Expression Patterns and Implications of LaminBl in Rat Cochleae

LaminB1,a major component of the nuclear lamina,is a potent regulator of cellular proliferation and senescence and also known to be essential for neuronal migration and brain development.However,the expression patterns of LaminB1 in the rat cochleae are still not fully revealed.Utilizing immunofluorescence.Western blotting,and quantitative real-time PCR,we identified the distribution and expression of LaminB1 in the rat cochleae.Immunofluorescence staining indicated that LaminBl was mainly localized in the auditory hair cells(HCs),spiral ganglion cells(SGC),stria vascularis(STV,including spiral ligament),Reissner's membrane(RM),and limbus laminae spiralis(LLS).Western blotting analysis illustrated that the distribution of LaminBl in rat cochleae was characterized by tissue specificity.The LaminB1 protein was expressed more in SGC and basilar membrane(BM)than in STV.Meanwhile,the mRNA expression of LaminB1 displayed difference in cochlear tissues.These observations preliminarily revealed the expression patterns of LaminB1,providing a theoretical basis for further study on the role of LaminB1 in auditory function.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.

Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells

The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechanisms responsible for initiating spontaneous ATP release have not been determined.Our previous study revealed that telomerase reverse transcriptase(TERT)is expressed in the basilar membrane during the first postnatal week.Its role in cochlear development remains unclear.In this study,we investigated the expression and role of TERT in postnatal cochlea supporting cells.Our results revealed that in postnatal cochlear Kölliker’s organ supporting cells,TERT shifts from the nucleus into the cytoplasm over time.We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo.Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis,suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions.We observed increased ATP synthesis,release and activation of purine signaling systems in supporting cells during the first 10 postnatal days.The phenomenon that TERT translocation coincided with changes in ATP synthesis,release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system.Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.

Comparing accuracy of cochlear measurements on magnetic resonance imaging and computed tomography:A step towards radiation-free cochlear implantation

Objective:Computed Tomography(CT)and Magnetic Resonance Imaging(MRI)are commonly employed in pre-operative evaluation for cochlear implant surgery.However,with a decrease in the age of implantation,even minor radiation exposure can cause detrimental effects on children over their lifetime.The current study compares different cochlear measurements from CT and MRI scans and evaluates the feasibility of using only an MRI scan for radiological evaluation before cochlear implantation.Methods:A longitudinal observational study was conducted on 94 ears/47 children,employing CT and MRI scans.The CT and MRI scan measurements include,A value,B value,Cochlear duct length(CDL),twoturn cochlear length,alpha and beta angles to look for cochlear orientation.Cochlear nerve diameter was measured using MRI.The values were compared.Results:The mean difference between measurements from CT and MRI scans for A value,B value,CDL,and two-turn cochlear length values was 0.567±0.413 mm,0.406±0.368 mm,2.365±1.675 mm,and 2.063±1.477 mm respectively without any significant difference.The alpha and beta angle measures were comparable,with no statistically significant difference.Conclusion:The study suggests that MRI scans can be the only radiological investigation needed with no radiation risk and reduces the cost of cochlear implant program in the paediatric population.There is no significant difference between the measurements obtained from CT and MRI scans.However,observed discrepancies in cochlear measurements across different populations require regionally or race-specific standardized values to ensure accurate diagnosis and precision in cochlear implant surgery.This aspect must be addressed to ensure positive outcomes for patients.

Efficient Authentication System Using Wavelet Embeddings of Otoacoustic Emission Signals

Biometrics,which has become integrated with our daily lives,could fall prey to falsification attacks,leading to security concerns.In our paper,we use Transient Evoked Otoacoustic Emissions(TEOAE)that are generated by the human cochlea in response to an external sound stimulus,as a biometric modality.TEOAE are robust to falsification attacks,as the uniqueness of an individual’s inner ear cannot be impersonated.In this study,we use both the raw 1D TEOAE signals,as well as the 2D time-frequency representation of the signal using Continuous Wavelet Transform(CWT).We use 1D and 2D Convolutional Neural Networks(CNN)for the former and latter,respectively,to derive the feature maps.The corresponding lower-dimensional feature maps are obtained using principal component analysis,which is then used as features to build classifiers using machine learning techniques for the task of person identification.T-SNE plots of these feature maps show that they discriminate well among the subjects.Among the various architectures explored,we achieve a best-performing accuracy of 98.95%and 100%using the feature maps of the 1D-CNN and 2D-CNN,respectively,with the latter performance being an improvement over all the earlier works.This performance makes the TEOAE based person identification systems deployable in real-world situations,along with the added advantage of robustness to falsification attacks.

