Bhlhe40 protects cochlear hair cell-like HEI-OC1 cells against H_(2)O_(2) ‑triggered oxidative injury

Background:Cochlear hair cell injury is a common pathological feature of hearing loss.The basic helix-loop-helix family,member e40(Bhlhe40),a gene belonging to the basic helix-loop-helix(bHLH)family,exhibits strong transcriptional repression activity.Methods:Oxidative damage,in House Ear Institute-Organ of Corti 1(HEI-OC1)cells,was caused using hydrogen peroxide(H2O2).The Ad-Bhlhe40 particles were constructed to overexpress Bhlhe40 in HEI-OC1 cells.Various assays including cell counting kit-8(CCK-8),terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay(TUNEL),flow cytometry,immunofluorescence,and corresponding commercial kits were employed to investigate the impacts of Bhlhe40 on cell viability,apoptosis,oxidative stress levels,mitochondrial membrane potential and cellular senescence.Additionally,a dual-luciferase reporter assay was performed to confirm the targeting of the histone deacetylases 2(Hdac2)by Bhlhe40.Results:The results revealed that Bhlhe40 was downregulated in H_(2)O_(2)-treated HEI-OC1 cells,but its overexpression improved cell viability and mitigated H_(2)O_(2)-induced oxidative injury in HEI-OC1 cells with increase of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)activities and decrease of reactive oxygen species(ROS)levels.Besides,overexpression of Bhlhe40 suppressed H_(2)O_(2)-triggered cell senescence,as evidenced by the fact that the upregulation of P53,P21,and P16 in HEI-OC1 cells treated with H2O2 were all alleviated by Bhlhe40 overexpression.And we further verified that overexpression of Bhlhe40 could inhibit the expression of Hdac2,which may be related to the repression of Hdac2 transcription.Conclusion:This study suggests that Bhlhe40 plays a protective role against senescence and oxidative damage in cochlear hair cells exposed to H2O2.

The role of Rho GTPase family in cochlear hair cells and hearing

Rho GTPases are essential regulators of the actin cytoskeleton.They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics,development,proliferation,survival,and regeneration.During the development of cochlear hair cells,Rho GTPases are activated by various extracellular signals through membrane receptors to further stimulate multiple downstream effectors.Specifically,RhoA,Cdc42,and Rac1,members of the classical subfamily of the Rho GTPase family,regulate the development and maintenance of cilia by inducing the polymerization of actin monomers and stabilizing actin filaments.In addition,they also regulate the normal morphology orientation of ciliary bundles in auditory hair cells,which is an important element of cell polarity regulation.Moreover,the actin-related pathways mediated by RhoA,Cdc42,and Rac1 also play a role in the motility of outer hair cells,indicating that the function of Rho GTPases is crucial in the highly polar auditory sensory system.In this review,we focus on the expression of RhoA,Cdc42,and Rac1 in cochlear hair cells and how these small molecules participate in ciliary bundle morphogenesis and cochlear hair cell movement.We also discuss the progress of current research investigating the use of these small molecules as drug targets for deafness treatment.

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.

Neurodynamics analysis of cochlear hair cell activity

There have been many studies on the effect of cochlea basal membrane movement on the resolution of different frequencies and intensities.However,these studies did not take into account the influence of power and energy consumption of the hair cells in the process of the electromotility movement,as well as the neurodynamic mechanism that produced this effect.This makes previous studies unable to fully clarify the function of outer hair cells(OHCs)and the mechanism of sound amplification.To this end,we introduce the gate conductance characteristics of the hair cells in the mechanical process of increasing frequency selectivity.The research finds that the low attenuation of OHCs membrane potential and the high gain in OHC power and energy consumption caused that OHC amplification is driven by electromotility.The research results show that the amplification of the OHCs is driven by low attenuation of membrane potential and high gain of power and energy consumption.This conclusion profoundly reveals the physiological mechanism of the electromotility movement.

Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds： supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein （EGFP） was used to track cell division during hair cell transformation.5-Bromo-2′-deoxyuridine （BrdU） was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Modulation of copper transporters in protection against cisplatin-induced cochlear hair cell damage

