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.

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.

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.

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.

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.

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.

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

Protective effects of the cochlear efferent system on the outer hair cells against intense sound:evidences from DPOAEs

It has been revealed in recent years that contralateral acoustic stimulation can affect cochlear active mechanisms through activating medial olivocochlear system (MOC) of the cochlear efferent nerve fibers. The MOC is therefore postulated to exert protective effects on outer hair cells (OHCs) under intense sound condition. In this study the effects of 4 kHz intense tone exposure on distortion product otoacoustic emissions (DPOAEs) in guinea pigs with and without contralateral white noise stimulation were observed so that to investigate the protective effects of MOC on OHCs. The results showed that DPOAEs obviously deceased after the intense tone exposure in all animals, while both the amplitude reduction and the affected frequency range of DPOAEs were smaller in animals with simultaneously delivered contralateral white noise during the tone exposure than that in animals without colltralateral acoustic stimulation. The above results may suggest some protective nature of the contralateral sound stimulating effects which might be mediated through the activity of MOC. These perhaps can serve as the evidence that the protective mechanism against intense sound operates in the outer hair cells which are strongly innervated by MOC

Basic flbroblast growth factor protects auditory neurons and hair cells from noise exposure and glutamate neurotoxicity

The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.

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.

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.

Quantitative Relations between Outer Hair Cell Electromotility and Nonlinear Capacitance

The electrically evoked somatic motility of outer hair cells （OHC）, briefly termed OHC electromotility, plays a crucial role in cochlear amplification that underlies the remarkably high sensitivity and frequency selectivity of the mammalian hearing. Accompanying OHC electromotility is a voltage-dependent gating charge movement within the cell lateral membrane, manifested as a measurable nonlinear capacitance （NLC） in OHCs. The electromotility and NLC of OHCs are highly correlated by sharing a common molecular substrate, the motor protein prestin. In this study, we systematically characterized the quantitative relationship between OHC electromotility and NLC in their voltage dependences for the purpose of further understanding the electromechanical transduction in OHCs. The results demonstrated that the two possess differing voltage dependences with the V^2 of electromotility consistently being -20 mV depolarized in comparison with that of NLC although their slope factors a are statistically identical. Further investigations showed that the initial state of OHCs influences the voltage dependence of electromotility but not that of NLC, indicating that some biophysical factors other than the motor protein per se are involved in the process of OHC length changes. We proposed that the cytoskeletal spectrin-actin framework underneath the OHC plasma membrane and the cell＇ s turgor are the two most probable factors that cause the voltage-dependence discrepancy between OHC electromotility and NLC.

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.

Apoptosis in inner ear sensory hair cells

Apoptosis, or controlled cell death, is a normal part of cellular lifespan. Cell death of cochlear hair cells causes deafness; an apoptotic process that is not well understood. Worldwide, 1.3 billion humans suffer some form of hearing loss, while 360 million suffer debilitating hearing loss as a direct result of the absence of these cochlear hair cells(Worldwide Hearing, 2014). Much is known about apoptosis in other systems and in other cell types thanks to studies done since the mid-20 th century. Here we review current literature on apoptosis in general, and causes of deafness and cochlear hair cells loss as a result of apoptosis. The family of B-cell lymphoma(Bcl) proteins are among the most studied and characterized. We will review current literature on the Bcl2 and Bcl6 protein interactions in relation to apoptosis and their possible roles in vulnerability and survival of cochlear hair cells.

Outer hair cells isolation from postnatal Sprague-Dawley rats

Outer hair cells (OHCs) damage is a general phenomenon in clinical disorders such as noise-induced hearing loss and drug-induced hearing loss.In order to elucidate the mechanism underlying these disorders,OHCs-its diseased region needs to be deeply investigated.However,OHCs array on the basilar membrane which contains massive cells with different types.Therefore,to isolate OHCs from this huge population is significant for revealing its pathological and molecular changes during disease processing.In the present study,we tried to isolate OHCs from the commonly used animal model-Sprague-Dawley (SD) rats.By separating outer hair cells from SD rats with different day ages,we found that 9 days after birth was a suitable time for the separation of the OHCs.At this time,the number of OHCs isolated from rats was large,and the cell morphology was typical of cylindrical shape.OHCs isolated using this method are histologically suitable and quantitatively adequate for molecular biological and electrophysiological analyses.

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

Phosphatidylserine-positive particles in the apical domain of sensory hair cells

Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles labeled by Annexin V in the apical portion of hair cells. The following characteristics of the PS-positive vesicles were noticed using scanning confocal fluorescence microscopy:(1) variable sizes around 200 nm; (2) variable distribution patterns (either uniformly along individual stereocilia in the hair bundle or irregular) in the stereocilia from cell to cell; (3) variable sizes and numbers at locations along the border of the cuticular plate (CP), with a large number of them located at the vestigal kinocilial location; (4) motility with some of the vesicles during the observation period; (5) increase in PS labeling and the number of PS-positive vesicles after loud sound stimulation; and (6) decreased PS labeling and PS-positive vesicle numbers following treatment with LY-294002, a PI3 -kinase inhibitor. These results suggest that the presence of PS-positive vesicles at the apical area of hair cells may be indicative of vesicle shedding or transportation of a protein or rafts.

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

30 and 32 kDa outer membrane proteins of Bordetella pertussis as a modulator on promoting degranulation of mast cells

The correlation between the activities of the outer membrane proteins (OMPs) of Bordetella pertussis and the IgE-mediated asthma was investigated in the present study, in which the OMPs of B.pertussis and their components were prepared by detergent treatment and chromatography, and the molecular weights of the OMPs components were determined by SDS-PAGE. The amounts of total as well as the ovalbumin (OVA)-specific IgE induced by dead B.pertussis whole bacterial vaccine on guinea pigs were detected by ELISA. Meanwhile, the effect of the OMPs and their components to promote the degranulation of guinea pig mast cells was observed by using the mast cell degranulation test, and ELISA assay was used to measure the histamine levels in the supernatants from the mast cell cultures. Histamine sensitive test was used to demonstrate the effects of the OMPs and their components to increase the histamine lethal sensitivity in mice. It was found that four components with molecular weights of 30, 32, 38 and 69 kDa could be obtained from the OMPs of B.pertussis, and the dead whole bacteria vaccine of B.pertussis had the ability to increase the levels of the total as well as the OVA-specific IgE in sera of guinea pigs. The OMPs and their 30 and 32 kDa components demonstrated significantly enhancing effect on the degranulation of guinea pig mast cells, and the histamine levels in the supernatants from the mast cell culture treated with OMPs and their 30 and 32 kDa components were also significantly increased. It is evident that the strong adjuvant activity and the enhancing effect to degranulation of mast cells and the release of histamine of certain outer membrane components of B.pertussis could be demonstrated as revealed by the results of the present study, suggesting the possibility of a close relationship between the infection of vaccination with B.pertussis and the IgE-mediated asthma.