鼓室注射庆大霉素治疗顽固性梅尼埃病

目前利用庆大霉素的耳毒性治疗梅尼埃病眩晕，得到越来越多耳科医师的关注。我科自2005年来对23例反复发作的顽固性梅尼埃病眩晕患者采用鼓室注射庆大霉素治疗，现报道如下。

耳蜗中的谷氨酸-谷氨酰胺循环

谷氨酸是耳蜗内主要的传入神经递质,其对听觉的产生具有重要的作用,同时过量释放的谷氨酸引起的兴奋性毒性作用与许多内耳疾病的发生有关。耳蜗中可能存在谷氨酸摄取系统,即谷氨酸-谷氨酰胺循环。本文对耳蜗中可能存在的谷氨酸-谷氨酰胺循环学说的来源、作用机理、及相关分子的分布特点、临床意义等进行简要综述。

A dosimetric comparison between 3D-Conformal radiation therapy and intensity modulated radiation therapy plans in the treatment of posterior fossa boost in children with high risk medulloblastom

Objective： The work is a comparative study between two modalities of radiation therapy, the aim of which is to compare 3D conformal radiation therapy （3D-CRT） and intensity modulated radiation therapy （IMRT） in treating posterior fossa boost in children with high risk medulloblastorna; dosimetrically evaluating and comparing both techniques as regard target coverage and doses to organs at risk （OAR）. Methods： Twenty patients with high risk medulloblastoma were treated by 3D-CRT technique. A dosimetric comparison was done by performing two plans for the posterior fossa boost, 3D-CRT and IMRT plans, for the same patient using Eclipse planning system （version 8.6）. Results： IMRT had a better conformity index compared to 3D-CRT plans （P value of 0.000）. As for the dose homogeneity it was also better in the IMRT plans, yet it hasn＇t reached the statistical significant value. Also, doses received by the cochleae, brainstem and spinal cord were significantly less in the IMRT plans than those of 3D-CRT （P value 〈 0.05）. Conclusion： IMRT technique was clearly able to improve conformity and homogeneity index, spare the cochleae, reduce dose to the brainstem and spinal cord in comparison to 3D- CRT technique.

耳蜗传出神经系统和氨基糖苷类抗生素耳中毒

耳蜗传出神经系统在正常听觉和氨基糖苷类抗生素耳中毒中发挥着重要的作用,本文复习近年来相关文献,对耳蜗传出神经系统的形态、生理、耳蜗机械特性和听觉反应的调控机制的研究近况以及氨基糖苷类抗生素对耳蜗传出神经系统的影响进行综述。

现实与希望

美国《Science》杂志评选的2008年度世界十大科技进展之首归属于“细胞重新编程”技术（iPS）。利用这项技术可以使已经发育成熟的成体细胞，逆向转化为具有多向分化潜能的干细胞，如利用患者自身皮肤细胞经诱导重新“编程”后，能够发育成完全不同的组织细胞，例如神经细胞和心脏细胞，甚至培育移植用器官，最终帮助患者解除病痛。

胆固醇增高患者畸变产物耳声发射检查

耳声发射作为一种客观、无创、敏感的耳科检查,已在临床广泛应用,它可以反映耳蜗功能的早期变化,提示耳蜗损害[1]。我们对40例（80耳）胆固醇增高患者进行畸变产物耳声发射（distortion product otoacoustic emisson，DPOAE）检查，分析其耳蜗功能状态及听力受损情况，为临床治疗提供参考。