Cisplatin belongs to platinum-based drugs and is widely used in cancer chemotherapy.Ototoxicity is one of the major dose limiting side-effects of cisplatin.For toxicity to occur cisplatin must first be transported from the bloodstream into cochlear cells.Three copper transporters are considered pathways for regulating the uptake and translocation of cisplatin into cells:Ctr1,ATP7A and ATP7B.Our recent study with cochlear organotypic cultures shows that cochlear hair cells can be destroyed by cisplatin at low concentrations from 10μm to 100μn.However,high doses of cisplatin cannot damage hair cells,maybe due to intrinsic feedback reactions that increase export of platinum by ATP7B when the platinum concentration is high in extracellular space.Cimitidine is a specific copper transporter inhibitor that can block the entrance of copper and platinum,and may prevent cisplatin-induced cochlear hair cell injury.To evaluate this hypothesis,we treated cochlear organotypic cultures with cisplatin (10 μm or 50 μm) alone,or cisplatin combined with cimitidine at concentrations ranging from 10-2000 μm for 48 hours.cisplatin at 10 μm damaged about 20% hair cells.In contrast,when cimitidine (10 μm,100 μm and 2000 μm) was added to the culture,near 100% cochlear hair cell survived.At higher concentration (50 μm),cisplatin destroyed about 80% of cochlear hair cells.However,100 μmcimitidine rescued about 50% hair cells from cisplatin damage,and 2000μm cimitidine protected about 80% hair cells.The data of western blot showed that CTR1 and ATP7B expressions were increased in cisplatin treated cochlear tissue,but cimitidine significantly reduced CTR1 and ATP7B.In addition,ATP7A expression was depressed a little after cisplatin treatment.Considering that Ctr1 is involved in copper and platinum influx,but the ATP7A and ATP7B are copper export transporters,the results suggest that cimitidine can effectively block the entrance by copper transporters and stop the influx of cisplatin.

Correlation of PDCD5 and Apoptosis in Hair Cells and Spiral Ganglion Neurons of Different Age of C57BL/6J Mice

This study examined the expression pattern of programmed cell death 5 (PDCD5) in co-chlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice.Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3,6,9 or 12 months).PDCD5 expression was detected by using immunohistochemistry,real-time PCR and Western blot.Morphological change of the cochleae was also evaluated by using immunoassay.The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice,as well as gradually increased apoptosis of cochlear hair cells and SGNs.In addition,we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing.It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs,and thereby plays a role in the pathogenesis of presbycusis.Thus,PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

Distribution of Prestin on Outer Hair Cell Basolateral Surface

Prestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility and is expressed on the OHC surface. Previous studies revealed that OHC electromotility and its associated nonlinear capacitance were mainly located at the OHC lateral wall and absent at the apical cuticular plate and the basal nucleus region. Immunofluorescent staining for prestin also failed to demonstrate prestin expression at the OHC basal ends in whole-mount preparation of the organ of Corti. However, there lacks a definitive demonstration of the pattern of prestin distribution. The OHC lateral wall has a trilaminate organization and is composed of the plasma membrane, cortical lattice, and subsurface cisternae. In this study, the location of prestin proteins in dissociated OHCs was examined using immunofluorescent staining and confocal microscopy. We found that prestin was uniformly expressed on the basolateral surface, including the basal pole. No staining was seen on the cuticular plate and stereocilia. When co-stained with a membrane marker di-8-ANEPPS, prestin-labeling was found to be in the outer layer of the OHC lateral wall. After separating the plasma membrane from the underlying subsurface cisternae using a hypotonic extracellular solution, prestin-labeling was found to be in the plasma membrane, not the subsurface cisternae. The data show that prestin is expressed in the plasma membrane on the entire OHC basolateral surface.

Mammalin cochlear supporting cells transdifferentiation into outer hair cells

Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection of cells that could take up BrdU. Results After 15 days of gentamicin treatment, all of the animals were proved to be deafened with significant increases of ABR thresholds, compared with control group. The findings in immunocytochemical stained samples and scanning electron microscopy revealed that BrdU labeled nuclei were observed in the cochlea in all of the deafened animals most commonly in the regions of the first-row and second-row Deiter’s cells (DCs) and occasionally in the regions of the third-row DCs. Conclusion We suggest that, under sufficient drug and enough time, the bat cochlear supporting cells can directly transdifferentiate into the outer hair cells after aminoglycoside exposure. This transdifferentation process is essential for repair of outer hair cells and recovery of normal function after gentamicin exposure.

Hopf Amplification Originated from the Force-Gating Channels of Auditory Hair Cells

The sense of mammalian hearing exhibits nonlinear phenomena which are most significant to hearing function, such as nonlinear dynamic compression, nonlinear tuning and combination tones. These nonlinear phenomena are suggested to originate from the Hopf amplification within the cochlea, while the mechanism underlying the Hopf amplification remains elusive. According to the experimental results of force-gating channel operation in hair cells, through a theoretic model, this work reveals a velocity-dependent open probability of force-gating channels in auditory hair cells, and a velocity-dependent active force produced by the force-gating channel operating, which makes sensors hear typical Hopf vibrators with nonlinear hearing phenomena.

Acetylcholine-induced calcium oscillation in isolated outer hair cells in guinea pig

Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.

