in this family.In addition,3 missense PI4 KB(p.Val434 Gly,p.Glu667 Lys,and p.Met739 Arg)mutations were identified in five patients with nonsyndromic SNHL from 57 sporadic cases.No such mutations were present within 600 Chinese controls,the 1000 genome project,gnom AD,or similar databases.Depleting pi4 kb m RNA expression in zebrafish caused inner ear abnormalities and audiosensory impairment,mimicking the patient phenotypes.Moreover,overexpression of 4 human missense PI4 KB mutant m RNAs in zebrafish embryos resulted in impaired hearing function,suggesting dominant-negative effects.Taken together,our results reveal that PI4 KB mutations can cause SNHL and inner ear malformation.PI4 KB should be included in neonatal deafness screening.

CRISPR/Cas9-mediated pou4f3 knockout induces defects in the development of the zebrafish inner ear

Objective:The zebrafish is an excellent model for studying gene function in auditory system development.Pou4f3 plays an important role in mouse hair cell formation.Here,we constructed apou4f3-knockoutTg(Brn3c:GFP)zebrafish to provide an efficient fluorescence-visualized model for studying the molecular mechanisms of ear development.Methods:Cas9/single guide RNAs targeting exon 2 ofpou4f3 were designed and injected into one-cell stage zebrafish embryos(G0 generation).The G0 generation were crossed withTg(Brn3c:GFP)zebrafish to obtainpou4f3-mutantTg(Brn3c:GFP)zebrafish.The targeting efficiency was detected by polymerase chain reaction amplification and Sanger sequencing.Zebrafish hair cells were observed by laser scanning confocal microscopyin vivo.The morphology of the otoliths and semicircular canals were analyzed.All animal experiments were approved by the Animal Care and Use Committee of Shandong Provincial Hospital,Cheeloo College of Medicine,Shandong University(approval No.2016-KY-040)on March 3,2016.Results:Thepou4f3-mutantTg(Brn3c:GFP)zebrafish line was successfully established.Fluorescence observation suggested that hair cell development was delayed inpou4f3-knockout zebrafish.Knockout ofpou4f3 also induced defects in the otoliths and semicircular canals and impaired ear function in zebrafish.Conclusion:A CRISPR/Cas9-mediatedpou4f3 mutantTg(Brn3c:GFP)zebrafish model was established for the first time to demonstrate the essential role ofpou4f3 in zebrafish ear development.Our study provides a highly efficient method for the establishment of a visualized model of gene knockout zebrafish and has the potential to allow high-throughput drug screening to explore therapeutics for related diseases.

HEARING SHIFT AND INNER EAR PATHOLOGY OF GUINEA PIGS EXPOSED TO OCTAVE BANDS OF NOICE CENTERED AT 63 Hz AND 4kHz

Twenty-four guinea pigs with normal Preyer's reflex were exposed to the octave bands of noise centered at 63 Hz and 4 kHz, 110 dB SPL. The duration of exposure was 4 and 8 hours respectively. The permanent shift of the guinea pigs at the above two frequencies was at 4-8 kHz. As the duration of exposure were 4 hours, the threshold shift at 63 Hz was smaller than that at 4 kHz. But as the duration of exposure was 8 hours, the threshold shift at 63 Hz and 4 kHz was almost the same. No morphological change of the inner ear was observed in the guinea pigs exposed for 4 hours at the octave bands of noise centered at 63 Hz and 4 kHz, but ultrastruetural abnormalities were noted. After the animals were exposed for 8 hours, morphological, nltrastructural changes in the inner ear were observed. The main pathologic changes were seen at the second torn of the cochlea. These changes in hearing physiology and pathology suggest that a high intensive low frequency noise can result in high frequency hearing loss and that the use of A-weighted levels of specific damage risk criteria for noise sources may be inappropriate.

A review of application of base editing for the treatment of inner ear disorders

The development of applications for the CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9)system has increased greatly in recent years,especially in the area of gene therapy by efficient in vivo genome editing.Although great success has been achieved in repairing and rewriting genomes through homology-directed repair coupled with Cas9 nuclease cleavage,its in vivo efficiency is insufficient for gene therapy.Base editing is a next-generation genome-editing tool that does not involve double-stranded DNA breaks and uses components of the CRISPR system together with other enzymes to make point mutations directly in cellular DNA or RNA.Base editors,composed of an engineered deaminase and a catalytically impaired CRISPR/Cas9 variant,are powerful tools for targeted base editing in cells and organisms.In non-dividing cells,base editors can directly transform one base or base pair into another,efficiently installing a point mutation.Undesired by-products of editing are seldom generated during this procedure.Herein we review the different base-editing platforms,including their deaminase recruitment strategies and editing outcomes,and the in vivo delivery of base editors.Additionally,we summarize therapeutic applications of base editing in disorders of the inner ear.

Inner Ear Size May Account for Spatial Orientation Differences Between Sexes

男性的空间方位感总体上比女性的要好,这是为什么呢?最近多伦多大学的研究者发现造成这种差别的主要原因是由于二者内耳的长度不同。

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。

突发性耳聋临床干预的队列研究

目的探讨甲泼尼松龙琥珀酸钠联合复方甘草酸苷药物在耳后注射给药方法在突发性耳聋患者中的临床效果。方法选取2020年1月至2022年6月门诊就诊的120例突发性耳聋患者为研究对象,随机数字表法分为两组,每组60例。两组均给予常规方法治疗,对照组采用耳后注射甲泼尼松龙琥珀酸钠治疗,观察组采用耳后注射甲泼尼松龙琥珀酸钠联合复方甘草酸苷治疗,比较两组纯音听阈值、血液流变学水平、同型半胱氨酸(Hcy)、D-二聚体(D-D)、耳内微循环、T淋巴细胞水平、药物安全性及复发率。结果观察组干预前后0.5、1、2及4 kHz下纯音听阈平均值的差值均好于对照组(P<0.01);观察组治疗2周后全血高切黏度、低切黏度、血浆黏度、Hcy及D-D干预前后平均值的差值均大于对照组(P<0.01);观察组治疗2周后脂质过氧化物(LPO)、胱抑素C(CysC)和内皮素(ET)水平低于对照组(P<0.01);超氧化物歧化酶(SOD)水平高于对照组(P<0.01);观察组治疗2周后CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)平均值在干预前后差值均好于对照组(P<0.01);CD8^(+)水平低于对照组(P<0.05);两组治疗过程中咽鼓管损伤、逆行感染、耳部不适、低血钾、高血压发生率差异无统计学意义(P>0.05);观察组随访6个月复发率低于对照组(P<0.05)。结论耳后注射甲泼尼松龙琥珀酸钠联合复方甘草酸苷用于突发性耳聋患者中,能降低纯音听阈值,有助于改善患者血液流变学水平和耳内微循环,降低Hcy及D-D水平,可调节患者T淋巴细胞水平,且药物安全性较高,能降低远期复发率,值得推广应用。