Gene expression in cisplatin ototoxicity and protection with p53 inhibitor

Cisplatin damages cochlear hair cells and spiral ganglion neurons through cell death signaling pathways that are not fully understood. We used focused apoptosis gene microarrays to study early changes in gene expres- sion in cochlear cultures from P3 neonatal rats treated with cisplatin (0.2 mM). After 12 hours of cisplatin treat- ment, more than 50% of the 96 genes on the array showed a significant decrease in expression, consistent with widespread cell death. However, after 3 hours of cisplatin treatment, 10 genes showed significant increase in ex- pression in total cochlear tissue. In experiments with subsets of cochlear tissues, at 3h, cisplatin induced increased expression of 12 genes in the cochlear sensory epithelium (basilar membrane) and 11 genes in the spiral ganglion (tissue of Rosenthal’s canal, containing the spiral ganglion). These included pro- and anti-apoptotic genes in- volved in the p53 signaling pathway, TNF receptor family, NF-kappaB pathway, death domain family, death effec- tor domain family, Bcl-2 family, CARD family, TRAF family, and GTP signal transduction. Although the changes in gene expression showed an overlap between basilar membrane and spiral ganglion, other changes, which may reflect the unique response of each tissue, were also observed. Pifithrin-α blocked cisplatin-induced up-regulation of genes in the p53 signaling pathway when assayed by both superarray and real time PCR. The data add to our understanding of the involvement of p53 in cisplatin-induced ototoxicity and otoprotection, conferred by the p53 inhibitor Pifithrin-α.

Lentivirus carrying the Atoh1 gene infects normal rat cochlea

Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.

Detection of apoptosis by RT-PCR array in mefloquine-induced cochlear damage

Objective To investigate the occurrence and possible mechanisms of apoptosis in cochlear epithelium and spiral ganglion neurons after mefloquine treatment. Methods We used quantitative RT-PCR apoptosis-focused gene arrays （96-well, 84 apoptosis related genes） to assess changes of gene expression in the cochlear basilar membrane （hair cells-supporting cells） and spiral ganglion neurons of rat cochlear organotypic cultures treated with 100 IxM mefloquine for 3 h. Results Significant up-or down-regulation in gene expression was detected in 23 genes in the cochlear basilar membrane, and in 32 genes in the spiral ganglion neurons compared with time-matched controls. The responding genes could be classified as pro-or anti-apoptotic, and were mainly implicated in the Bcl-2, Caspase, Card, IAP, TNF ligand / TNF receptor, Death domain / Death effector domain, DNA damage / p53, and NF-kappa B families. Synthetic analysis suggested that these families could be revised to two major pathways mainly involved in t]he death receptor-mediated signaling pathway and apoptotic mitochondrial pathway. In addition, it was found that numerous anti-apoptotic genes such as Bcl2al, Birclb, Birc3, Birc4, Bnipl, Cflar, II10, Lhx4, Mcll, Nfkbl, Prlr, Prok2, and TNF were greatly up-regulated in the cochlear tissue, which might imply the co-existence of protective response in the ceils at the early stage of mefloquine-induced damage.

Lead neurotoxicity in rat cochlear organotypic cultures

Lead is a major environmental toxicant throughout the world.Lead can induce severe neurotoxicity including irreversible hearing impairment.Many in vivo studies have shown that lead damages the auditory nervous system,but has little or no effect on cochlear sensory hair cells.To gain insights on lead ototoxic and neurotoxic effects in vitro,lead acetate (LA) was applied to postnatal day 3-4 rat cochlear organotypic cultures for 24 or 72 h with doses of 0.1,0.5,1,2 or 4 mM.After 24 or 72 h treatment with lead acetate,nearly all of cochlear sensory hair cells were intact.However,after 72 h treatment,the peripheral auditory nerve fibers projecting to the hair cells and the spiral ganglion neurons (SGN) were damaged when lead concentration exceeded 2 mM.Our results indicated that 72 h treatment with only the high doses (> 2 mM) of lead actate damaged SGNs and peripheral nerve fibers;hair cells remained structurally intact even after 4 mM treatment.These results show that lead primarily damages cochlear nerve fibers andSGNratherthanhaircells.