Inner ear hair cell regeneration A look from the past to the future

Most recent studies on regeneration of inner ear hair cells focus on use of stem cells, gene therapy and neurotrophic factors. Cochlear gene therapy has been successfully used in the treatment of neu- rosensory hearing loss. This suggests that cochlear hair cell regeneration is possible. The objective of this paper is to review research and clinical application of inner near hair cell regeneration.

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.

HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possible utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through mor-phology observation, immunnocytochemical staining and RT-PCR analysis. The Hath1 gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1(a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differentiate into hair cell-like cells when forced to express Hath1. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

Noise exposure induced cochlear hair cell death pathways in guinea pig

Objective To understand the mechanism of noise exposure induced outer hair cells(OHCs) death pathways. Methods Thirty two guinea pigs were used in this study. The animals were either exposed for 4 h/day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused with MNNG. After auditory test, the cochleae of animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. F-actin staining was used to determine missing OHCs. Caspase-3 was detected in living organ of Corti whole mounts using the fluorescent probe. The single strand DNA (ssDNA) in apoptotic OHCs in guinea pigs and apoptosis inducing factor (AIF) in hair cells in guinea pigs were examined by immunohistology method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. Results (1) Both apoptotic and necrotic hair cells appeared following noise exposure. (2) Noise exposure induced single strand DNA in apoptotic OHCs but not in the normal OHCs. (3) Either after noise exposure or after MNNG perfusion, apoptotic OHCs were featured by nuclear condensation or fragmentation with caspase-3 activation, whereas necrotic OHCs were characterized by nuclear swelling without caspase-3 activation. (4) In normal organ of Corti, AIF was located in the mitochondria areas. After noise exposure, AIF was translocated from mitochondria in apoptotic and necrotic OHCs. Conclusion These findings indicate that noise exposure damages DNA in the OHC, which triggers action of Caspase-3. Subsequently, AIF is translocated to the nucleus, leading to DNA damage and OHCs death.

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

In vitro Differentiation of Adipose-Derived Stem Cells into Hair Cell-Like Cells in Guinea Pigs

Objective Adipose-derived stem cells(ADSCs) are suggested to possess a highly plastic ability to differentiate into several specific cell types in addition to adipocyte lineages, including germ layer tissue-specific cell lineages such as chondrocyte, myocyte, neuronal, and osteoblast lineages. The aim of this study is to establish an in vitro culture technique for ADSCs in an adult guinea pig model that facilitate their differentiation into hair cell-like cells. Materials and Methods Cells from inguinal fat pads in adult guinea pigs were cultured with β-mercaptoethanol, RA, Forskorin, Heregulin, bFGF, BDNF and EGF. Cellular differentiation was examined using immunocytochemistry techniques. Results The ADSCs demonstrated hair cell immunophenotypes with expression of epitopes of the hair cell marker protein myosin Ⅶa. Conclusion ADSCs from adult guinea pig adipose tissue can differentiate into hair cell-like cells when cultured in vitro. ADSCs may serve as seed cells for tissue engineering.

EXPERIMENTAL STUDY OF COCHLEAR HAIR CELL LOSS IN THE GUINEA PIG

The damage to spiral organs related to age and sex in guinea pigs was studied by the surface preparation technique. Hair cell loss of spiral organs of the following four age groups: 8month, 1, 2, and 2.5-year old pigs, was assessed. Each group was divided Into two subgroups according to different sexes. The decrease of hair cell population was linearly related to age, but not related to sex. The damage rate was greater in outer hair cells than in inner hair calls. In all of animals of different ages and both sexes, outer hair cell loss was prominent at the apex of cochlea. Less damage to outer hair cell was found at the base of cochlea. Damage to inner hair cell was also increased with age, but to a lesser extent.

Muscarinic Acetylcholine Receptor Subtype Expression in Type Vestibular Hair Cells of Guinea Pigs

Recent studies have demonstrated that five subtypes （M1-M5） of muscarinic acetylcholine receptor （mAChR） are expressed in the vestibular periphery. However, the exact cellular location of the mAChRs is not clear. In this study, we investigated whether there is the expression of M1-M5 muscarinic receptor mRNA in isolated type Ⅱ vestibular hair cells of guinea pig by using single-cell RT-PCR. In vestibular end-organ, cDNA of the expected size was obtained by RT-PCR. Moreover, mRNA was identified by RT-PCR from individually isolated type Ⅱ vestibular hair cells （single-cell RT-PCR）. Sequence analysis confirmed that the products were M1-M5 mAChR. These results dem-onstrated that M1-M5 mAChR was expressed in the typeⅡvestibular hair cells of the guinea pig, which lends further support for the role of M1-M5 mAChR as a mediator of efferent cholinergic signalling pathway in vestibular hair cells.