Calpain mediated cisplatin-induced ototoxicity in mice

Ototoxic drug-induced apoptosis of inner ear cells has been shown to be associated with calpain expression. Cisplatin has severe ototoxicity, and can induce cochlear cell apoptosis. This study assumed that cisplatin activated calpain expression in apoptotic cochlear cells. A mouse model of cisplatin-induced ototoxicity was established by intraperitoneal injection with cisplatin （2.5, 3.5, 4.5, 5.5 mg/kg）. Immunofluorescence staining, image analysis and western blotting were used to detect the expression of calpain 1 and calpain 2 in the mouse cochlea. At the same time, the auditory brainstem response was measured to observe the change in hearing. Results revealed that after intraperitoneal injection with cisplatin for 5 days, the auditory brainstem response threshold shifts increased in mice. Calpain 1 and calpain 2 expression significantly increased in outer hair cells, the spiral ganglion and stria vascularis. Calpain 2 protein expression markedly increased with an increased dose of cisplatin. Results suggested that calpain 1 and calpain 2 mediated cisplatin-induced ototoxicity in BALB/c mice. During this process, calpain 2 plays a leading role.

Ligustrazine facilitates hair cell regeneration in the cochlea following gentamicin ototoxicity

BACKGROUND： Ligustrazine （tetramethylpyrazine） decreases ototoxicity induced by gentamicin and facilitates hair cell regeneration and repair, but the precise mechanisms remain controversial.OBJECTIVE： To explore the protective effects of ligustrazine on gentamicin ototoxicity by determining heat shock protein 70 mRNA and protein expression in the cochlear stria vascularis of guinea pigs at different time points.DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Laboratory of Physiology, Shenyang Medical College of China in 2007.MATERIALS： Ligustrazine parenteral solution （Qiqihar Pharmaceutical Factory, China） and gentamicin sulfate （Shenyang First Pharmaceutical Factory, China） were used in this experiment.METHODS： White guinea pigs with red eyes were randomly intraperitoneally administered gentamicin sulfate injection ＋ saline, gentamicin sulfate injection ＋ ligustrazine, and ligustrazine ＋ saline, respectively.MAIN OUTCOME MEASURES： Auditory brains tern response threshold was measured. Immunohistochemistry, in situ hybridization, and image analyzing techniques were utilized to determine heat shock protein 70 mRNA and protein expression in cochlear stria vascularis of guinea pigs.RESULTS： Following gentamicin ototoxicity, the auditory brainstem response threshold increased, peaked on day 3, and then decreased with increased time after drug withdrawal. The auditory brainstem response threshold was significantly diminished following ligustrazine intervention, but recovered to normal on day 30 （P〉0.05）. Heat shock protein 70 expression also increased, peaked on day 3, and then decreased in the cochlear stria vascularis of guinea pigs following gentamicin ototoxicity. Ligustrazine intervention resulted in decreased heat shock protein 70 expression in the cochlear stria vascularis, which recovered to normal on day 14. Heat shock protein 70 mRNA expression increased in the cochlear stria vascularis following gentamicin ototoxicity, but ligustrazine intervention resulted in decreased levels. CONCLUSION： Ligustrazine significantly ameliorated gentamicin ototoxicity by reducing heat shock protein 70 mRNA and protein expression in the cochlear stria vascularis.

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCⅡ and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy(SR-SIM) were used in the study. CD4^+ and CD8^+ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons,CD4^+ and CD8^+ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus,macrophages, CD4^+ and CD8^+ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4^+ and CD8^+ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.

Backward Propagation of Otoacoustic Emissions

Normal mammalian ears not only detect but also generate sounds. The ear-generated sounds, i.e., otoacoustic emissions (OAEs), can be measured in the external ear canal using a tiny sensitive microphone. In spite of wide applications of OAEs in diagnosis of hearing disorders and in studies of cochlear functions, the question of how the cochlea emits sounds remains unclear. The current dominating theory is that the OAE reaches the cochlear base through a backward traveling wave. However, recently published works, including experimental data on the spatial pattern of basilar membrane vibrations at the emission frequency, demonstrated only forward traveling waves and no signs of backward traveling waves. These new findings indicate that the cochlea emits sounds through cochlear fluids as compression waves rather than through the basilar membrane as backward traveling waves. This article reviews different mechanisms of the backward propagation of OAEs and summarizes recent experimental results.